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    Thermo Fisher flow cytometry analysis
    Characterization of Zscan4 knockdown cells a , Confirmation of Zscan4 knockdown and rescue by qPCR analysis using a common primer for all Zscan4 paralogs (top) and Western blot analysis (bottom). Cells were cultured for 3 days in Dox+ or Dox− conditions. shCont (control shRNA in the same parental cells) was used to exclude off-target effects. See Supplementary Fig 7 and 8 for additional controls. b , Reduction of proliferation by Zscan4 knockdown, until cells died abruptly at passage 8 (P+8). Rescue improved proliferation. Assays were done in triplicate in four independent experiments. c , Annexin-V Apoptosis assay performed by flow <t>cytometry.</t> d , Karyotype deterioration seen after Zscan4 knockdown.
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    Images

    1) Product Images from "Zscan4 regulates telomere elongation and genomic stability in ES cells"

    Article Title: Zscan4 regulates telomere elongation and genomic stability in ES cells

    Journal: Nature

    doi: 10.1038/nature08882

    Characterization of Zscan4 knockdown cells a , Confirmation of Zscan4 knockdown and rescue by qPCR analysis using a common primer for all Zscan4 paralogs (top) and Western blot analysis (bottom). Cells were cultured for 3 days in Dox+ or Dox− conditions. shCont (control shRNA in the same parental cells) was used to exclude off-target effects. See Supplementary Fig 7 and 8 for additional controls. b , Reduction of proliferation by Zscan4 knockdown, until cells died abruptly at passage 8 (P+8). Rescue improved proliferation. Assays were done in triplicate in four independent experiments. c , Annexin-V Apoptosis assay performed by flow cytometry. d , Karyotype deterioration seen after Zscan4 knockdown.
    Figure Legend Snippet: Characterization of Zscan4 knockdown cells a , Confirmation of Zscan4 knockdown and rescue by qPCR analysis using a common primer for all Zscan4 paralogs (top) and Western blot analysis (bottom). Cells were cultured for 3 days in Dox+ or Dox− conditions. shCont (control shRNA in the same parental cells) was used to exclude off-target effects. See Supplementary Fig 7 and 8 for additional controls. b , Reduction of proliferation by Zscan4 knockdown, until cells died abruptly at passage 8 (P+8). Rescue improved proliferation. Assays were done in triplicate in four independent experiments. c , Annexin-V Apoptosis assay performed by flow cytometry. d , Karyotype deterioration seen after Zscan4 knockdown.

    Techniques Used: Real-time Polymerase Chain Reaction, Western Blot, Cell Culture, shRNA, Apoptosis Assay, Flow Cytometry, Cytometry

    Zscan4 is transiently expressed in ES cells a , Zscan4 expression visualized by RNA in situ hybridization and GFP-Emerald reporter gene ( green ). b , FACS analysis of pZscan4-Emerald cells. c , Time-course fluorescent/DIC images of pZscan4-Emerald cells after sorting and culturing only Em+ cells or Em− cells. d , Flow cytometry analysis of cells shown in c . e , Images of pZscan4-CreERT2 cells maintained in tamoxifen and stained with X-gal at each passage. Inserts: a single colony. f , A % fraction of LacZ+ cells measured by flow cytometry analysis using CMFDG fluorescence LacZ assay: tamoxifen was present continuously (Tam+: P1–P9), only for the first three passages (Tam+: P1–P3), or absent (Tam−, control).
    Figure Legend Snippet: Zscan4 is transiently expressed in ES cells a , Zscan4 expression visualized by RNA in situ hybridization and GFP-Emerald reporter gene ( green ). b , FACS analysis of pZscan4-Emerald cells. c , Time-course fluorescent/DIC images of pZscan4-Emerald cells after sorting and culturing only Em+ cells or Em− cells. d , Flow cytometry analysis of cells shown in c . e , Images of pZscan4-CreERT2 cells maintained in tamoxifen and stained with X-gal at each passage. Inserts: a single colony. f , A % fraction of LacZ+ cells measured by flow cytometry analysis using CMFDG fluorescence LacZ assay: tamoxifen was present continuously (Tam+: P1–P9), only for the first three passages (Tam+: P1–P3), or absent (Tam−, control).

    Techniques Used: Expressing, RNA In Situ Hybridization, FACS, Flow Cytometry, Cytometry, Staining, Fluorescence

    2) Product Images from "Effects of the NUP98-DDX10 oncogene on primary human CD34+ cells: Role of a conserved helicase motif"

    Article Title: Effects of the NUP98-DDX10 oncogene on primary human CD34+ cells: Role of a conserved helicase motif

    Journal: Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, U.K

    doi: 10.1038/leu.2010.42

    NUP98-DDX10 disrupts myeloid and erythroid differentiation of primary human CD34+ cells. Human CD34+ cells were retrovirally transduced with either control MSCV-IRES-GFP vector or vector expressing NUP98-DDX10, and cells were sorted for GFP positivity. ( a ) One thousand cells were seeded into each of two duplicate plates for CFC assay and the experiment was repeated 6 independent times. Representative plates without magnification (upper panels) and low power photomicrographs of representative erythroid colonies (middle panels) are shown. Cytospin smears were prepared from the CFC plates and stained with Giemsa (lower panels). Photomicrographs were taken from representative fields with a 60X oil objective. Representative cells are indicated by arrows. B: blast; MM: mature myeloid; IM: intermediate myeloid; ME: mature erythroid; IE: intermediate erythroid. ( b ) 500 cell differential counts were performed to distinguish 5 groups of cells. Cells with blast and promyelocyte morphology were counted as primitive; those with myelocyte/metamyelocyte morphology as intermediate myeloid; those with band, segmented neutrophil, monocyte, and macrophage morphology as mature myeloid; those with intermediate hemoglobinization as intermediate erythroid; and those with full hemoglobinization as mature erythroid. Averages from 6 independent experiments are shown; error bars represent standard deviations. The P value was obtained by comparing to control using a two-sample, equal variance, two-tailed distribution t-test. ( c ) Flow cytometry for myeloid differentiation: Cells from the CFC plates were harvested and stained with CD45, CD33 and CD11b; the CD33+ gate was plotted on a histogram to show CD11b expression compared to control (lower panel). ( d ) Flow cytometry for erythroid differentiation: Cells from the CFC plates were harvested and stained with antibodies to CD45 and CD235a. The CD235a + gate was plotted on a histogram (lower panel) to show the expression of CD235a relative to control cells. The percentages of cells falling within each gate are shown.
    Figure Legend Snippet: NUP98-DDX10 disrupts myeloid and erythroid differentiation of primary human CD34+ cells. Human CD34+ cells were retrovirally transduced with either control MSCV-IRES-GFP vector or vector expressing NUP98-DDX10, and cells were sorted for GFP positivity. ( a ) One thousand cells were seeded into each of two duplicate plates for CFC assay and the experiment was repeated 6 independent times. Representative plates without magnification (upper panels) and low power photomicrographs of representative erythroid colonies (middle panels) are shown. Cytospin smears were prepared from the CFC plates and stained with Giemsa (lower panels). Photomicrographs were taken from representative fields with a 60X oil objective. Representative cells are indicated by arrows. B: blast; MM: mature myeloid; IM: intermediate myeloid; ME: mature erythroid; IE: intermediate erythroid. ( b ) 500 cell differential counts were performed to distinguish 5 groups of cells. Cells with blast and promyelocyte morphology were counted as primitive; those with myelocyte/metamyelocyte morphology as intermediate myeloid; those with band, segmented neutrophil, monocyte, and macrophage morphology as mature myeloid; those with intermediate hemoglobinization as intermediate erythroid; and those with full hemoglobinization as mature erythroid. Averages from 6 independent experiments are shown; error bars represent standard deviations. The P value was obtained by comparing to control using a two-sample, equal variance, two-tailed distribution t-test. ( c ) Flow cytometry for myeloid differentiation: Cells from the CFC plates were harvested and stained with CD45, CD33 and CD11b; the CD33+ gate was plotted on a histogram to show CD11b expression compared to control (lower panel). ( d ) Flow cytometry for erythroid differentiation: Cells from the CFC plates were harvested and stained with antibodies to CD45 and CD235a. The CD235a + gate was plotted on a histogram (lower panel) to show the expression of CD235a relative to control cells. The percentages of cells falling within each gate are shown.

    Techniques Used: Transduction, Plasmid Preparation, Expressing, Staining, Two Tailed Test, Flow Cytometry, Cytometry

    Mutation of the YIHRAGTAR motif diminishes the ability of NUP98-DDX10 to disrupt the differentiation of primary human CD34+ cells. Primary human CD34+ cells were retrovirally transduced with either control MSCV-IRES-GFP vector or vector expressing NUP98-DDX10 or vector expressing NUP98-DDX10/3Q, and cells were sorted for GFP positivity. ( a ) One thousand cells were seeded into each of two duplicate plates for CFC assay and the experiment was repeated 3 independent times. Representative plates without magnification (upper panel) and low power photomicrographs of representative erythroid colonies (lower panel). ( b ) 500 cell differential counts were performed to distinguish 5 groups of cells. Averages from 3 – 6 independent experiments are shown; error bars represent standard deviations. The P value was obtained by comparing the samples using a two-sample, equal variance, two-tailed distribution t-test. ( c ) Flow cytometry for myeloid differentiation: Cells from the CFC plates were harvested and stained with CD45, CD33 and CD11b; the CD33+ gate was plotted on a histogram to show CD11b expression (lower panel). ( d ) Flow cytometry for erythroid differentiation: Cells from the CFC plates were harvested and stained with antibodies to CD45 and CD235a. The CD235a + gate was plotted on a histogram (lower panel) to show the expression of CD235a. The percentages of cells falling within each gate are shown. The mean fluorescence intensity (MFI) is the arithmetic mean of the linear scaled fluorescence intensity.
    Figure Legend Snippet: Mutation of the YIHRAGTAR motif diminishes the ability of NUP98-DDX10 to disrupt the differentiation of primary human CD34+ cells. Primary human CD34+ cells were retrovirally transduced with either control MSCV-IRES-GFP vector or vector expressing NUP98-DDX10 or vector expressing NUP98-DDX10/3Q, and cells were sorted for GFP positivity. ( a ) One thousand cells were seeded into each of two duplicate plates for CFC assay and the experiment was repeated 3 independent times. Representative plates without magnification (upper panel) and low power photomicrographs of representative erythroid colonies (lower panel). ( b ) 500 cell differential counts were performed to distinguish 5 groups of cells. Averages from 3 – 6 independent experiments are shown; error bars represent standard deviations. The P value was obtained by comparing the samples using a two-sample, equal variance, two-tailed distribution t-test. ( c ) Flow cytometry for myeloid differentiation: Cells from the CFC plates were harvested and stained with CD45, CD33 and CD11b; the CD33+ gate was plotted on a histogram to show CD11b expression (lower panel). ( d ) Flow cytometry for erythroid differentiation: Cells from the CFC plates were harvested and stained with antibodies to CD45 and CD235a. The CD235a + gate was plotted on a histogram (lower panel) to show the expression of CD235a. The percentages of cells falling within each gate are shown. The mean fluorescence intensity (MFI) is the arithmetic mean of the linear scaled fluorescence intensity.

    Techniques Used: Mutagenesis, Transduction, Plasmid Preparation, Expressing, Two Tailed Test, Flow Cytometry, Cytometry, Staining, Fluorescence

    3) Product Images from "Signaling from the Sympathetic Nervous System Regulates Hematopoietic Stem Cell Emergence during Embryogenesis"

    Article Title: Signaling from the Sympathetic Nervous System Regulates Hematopoietic Stem Cell Emergence during Embryogenesis

    Journal: Cell Stem Cell

    doi: 10.1016/j.stem.2012.07.002

    Gata3 Regulation of AGM HSCs Is Secondary to Its Role in the Sympathetic Nervous System (A) In situ hybridization with riboprobes for Th , Gata2 , and Hand1 on Gata3 +/+ and Gata3 −/− E11.5 embryo sections. (B) Summary of repopulation analysis of recipients injected with uncultured E11/11.5 AGM cells (1 ee) from Gata3 +/+ , Gata3 +/− , and Gata3 −/− embryos that had received catecholamine derivatives in vivo through the drinking water from E8.5. See also Figure S2 . (C) Summary of repopulation analysis of recipients injected with uncultured E11/11.5 AGM cells (1 ee) from Th +/+ , Th +/− , and Th −/− embryos. See also Figure S2 . (D) Percentage of recipients repopulated with wild-type E11.5 AGMs that had been cultured in the presence or absence of a Th inhibitor. The number of repopulated/total recipients is indicated above each bar. (E and F) Flow cytometry analysis of ckit and CD45 expression on cells from E11.5 wild-type AGMs cultured in the absence (E) or presence (F) of a Th inhibitor. The percentage of apoptotic cells within the ckit+CD45+ population is shown.
    Figure Legend Snippet: Gata3 Regulation of AGM HSCs Is Secondary to Its Role in the Sympathetic Nervous System (A) In situ hybridization with riboprobes for Th , Gata2 , and Hand1 on Gata3 +/+ and Gata3 −/− E11.5 embryo sections. (B) Summary of repopulation analysis of recipients injected with uncultured E11/11.5 AGM cells (1 ee) from Gata3 +/+ , Gata3 +/− , and Gata3 −/− embryos that had received catecholamine derivatives in vivo through the drinking water from E8.5. See also Figure S2 . (C) Summary of repopulation analysis of recipients injected with uncultured E11/11.5 AGM cells (1 ee) from Th +/+ , Th +/− , and Th −/− embryos. See also Figure S2 . (D) Percentage of recipients repopulated with wild-type E11.5 AGMs that had been cultured in the presence or absence of a Th inhibitor. The number of repopulated/total recipients is indicated above each bar. (E and F) Flow cytometry analysis of ckit and CD45 expression on cells from E11.5 wild-type AGMs cultured in the absence (E) or presence (F) of a Th inhibitor. The percentage of apoptotic cells within the ckit+CD45+ population is shown.

    Techniques Used: In Situ Hybridization, Injection, In Vivo, Cell Culture, Flow Cytometry, Cytometry, Expressing

    Catecholamines Can Rescue HSC Activity In Vitro in the Absence of Circulation (A) Ventral halves of dorsal aortae from E11 Gata3 +/+ and Gata3 −/− embryos were analyzed for Nos3 expression by quantitative RT-PCR. Data is representative of two independent experiments. (B) E11.5 Gata3 +/+ and Gata3 −/− embryo sections were stained for Nos3 (red/Alexa 555) and counterstained with DAPI (blue). Ventral, down; 20×/0.45 objective. (C) Schematic outline of catecholamine treatment of AGMs in explant culture. (D) Summary of repopulation analysis of recipients injected with cells (1 ee) from Gata3 +/+ , Gata3 +/− , and Gata3 −/− E11/11.5 AGMs that had been exposed to catecholamine derivatives in explant cultures. See also Figure S3 . (E) Quantitative RT-PCR expression analysis of the α1d- ( Adra1d ), β2- ( Adrb2 ), and β3- ( Adrb3 ) adrenergic receptors in mesenchymal (MC), endothelial (EC), and hematopoietic stem cell (HSC) populations sorted from E11.5 aorta-mesenchymes (normalized to Actb and Tbp ). (F) Flow cytometry analysis of the expression of the β2-adrenergic receptor on CD34+ or CD45+ E11.5 AGM cells. (G–I) Immunohistochemistry on E11.5 wild-type embryo sections with an antibody to the β2-adrenergic receptor (red, Cy3). Nuclear DAPI staining is shown (blue) in (G). Arrows in (H) and (I) highlight expression on endothelial cells. Ventral, down; objectives were 10×/0.25 (G) or 20×/0.45 (H and I). ao, dorsal aorta; drg, dorsal root ganglia; fl, fetal liver; m, myotome; nt, neural tube.
    Figure Legend Snippet: Catecholamines Can Rescue HSC Activity In Vitro in the Absence of Circulation (A) Ventral halves of dorsal aortae from E11 Gata3 +/+ and Gata3 −/− embryos were analyzed for Nos3 expression by quantitative RT-PCR. Data is representative of two independent experiments. (B) E11.5 Gata3 +/+ and Gata3 −/− embryo sections were stained for Nos3 (red/Alexa 555) and counterstained with DAPI (blue). Ventral, down; 20×/0.45 objective. (C) Schematic outline of catecholamine treatment of AGMs in explant culture. (D) Summary of repopulation analysis of recipients injected with cells (1 ee) from Gata3 +/+ , Gata3 +/− , and Gata3 −/− E11/11.5 AGMs that had been exposed to catecholamine derivatives in explant cultures. See also Figure S3 . (E) Quantitative RT-PCR expression analysis of the α1d- ( Adra1d ), β2- ( Adrb2 ), and β3- ( Adrb3 ) adrenergic receptors in mesenchymal (MC), endothelial (EC), and hematopoietic stem cell (HSC) populations sorted from E11.5 aorta-mesenchymes (normalized to Actb and Tbp ). (F) Flow cytometry analysis of the expression of the β2-adrenergic receptor on CD34+ or CD45+ E11.5 AGM cells. (G–I) Immunohistochemistry on E11.5 wild-type embryo sections with an antibody to the β2-adrenergic receptor (red, Cy3). Nuclear DAPI staining is shown (blue) in (G). Arrows in (H) and (I) highlight expression on endothelial cells. Ventral, down; objectives were 10×/0.25 (G) or 20×/0.45 (H and I). ao, dorsal aorta; drg, dorsal root ganglia; fl, fetal liver; m, myotome; nt, neural tube.

    Techniques Used: Activity Assay, In Vitro, Expressing, Quantitative RT-PCR, Staining, Injection, Flow Cytometry, Cytometry, Immunohistochemistry

    4) Product Images from "Genome-wide gene expression profiling of human mast cells stimulated by IgE or Fc?RI-aggregation reveals a complex network of genes involved in inflammatory responses"

    Article Title: Genome-wide gene expression profiling of human mast cells stimulated by IgE or Fc?RI-aggregation reveals a complex network of genes involved in inflammatory responses

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-7-210

    Purity of Mast cells . A. Flow cytometry analysis of intracellular chymase expression . Human cord blood-derived mast cells labelled for intracellular mast-cell chymase expression (solid line), isotype control (Black area). Results shown are representative of four separate experiments. B. Toluidine blue staining of mast cells . Fluorescence microscopy of human cord blood-derived mast cells stained with Toluidine blue. Results shown are representative of at least three separate experiments. C. Flow cytometry analysis of cell surface expression of c-kit and FcεRI . Human cord blood-derived mast cells labelled for cell-surface expression of c-kit and FceRI (left panel), and isotype controls (right panel). Results shown are representative of four separate experiments.
    Figure Legend Snippet: Purity of Mast cells . A. Flow cytometry analysis of intracellular chymase expression . Human cord blood-derived mast cells labelled for intracellular mast-cell chymase expression (solid line), isotype control (Black area). Results shown are representative of four separate experiments. B. Toluidine blue staining of mast cells . Fluorescence microscopy of human cord blood-derived mast cells stained with Toluidine blue. Results shown are representative of at least three separate experiments. C. Flow cytometry analysis of cell surface expression of c-kit and FcεRI . Human cord blood-derived mast cells labelled for cell-surface expression of c-kit and FceRI (left panel), and isotype controls (right panel). Results shown are representative of four separate experiments.

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Derivative Assay, Staining, Fluorescence, Microscopy

    5) Product Images from "Mammalian deubiquitinating enzyme inhibitors display in vitro and in vivo activity against malaria parasites and potentiate artemisinin action"

    Article Title: Mammalian deubiquitinating enzyme inhibitors display in vitro and in vivo activity against malaria parasites and potentiate artemisinin action

    Journal: bioRxiv

    doi: 10.1101/2020.08.13.249425

    In vivo activity of b-AP15 alone and or in combination with ART. A Mice (4 groups of 3 mice each) were infected with 10 5 parasites on day 1 and treated with indicated drug doses ∼1 hour post infection for four consecutive days (indicated by arrows). Parasitaemia was monitored daily by flow cytometry and analysis of Giemsa stained smears. B, C . Percentage suppressions on day 4 ( B ) and bar of parasitaemias on day 4 and day 5 ( C ). D, E . Combination of ART and b-AP15 in established mouse infections. ART at 5mg/kg ( D ) or 10mg/kg ( E ) combined with b-AP15 (1mg/kg) administered in established mice infections at a parasitaemia of 2-2.5% for three consecutive days (indicated by arrows). Parasitaemia was monitored daily. ART at 20mg/kg was used as a curative control. Significant differences were calculated using one-way ANOVA alongside the Dunnet’s multiple comparison test. Significance is indicated with asterisks; *p
    Figure Legend Snippet: In vivo activity of b-AP15 alone and or in combination with ART. A Mice (4 groups of 3 mice each) were infected with 10 5 parasites on day 1 and treated with indicated drug doses ∼1 hour post infection for four consecutive days (indicated by arrows). Parasitaemia was monitored daily by flow cytometry and analysis of Giemsa stained smears. B, C . Percentage suppressions on day 4 ( B ) and bar of parasitaemias on day 4 and day 5 ( C ). D, E . Combination of ART and b-AP15 in established mouse infections. ART at 5mg/kg ( D ) or 10mg/kg ( E ) combined with b-AP15 (1mg/kg) administered in established mice infections at a parasitaemia of 2-2.5% for three consecutive days (indicated by arrows). Parasitaemia was monitored daily. ART at 20mg/kg was used as a curative control. Significant differences were calculated using one-way ANOVA alongside the Dunnet’s multiple comparison test. Significance is indicated with asterisks; *p

    Techniques Used: In Vivo, Activity Assay, Mouse Assay, Infection, Flow Cytometry, Staining

    pre-exposure of malaria parasites to UPS inhibitors alone or in combination enhances DHA action. A pre-treatment of the PB 507 line (1.5 hours old rings) with b-AP15 at IC 50 (1.5µM) for 3 hours followed by a wash and then DHA for another 3 hours. Median GFP intensity quantified by flow cytometry at 6 hours, 18hours and 24 hours. b-AP15 at IC 50 readded after DHA wash off in one experimental condition (green plot) while b-AP15 alone used as an additional control. Results are representative of three independent experiments. B . DHA dose response viability plots and lethal dose (LD 50 ) comparisons at 66 hours after pre-exposure of 0-3 hours old rings of the 3D7 line to DMSO (0.1%) or b-AP15 at half IC 50 (0.75µM), IC 50 (1.5µM) or 4X IC 50 (6µM) followed by DHA for 4 hours. C, D . DHA dose response viability plots and lethal dose (LD 50 ) comparisons at 66 hours after pre-exposure of 0-3 hours old rings of the 3D7 line ( C ) and ART resistant Kelch-13 C580Y line ( D ) to DMSO (0.1%) or epoxomicin at 0.2x IC 50 (2nM), IC 50 (12nM) or a combination of b-AP15 and epoxomicin at half IC 50 followed by DHA for 4 hours. Data from three independent experimental repeats. Significant differences between the conditions were calculated using one-way ANOVA alongside the Dunnet’s multiple comparison test. Significance is indicated with asterisks; ****p
    Figure Legend Snippet: pre-exposure of malaria parasites to UPS inhibitors alone or in combination enhances DHA action. A pre-treatment of the PB 507 line (1.5 hours old rings) with b-AP15 at IC 50 (1.5µM) for 3 hours followed by a wash and then DHA for another 3 hours. Median GFP intensity quantified by flow cytometry at 6 hours, 18hours and 24 hours. b-AP15 at IC 50 readded after DHA wash off in one experimental condition (green plot) while b-AP15 alone used as an additional control. Results are representative of three independent experiments. B . DHA dose response viability plots and lethal dose (LD 50 ) comparisons at 66 hours after pre-exposure of 0-3 hours old rings of the 3D7 line to DMSO (0.1%) or b-AP15 at half IC 50 (0.75µM), IC 50 (1.5µM) or 4X IC 50 (6µM) followed by DHA for 4 hours. C, D . DHA dose response viability plots and lethal dose (LD 50 ) comparisons at 66 hours after pre-exposure of 0-3 hours old rings of the 3D7 line ( C ) and ART resistant Kelch-13 C580Y line ( D ) to DMSO (0.1%) or epoxomicin at 0.2x IC 50 (2nM), IC 50 (12nM) or a combination of b-AP15 and epoxomicin at half IC 50 followed by DHA for 4 hours. Data from three independent experimental repeats. Significant differences between the conditions were calculated using one-way ANOVA alongside the Dunnet’s multiple comparison test. Significance is indicated with asterisks; ****p

    Techniques Used: Flow Cytometry

    6) Product Images from "Cocaine Enhances HIV-1 Transcription in Macrophages by Inducing p38 MAPK Phosphorylation"

    Article Title: Cocaine Enhances HIV-1 Transcription in Macrophages by Inducing p38 MAPK Phosphorylation

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2016.00823

    Induction of p38 MAPK/MSK1 signaling cascade in uninfected THP-1macs by cocaine can be abrogated by pharmacological inhibitors of p38 MAPK and MSK1. THP-1macs were pretreated with 10 μM p38 MAPK inhibitor (SB-203580) or 10 μM MSK1 inhibitor (SB-747651A) for 1 h prior to overnight treatment with cocaine. Cells were then harvested, stained with appropriate antibodies or isotype controls, and analyzed by flow cytometry. (A) Levels of phospho-MSK1 in cocaine-treated cells. Levels of phospho-MSK1 in cocaine- and p38 MAPK inhibitor-treated cells (B) , and in cocaine- and MSK1 inhibitor-treated cells (C) .
    Figure Legend Snippet: Induction of p38 MAPK/MSK1 signaling cascade in uninfected THP-1macs by cocaine can be abrogated by pharmacological inhibitors of p38 MAPK and MSK1. THP-1macs were pretreated with 10 μM p38 MAPK inhibitor (SB-203580) or 10 μM MSK1 inhibitor (SB-747651A) for 1 h prior to overnight treatment with cocaine. Cells were then harvested, stained with appropriate antibodies or isotype controls, and analyzed by flow cytometry. (A) Levels of phospho-MSK1 in cocaine-treated cells. Levels of phospho-MSK1 in cocaine- and p38 MAPK inhibitor-treated cells (B) , and in cocaine- and MSK1 inhibitor-treated cells (C) .

    Techniques Used: Staining, Flow Cytometry, Cytometry

    Induction of p38 MAPK/MSK1 signaling cascade in MDMs. MDMs were cultured in the absence or presence of 25 μM cocaine and analyzed by flow cytometry to determine the levels of (A) phosphorylated p38 MAPK and (B) phosphorylated MSK1. To determine that cocaine specifically targets p38 MAPK/MSK1 pathway, MDMs were pretreated with 10 μM p38 MAPK inhibitor (SB-203580) or 10 μM MSK1 inhibitor (SB-747651A) prior to cocaine treatment. Thereafter, cells were harvested and analyzed by flow cytometry to determine the (C) effects of p38 MAPK inhibitor on MSK1 phosphorylation, and (D) effects of MSK1 inhibitor on MSK1 phosphorylation.
    Figure Legend Snippet: Induction of p38 MAPK/MSK1 signaling cascade in MDMs. MDMs were cultured in the absence or presence of 25 μM cocaine and analyzed by flow cytometry to determine the levels of (A) phosphorylated p38 MAPK and (B) phosphorylated MSK1. To determine that cocaine specifically targets p38 MAPK/MSK1 pathway, MDMs were pretreated with 10 μM p38 MAPK inhibitor (SB-203580) or 10 μM MSK1 inhibitor (SB-747651A) prior to cocaine treatment. Thereafter, cells were harvested and analyzed by flow cytometry to determine the (C) effects of p38 MAPK inhibitor on MSK1 phosphorylation, and (D) effects of MSK1 inhibitor on MSK1 phosphorylation.

    Techniques Used: Cell Culture, Flow Cytometry, Cytometry

    7) Product Images from "HER2 Targeting Peptides Screening and Applications in Tumor Imaging and Drug Delivery"

    Article Title: HER2 Targeting Peptides Screening and Applications in Tumor Imaging and Drug Delivery

    Journal: Theranostics

    doi: 10.7150/thno.14302

    Sequences alignment of key binding residues in HER1, HER2, HER3 and HER4 (A) and cellular analyses of peptides affinity for HER2 binding. Alignment of the key binding residues, the sequences of 13-mer and 16-mer peptides selected to synthesize (B, C). Immunocytochemistry analysis of peptides PS1, PS2, PS3, PS4, WP1, WP2, WP3 and WP4 (labeled with FITC, green) in HER2 positive SKBR3 cells (D and E), colocalization analysis of WP1 (labeled with FITC, green) and anti-HER2 antibody (labeled with PE, red) in HER2 positive SKBR3 cells (F). Hoechst 33342-stained cell nuclei are in blue. (G, H) Flow cytometry analysis of WP1, WP2, WP3, WP4 and phosphate buffer saline (PBS) control for SKBR3 cell line and the binding percentages are plotted as bars in panel h, respectively (n = 3).
    Figure Legend Snippet: Sequences alignment of key binding residues in HER1, HER2, HER3 and HER4 (A) and cellular analyses of peptides affinity for HER2 binding. Alignment of the key binding residues, the sequences of 13-mer and 16-mer peptides selected to synthesize (B, C). Immunocytochemistry analysis of peptides PS1, PS2, PS3, PS4, WP1, WP2, WP3 and WP4 (labeled with FITC, green) in HER2 positive SKBR3 cells (D and E), colocalization analysis of WP1 (labeled with FITC, green) and anti-HER2 antibody (labeled with PE, red) in HER2 positive SKBR3 cells (F). Hoechst 33342-stained cell nuclei are in blue. (G, H) Flow cytometry analysis of WP1, WP2, WP3, WP4 and phosphate buffer saline (PBS) control for SKBR3 cell line and the binding percentages are plotted as bars in panel h, respectively (n = 3).

    Techniques Used: Binding Assay, Immunocytochemistry, Labeling, Staining, Flow Cytometry, Cytometry

    Flow cytometry analysis shows P51 and P25 have high affinity and specificity for SKBR3 (HER2 high expression) cells. The fluorescence intensities of cells bound with peptides or antibody for SKBR3 and 293A cell lines are shown in A and C, respectively, and the binding percentages in two cell lines are plotted as bars in panel B and D, respectively (n = 3). Panel E shows the colocalization analysis of peptides and antibodies in HER2 or HER1 high expression cell lines, both peptides showed significant fluorescence signals and overlap in SKBR3 cell surface, but very weak signals are detected in 468 cells. HER2 high expression SKBR3 cells were treated with peptides (labeled with FITC, green) and anti-HER2 antibody (labeled with PE, red), and HER2 low expression but HER1 high expression 468 cells were treated with peptides (labeled with FITC, green) and anti-HER1 antibody (labeled with AlexaFluor555, red).
    Figure Legend Snippet: Flow cytometry analysis shows P51 and P25 have high affinity and specificity for SKBR3 (HER2 high expression) cells. The fluorescence intensities of cells bound with peptides or antibody for SKBR3 and 293A cell lines are shown in A and C, respectively, and the binding percentages in two cell lines are plotted as bars in panel B and D, respectively (n = 3). Panel E shows the colocalization analysis of peptides and antibodies in HER2 or HER1 high expression cell lines, both peptides showed significant fluorescence signals and overlap in SKBR3 cell surface, but very weak signals are detected in 468 cells. HER2 high expression SKBR3 cells were treated with peptides (labeled with FITC, green) and anti-HER2 antibody (labeled with PE, red), and HER2 low expression but HER1 high expression 468 cells were treated with peptides (labeled with FITC, green) and anti-HER1 antibody (labeled with AlexaFluor555, red).

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Fluorescence, Binding Assay, Labeling

    8) Product Images from "Upregulation of Akt/NF-κB-regulated inflammation and Akt/Bad-related apoptosis signaling pathway involved in hepatic carcinoma process: suppression by carnosic acid nanoparticle"

    Article Title: Upregulation of Akt/NF-κB-regulated inflammation and Akt/Bad-related apoptosis signaling pathway involved in hepatic carcinoma process: suppression by carnosic acid nanoparticle

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S101285

    The effect of carnosic acid on apoptosis in Akt-deficient liver cancer cells. Notes: ( A ) The number of cells was detected through terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis in MHCC97-H cells and in Bel7402 cells. ( B ) Annexin V/PI analysis for the evaluation of apoptosis via flow cytometry in MHCC97-H cells and in Bel7402 cells. ( C ) Cell proliferation was determined by Western blot in different liver cancer cell lines. ( D ) The histogram analysis based on Western blot in MHCC97-H cells and Bel7402 cells. ( E ) Effects of carnosic acid on cell cycle progression in MHCC97-H cells. ( F ) Effects of carnosic acid on cell cycle progression in Bel7402 cells. Data are expressed as the mean ± standard error of the mean. * P
    Figure Legend Snippet: The effect of carnosic acid on apoptosis in Akt-deficient liver cancer cells. Notes: ( A ) The number of cells was detected through terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis in MHCC97-H cells and in Bel7402 cells. ( B ) Annexin V/PI analysis for the evaluation of apoptosis via flow cytometry in MHCC97-H cells and in Bel7402 cells. ( C ) Cell proliferation was determined by Western blot in different liver cancer cell lines. ( D ) The histogram analysis based on Western blot in MHCC97-H cells and Bel7402 cells. ( E ) Effects of carnosic acid on cell cycle progression in MHCC97-H cells. ( F ) Effects of carnosic acid on cell cycle progression in Bel7402 cells. Data are expressed as the mean ± standard error of the mean. * P

    Techniques Used: End Labeling, TUNEL Assay, Flow Cytometry, Cytometry, Western Blot

    9) Product Images from "MicroRNA-495 downregulates FOXC1 expression to suppress cell growth and migration in endometrial cancer"

    Article Title: MicroRNA-495 downregulates FOXC1 expression to suppress cell growth and migration in endometrial cancer

    Journal: Tumour Biology

    doi: 10.1007/s13277-015-3686-6

    FOXC1 rescues miR-495-induced cellular phenotypes in endometrial cancer. a A colony formation assay was performed after transfection. b Flow cytometry analysis was used to detect cell apoptosis in AN3CA and KLE cells. c The ectopic expression of FOXC1 without a 3′UTR rescued AN3CA and KLE cell migration (* p
    Figure Legend Snippet: FOXC1 rescues miR-495-induced cellular phenotypes in endometrial cancer. a A colony formation assay was performed after transfection. b Flow cytometry analysis was used to detect cell apoptosis in AN3CA and KLE cells. c The ectopic expression of FOXC1 without a 3′UTR rescued AN3CA and KLE cell migration (* p

    Techniques Used: Colony Assay, Transfection, Flow Cytometry, Cytometry, Expressing, Migration

    10) Product Images from "Thymic resident NKT cell subsets show differential requirements for CD28 co-stimulation during antigenic activation"

    Article Title: Thymic resident NKT cell subsets show differential requirements for CD28 co-stimulation during antigenic activation

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-65129-3

    PLZF and CD28 expression are altered following stimulation. ( A ) Representative plots of NKT cell populations stratified by NK1.1 and PLZF expression. Quadrants represent the following populations: U, Stage 3, NK1.1 + , L, Stage 2, NK1.1- ( B ) The MFI of PLZF in bulk stage 2 (grey bars) and stage 3 (red bars) NKT cells. ( C ) The MFI of PLZF in undivided stage 2 (grey bars) and stage 3 (red bars) NKT cells. ( D ) The MFI of CD28 on undivided (red bars) and divided (grey bars) NKT cells. ( E ) The MFI of CD69 on NKT cells. All data displayed in graphs correspond to mean +/− SEM of 3 biological replicates. Statistical significance was determined using one-way ANOVA followed by Bonferroni tests. Relevant statistical analyses are discussed in the text. Flow cytometry gating strategy outlined in Materials and Methods.
    Figure Legend Snippet: PLZF and CD28 expression are altered following stimulation. ( A ) Representative plots of NKT cell populations stratified by NK1.1 and PLZF expression. Quadrants represent the following populations: U, Stage 3, NK1.1 + , L, Stage 2, NK1.1- ( B ) The MFI of PLZF in bulk stage 2 (grey bars) and stage 3 (red bars) NKT cells. ( C ) The MFI of PLZF in undivided stage 2 (grey bars) and stage 3 (red bars) NKT cells. ( D ) The MFI of CD28 on undivided (red bars) and divided (grey bars) NKT cells. ( E ) The MFI of CD69 on NKT cells. All data displayed in graphs correspond to mean +/− SEM of 3 biological replicates. Statistical significance was determined using one-way ANOVA followed by Bonferroni tests. Relevant statistical analyses are discussed in the text. Flow cytometry gating strategy outlined in Materials and Methods.

    Techniques Used: Expressing, Flow Cytometry

    NKT cell activation is not affected by CTLA-4 blockade or activation, but is inhibited by CD80/86 blockade. ( A ) Representative plots of NKT cell populations (αGC:CD1d tetramer+TCRβ+) after 3-day co-culture. ( B ) Representative plots of NKT cell population proliferation with division bins denoted after 3-day co-culture. ( C ) NKT cell percentage ( D ) Total NKT cell number, and ( E ) NKT cell fold expansion post stimulation. ( F ) The precursor proliferation curve. ( G ) The mean division number of proliferating precursors. All data displayed in graphs correspond to mean +/− SEM of 3 biological replicates. Statistical significance was determined using one-way ANOVA followed by Bonferroni tests. Relevant statistical analyses are discussed in the text. Flow cytometry gating strategy outlined in Materials and Methods.
    Figure Legend Snippet: NKT cell activation is not affected by CTLA-4 blockade or activation, but is inhibited by CD80/86 blockade. ( A ) Representative plots of NKT cell populations (αGC:CD1d tetramer+TCRβ+) after 3-day co-culture. ( B ) Representative plots of NKT cell population proliferation with division bins denoted after 3-day co-culture. ( C ) NKT cell percentage ( D ) Total NKT cell number, and ( E ) NKT cell fold expansion post stimulation. ( F ) The precursor proliferation curve. ( G ) The mean division number of proliferating precursors. All data displayed in graphs correspond to mean +/− SEM of 3 biological replicates. Statistical significance was determined using one-way ANOVA followed by Bonferroni tests. Relevant statistical analyses are discussed in the text. Flow cytometry gating strategy outlined in Materials and Methods.

    Techniques Used: Activation Assay, Co-Culture Assay, Flow Cytometry

    NKT cell subtypes are differentially affected by CD80/86 blockade. ( A ) Representative plots of NKT cell populations stratified by NK1.1 expression and proliferation dye dilution. Quadrants represent the following populations: UR, Undivided Stage 3, UL, Divided Stage 3, LR, Undivided Stage 2, LL, Divided Stage 2. ( B ) Representative plots of NKT cell populations stratified by PLZF expression and proliferation dye dilution. Quadrants represent the following populations: UR, Undivided PLZF-Hi, UL, Divided PLZF-Hi, LR, Undivided PLZF-Lo, LL, Divided PLZF-Lo. ( C ) The number of NKT cells present in the following bins: Undivided Stage 3 (white bars), Divided Stage 3 (red bars), Undivided Stage 2 (yellow bars), Divided Stage 2 (grey bars). ( D ) The number of NKT cells present in the following bins: Undivided PLZF-Hi (white bars), Divided PLZF-Hi (red bars), Undivided PLZF-Lo (yellow bars), Divided PLZF-Lo (grey bars). ( E ) The precursor proliferation curve of stage 2 NKT cells. ( F ) The precursor proliferation curve of stage 3 NKT cells. ( G ) The mean division number of stage 2 and stage 3 precursor cells. ( H ) Fold expansion of FACS-isolated stage 2 and stage 3 NKT cells after co-culture with splenocytes loaded with 10 ng/mL α-GalCer. ( I ) Mean division score of sorted stage 2 and stage 3 NKT cells after co-culture with splenocytes loaded with 10 ng/mL α-GalCer. ( J ) Precursor proliferation curve of sorted NKT cells after co-culture with splenocytes loaded with 10 ng/mL α-GalCer. All data displayed in graphs correspond to mean +/− SEM of 3 biological replicates. Statistical significance was determined using one-way ANOVA followed by Bonferroni tests. Relevant statistical analyses are discussed in the text. Flow cytometry gating strategy outlined in Materials and Methods.
    Figure Legend Snippet: NKT cell subtypes are differentially affected by CD80/86 blockade. ( A ) Representative plots of NKT cell populations stratified by NK1.1 expression and proliferation dye dilution. Quadrants represent the following populations: UR, Undivided Stage 3, UL, Divided Stage 3, LR, Undivided Stage 2, LL, Divided Stage 2. ( B ) Representative plots of NKT cell populations stratified by PLZF expression and proliferation dye dilution. Quadrants represent the following populations: UR, Undivided PLZF-Hi, UL, Divided PLZF-Hi, LR, Undivided PLZF-Lo, LL, Divided PLZF-Lo. ( C ) The number of NKT cells present in the following bins: Undivided Stage 3 (white bars), Divided Stage 3 (red bars), Undivided Stage 2 (yellow bars), Divided Stage 2 (grey bars). ( D ) The number of NKT cells present in the following bins: Undivided PLZF-Hi (white bars), Divided PLZF-Hi (red bars), Undivided PLZF-Lo (yellow bars), Divided PLZF-Lo (grey bars). ( E ) The precursor proliferation curve of stage 2 NKT cells. ( F ) The precursor proliferation curve of stage 3 NKT cells. ( G ) The mean division number of stage 2 and stage 3 precursor cells. ( H ) Fold expansion of FACS-isolated stage 2 and stage 3 NKT cells after co-culture with splenocytes loaded with 10 ng/mL α-GalCer. ( I ) Mean division score of sorted stage 2 and stage 3 NKT cells after co-culture with splenocytes loaded with 10 ng/mL α-GalCer. ( J ) Precursor proliferation curve of sorted NKT cells after co-culture with splenocytes loaded with 10 ng/mL α-GalCer. All data displayed in graphs correspond to mean +/− SEM of 3 biological replicates. Statistical significance was determined using one-way ANOVA followed by Bonferroni tests. Relevant statistical analyses are discussed in the text. Flow cytometry gating strategy outlined in Materials and Methods.

    Techniques Used: Expressing, FACS, Isolation, Co-Culture Assay, Flow Cytometry

    Depletion of CD8α and CD24 enriches for mature thymic NKT cells without altering their composition. ( A ) Schematic of the thymic NKT cell proliferation assay. ( B ) NKT cell populations (αGC:CD1d tetramer+TCRβ+) pre- and post-enrichment with unloaded tetramer shown as a control. ( C ) Pre- and post-enrichment NKT cell populations subdivided into stage 2 (CD44 + NK1.1−) and stage 3 (CD44 + NK1.1+). ( D ) The percentage of stage 2 and stage 3 NKT cells pre- and post-enrichment. Relevant statistical analyses are discussed in the text. Data correspond to mean+/− SEM of 3 biological replicates. Statistical significance determined by student’s t test. Flow cytometry gating strategy is outlined in the Materials and Methods.
    Figure Legend Snippet: Depletion of CD8α and CD24 enriches for mature thymic NKT cells without altering their composition. ( A ) Schematic of the thymic NKT cell proliferation assay. ( B ) NKT cell populations (αGC:CD1d tetramer+TCRβ+) pre- and post-enrichment with unloaded tetramer shown as a control. ( C ) Pre- and post-enrichment NKT cell populations subdivided into stage 2 (CD44 + NK1.1−) and stage 3 (CD44 + NK1.1+). ( D ) The percentage of stage 2 and stage 3 NKT cells pre- and post-enrichment. Relevant statistical analyses are discussed in the text. Data correspond to mean+/− SEM of 3 biological replicates. Statistical significance determined by student’s t test. Flow cytometry gating strategy is outlined in the Materials and Methods.

    Techniques Used: Proliferation Assay, Flow Cytometry

    11) Product Images from "Lack of miR-378 attenuates muscular dystrophy in mdx mice"

    Article Title: Lack of miR-378 attenuates muscular dystrophy in mdx mice

    Journal: JCI Insight

    doi: 10.1172/jci.insight.135576

    The effect of miR-378 deficiency in 6-month-old animals. ( A ) Muscle performance test indicating the increased running capacity of miR-378 –/– animals in comparison with WT mice; downhill running treadmill test presented as the percentage of the running distance to exhaustion compared with WT animals; n = 4–5/group. ( B ) Increased body weight of mdx animals without the impact of miR-378 loss; n = 11–15/group. ( C ) Gastrocnemius and tibialis anterior muscle mass calculated per kg BW showing increased muscle mass in 6-month-old mdx mice with no effect of miR-378 deficiency; n = 6–10/group. ( D ) Elevated LDH activity in dystrophic animals with no changes in mice lacking miR-378; activity assay; n = 10–16/group. ( E ) Semiquantitative analysis of collagen deposition based on Masson’s trichrome staining of the gastrocnemius muscle, indicating no effect of miR-378 loss. Microscopic assessment using Nikon Eclipse microscope; n = 5–6/group. ( F ) Increased number of WBC in the peripheral blood; blood cell count; n = 13–19/group. ( G ) Semiquantitative analysis of inflammation extent (based on H E staining) showing increased inflammatory cell infiltration in mdx mice with no effect of miR-378 deficiency; microscopic assessment using Nikon Eclipse microscope; n = 4–10/group. ( H and I ) The analysis of inflammatory cells in hind limb muscles showing the percentage of leukocytes (CD45 + cells) ( H ) and macrophages (CD45 + F4/80 + CD11b + cells) ( I ); flow cytometry analysis calculated as the percentage of Hoechst + cells; n = 5–6/group. Data are presented as mean ± SEM. * P
    Figure Legend Snippet: The effect of miR-378 deficiency in 6-month-old animals. ( A ) Muscle performance test indicating the increased running capacity of miR-378 –/– animals in comparison with WT mice; downhill running treadmill test presented as the percentage of the running distance to exhaustion compared with WT animals; n = 4–5/group. ( B ) Increased body weight of mdx animals without the impact of miR-378 loss; n = 11–15/group. ( C ) Gastrocnemius and tibialis anterior muscle mass calculated per kg BW showing increased muscle mass in 6-month-old mdx mice with no effect of miR-378 deficiency; n = 6–10/group. ( D ) Elevated LDH activity in dystrophic animals with no changes in mice lacking miR-378; activity assay; n = 10–16/group. ( E ) Semiquantitative analysis of collagen deposition based on Masson’s trichrome staining of the gastrocnemius muscle, indicating no effect of miR-378 loss. Microscopic assessment using Nikon Eclipse microscope; n = 5–6/group. ( F ) Increased number of WBC in the peripheral blood; blood cell count; n = 13–19/group. ( G ) Semiquantitative analysis of inflammation extent (based on H E staining) showing increased inflammatory cell infiltration in mdx mice with no effect of miR-378 deficiency; microscopic assessment using Nikon Eclipse microscope; n = 4–10/group. ( H and I ) The analysis of inflammatory cells in hind limb muscles showing the percentage of leukocytes (CD45 + cells) ( H ) and macrophages (CD45 + F4/80 + CD11b + cells) ( I ); flow cytometry analysis calculated as the percentage of Hoechst + cells; n = 5–6/group. Data are presented as mean ± SEM. * P

    Techniques Used: Mouse Assay, Activity Assay, Staining, Microscopy, Cell Counting, Flow Cytometry

    Muscle damage markers in serum and inflammation in gastrocnemius muscle are decreased in 3-month-old dystrophic mice lacking miR-378. ( A ) Lower serum activity of LDH in mdx animals lacking miR-378; activity assay; n = 10–14/group. ( B ) Increased serum CK activity in mdx mice with a tendency to be decreased by miR-378-KO, activity assay; n = 11–13/group. ( C ) Necrosis assessment by immunofluorescent staining of IgM and IgG (green) binding and its calculation indicating no differences between groups; n = 9–10/group. Scale bar: 100 μm. ( D ) Representative pictures of H E staining of gastrocnemius muscle with semiquantitative analysis of inflammation extent showing a tendency in decreased inflammatory cell infiltration in dKO mice; microscopic assessment using Nikon Eclipse microscope. Scale bar: 100 μm; n = 4–6/group. ( E ) Decreased number of WBC in the peripheral blood in dKO mice; blood cell count; n = 5–6/group. ( F–J ) The analysis of inflammatory cells in hind limb muscles with special emphasis on macrophage subpopulations; flow cytometry analysis calculated as the percentage of Hoechst + cells; n = 5/group. The percentage of CD45 + cells ( F ), macrophages (CD45 + F4/80 + CD11b + cells) ( G ), M1-like macrophages (CD45 + F4/80 + CD11b + MHCII hi CD206 lo cells) ( H ), M2-like macrophages (CD45 + F4/80 + CD11b + MHCII lo CD206 hi cells) ( I ), and eosinophils (CD45 + F4/80 + CD86 + cells) ( J ) showing significant decrease in dKO mice. ( K ) The decreased HO-1 protein level in dKO animals assessed by Western blot; GAPDH used as loading control. Representative result of 2 independent experiments; n = 4–5/group. Data are presented as mean ± SEM. * P
    Figure Legend Snippet: Muscle damage markers in serum and inflammation in gastrocnemius muscle are decreased in 3-month-old dystrophic mice lacking miR-378. ( A ) Lower serum activity of LDH in mdx animals lacking miR-378; activity assay; n = 10–14/group. ( B ) Increased serum CK activity in mdx mice with a tendency to be decreased by miR-378-KO, activity assay; n = 11–13/group. ( C ) Necrosis assessment by immunofluorescent staining of IgM and IgG (green) binding and its calculation indicating no differences between groups; n = 9–10/group. Scale bar: 100 μm. ( D ) Representative pictures of H E staining of gastrocnemius muscle with semiquantitative analysis of inflammation extent showing a tendency in decreased inflammatory cell infiltration in dKO mice; microscopic assessment using Nikon Eclipse microscope. Scale bar: 100 μm; n = 4–6/group. ( E ) Decreased number of WBC in the peripheral blood in dKO mice; blood cell count; n = 5–6/group. ( F–J ) The analysis of inflammatory cells in hind limb muscles with special emphasis on macrophage subpopulations; flow cytometry analysis calculated as the percentage of Hoechst + cells; n = 5/group. The percentage of CD45 + cells ( F ), macrophages (CD45 + F4/80 + CD11b + cells) ( G ), M1-like macrophages (CD45 + F4/80 + CD11b + MHCII hi CD206 lo cells) ( H ), M2-like macrophages (CD45 + F4/80 + CD11b + MHCII lo CD206 hi cells) ( I ), and eosinophils (CD45 + F4/80 + CD86 + cells) ( J ) showing significant decrease in dKO mice. ( K ) The decreased HO-1 protein level in dKO animals assessed by Western blot; GAPDH used as loading control. Representative result of 2 independent experiments; n = 4–5/group. Data are presented as mean ± SEM. * P

    Techniques Used: Mouse Assay, Activity Assay, Staining, Binding Assay, Microscopy, Cell Counting, Flow Cytometry, Western Blot

    Fibrosis extent is diminished in gastrocnemius muscle of 3-month-old dystrophic mice lacking miR-378. ( A ) Representative photos of Masson’s trichrome staining with semiquantitative analysis of collagen deposition showing the decreased extent of fibrosis in dKO mice; microscopic assessment using Nikon Eclipse microscope. Scale bar: 100 μm; n = 5–6/group. ( B and C ) Decreased expression of fibrotic markers in dKO mice: Col1a1 ( B ) and Fn1 ( C ), qPCR; n = 5–8/group. ( D ) The diminished abundance of FAPs identified as CD45 – CD31 – Sca1 + α7i – CD34 + cells in hind limb muscles of dKO mice; flow cytometry analysis calculated as the percentage of Hoechst + cells; n = 5/group. Data are presented as mean ± SEM. * P
    Figure Legend Snippet: Fibrosis extent is diminished in gastrocnemius muscle of 3-month-old dystrophic mice lacking miR-378. ( A ) Representative photos of Masson’s trichrome staining with semiquantitative analysis of collagen deposition showing the decreased extent of fibrosis in dKO mice; microscopic assessment using Nikon Eclipse microscope. Scale bar: 100 μm; n = 5–6/group. ( B and C ) Decreased expression of fibrotic markers in dKO mice: Col1a1 ( B ) and Fn1 ( C ), qPCR; n = 5–8/group. ( D ) The diminished abundance of FAPs identified as CD45 – CD31 – Sca1 + α7i – CD34 + cells in hind limb muscles of dKO mice; flow cytometry analysis calculated as the percentage of Hoechst + cells; n = 5/group. Data are presented as mean ± SEM. * P

    Techniques Used: Mouse Assay, Staining, Microscopy, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry

    The KO of miR-378 affects the phenotype and properties of dystrophic muscle satellite cells (mSCs). ( A ) The abundance of mSCs within the gastrocnemius muscle of 3-month-old mdx mice without the apparent influence of miR-378, as quantified based on Pax7 + nuclei per myofibers. Immunofluorescent staining with representative pictures; confocal microscope LSM-510, Carl Zeiss. Scale bar: 50 μm; n = 3–4/group. Arrows indicate Pax7 + cells (green) colocalizing with nuclei stained with Hoechst (blue). ( B–D ) The analysis of mSCs in hind limb muscles of 10-week-old mice; flow cytometry analysis; n = 5/group. ( B ) quiescent (CD34 + ) and activated (CD34 – ) cells contribution within mSCs (CD45 – CD31 – Sca1 – α7i + ) population showing a decrease in CD34 – cells in dKO mice. The percentage of CD34 + ( C ) and CD34 – ( D ) mSCs in S + G2 + M phases of the cell cycle, revealing a decreased percentage of mSCs in the proliferative state of the cell cycle in dKO mice. ( E ) Representative pictures of MyHC (green) and Hoechst (blue) immunofluorescent staining of mSCs isolated from hind limb muscles of 10-week-old mice differentiating for 3 days ex vivo. Nikon Eclipse microscope. Scale bar: 100 μm; n = 9–10/group. ( F ) Fusion index determined by the percentage of MyHC + fibers containing 3 or more nuclei among the total number of nuclei showing a significant decrease in the fusion index in dKO mice; n = 9–10/group. Data are presented as mean ± SEM. ** P
    Figure Legend Snippet: The KO of miR-378 affects the phenotype and properties of dystrophic muscle satellite cells (mSCs). ( A ) The abundance of mSCs within the gastrocnemius muscle of 3-month-old mdx mice without the apparent influence of miR-378, as quantified based on Pax7 + nuclei per myofibers. Immunofluorescent staining with representative pictures; confocal microscope LSM-510, Carl Zeiss. Scale bar: 50 μm; n = 3–4/group. Arrows indicate Pax7 + cells (green) colocalizing with nuclei stained with Hoechst (blue). ( B–D ) The analysis of mSCs in hind limb muscles of 10-week-old mice; flow cytometry analysis; n = 5/group. ( B ) quiescent (CD34 + ) and activated (CD34 – ) cells contribution within mSCs (CD45 – CD31 – Sca1 – α7i + ) population showing a decrease in CD34 – cells in dKO mice. The percentage of CD34 + ( C ) and CD34 – ( D ) mSCs in S + G2 + M phases of the cell cycle, revealing a decreased percentage of mSCs in the proliferative state of the cell cycle in dKO mice. ( E ) Representative pictures of MyHC (green) and Hoechst (blue) immunofluorescent staining of mSCs isolated from hind limb muscles of 10-week-old mice differentiating for 3 days ex vivo. Nikon Eclipse microscope. Scale bar: 100 μm; n = 9–10/group. ( F ) Fusion index determined by the percentage of MyHC + fibers containing 3 or more nuclei among the total number of nuclei showing a significant decrease in the fusion index in dKO mice; n = 9–10/group. Data are presented as mean ± SEM. ** P

    Techniques Used: Mouse Assay, Staining, Microscopy, Flow Cytometry, Isolation, Ex Vivo

    12) Product Images from "Senescence-Induced Vascular Remodeling Creates Therapeutic Vulnerabilities in Pancreas Cancer"

    Article Title: Senescence-Induced Vascular Remodeling Creates Therapeutic Vulnerabilities in Pancreas Cancer

    Journal: Cell

    doi: 10.1016/j.cell.2020.03.008

    Therapy-Induced Senescence Leads to T Cell Activation and Exhaustion (A–D) Flow cytometry analysis of KPC mut cell transplant tumors following 2-week treatment with vehicle, trametinib (1 mg/kg) and/or palbociclib (100 mg/kg) (n = 3–5). (A) Percentage of CD69 + CD8 + T cells. (B) Percentage of CD44 + CD8 + T cells. (C) Representative histograms (left) and quantification of mean fluorescent intensity (MFI) of MHC-I (H-2k b ) expression on tumor cells. (D) Percentage of CD107a + T cells. (E) Kaplan-Meier survival curve of KPC mut cell transplant mice treated with vehicle or trametinib (1 mg/kg) and palbociclib (100 mg/kg) in the presence or absence of a CD8 (2.43; 200 μg) or CD4 (GK1.5; 200 μg) depleting antibody (n ≥ 6). (F and G) Flow cytometry analysis of KPC mut cell transplant tumors following treatment as in (A). (F) Representative flow cytometry plots and MFI for PD-1 (p = 0.016), 2B4 (p
    Figure Legend Snippet: Therapy-Induced Senescence Leads to T Cell Activation and Exhaustion (A–D) Flow cytometry analysis of KPC mut cell transplant tumors following 2-week treatment with vehicle, trametinib (1 mg/kg) and/or palbociclib (100 mg/kg) (n = 3–5). (A) Percentage of CD69 + CD8 + T cells. (B) Percentage of CD44 + CD8 + T cells. (C) Representative histograms (left) and quantification of mean fluorescent intensity (MFI) of MHC-I (H-2k b ) expression on tumor cells. (D) Percentage of CD107a + T cells. (E) Kaplan-Meier survival curve of KPC mut cell transplant mice treated with vehicle or trametinib (1 mg/kg) and palbociclib (100 mg/kg) in the presence or absence of a CD8 (2.43; 200 μg) or CD4 (GK1.5; 200 μg) depleting antibody (n ≥ 6). (F and G) Flow cytometry analysis of KPC mut cell transplant tumors following treatment as in (A). (F) Representative flow cytometry plots and MFI for PD-1 (p = 0.016), 2B4 (p

    Techniques Used: Activation Assay, Flow Cytometry, Expressing, Mouse Assay

    SASP Factors Contribute to Vascular Remodeling in PDAC (A) Heatmap of cytokine array results from KPC mut cells following 8-day treatment with trametinib (25 nM) and/or palbociclib (500 nM). Data presented as mean of three biological replicates. (B) Cell growth analysis of 3B11 cells cultured in serum-free (basal) or conditioned media (CM) from KPC mut cells treated as in (A) (n = 3). (C) Endothelial tube formation analysis of 3B11 cells cultured in CM from (B). Quantification of total tube length is shown (n = 2; V versus T/P, p = 0.01). (D) Heatmap of cytokine array results from KPC mut cells harboring control Renilla ( Ren ) or p65 shRNAs and treated as in (A). Data presented as mean of three biological replicates. (E) IHC staining and quantification of blood vessels per field in KPC mut organoid transplant tumors harboring Ren or p65 shRNAs and treated for 2 weeks with vehicle or trametinib (1 mg/kg) and palbociclib (100 mg/kg) (n = 3; Ren T/P versus p65 T/P, p = 0.003). Arrowhead, collapsed vessel; arrow, visible lumen. (F) IHC and IF staining of tumor samples in (E). Quantification of HA staining and αSMA and VCAM-1 colocalization with blood vessels are shown (n = 2–3; Ren V versus Ren T/P, p ≤ 0.05; Ren T/P versus p65 T/P, p ≤ 0.05). (G) IHC staining and quantification of blood vessels per field in KPC mut organoid transplant tumors treated for 2 weeks with vehicle, trametinib (1 mg/kg), palbociclib (100 mg/kg), and/or a VEGFR-2 blocking antibody (DC101; 800 μg) (n = 3; T/P versus T/P+DC101, p = 0.003). (H) Flow cytometry analysis of VCAM-1 expression on endothelial cells in KPC mut cell transplant tumors treated with vehicle, trametinib (1 mg/kg), palbociclib (100 mg/kg), and/or monoclonal antibodies (mAbs) targeting CCL5 (MAB478; 50 μg), CXCL1 (MAB453; 25 μg), TGF-β (1D11; 300 μg), IL-1β (B122; 200 μg), IL-6 (MP5-20F3; 200 μg), or VEGFR-2 (DC101; 800 μg) for 2 weeks (n ≥ 4). Two-way ANOVA (B). One-way ANOVA (F and H). Error bars, mean ± SEM. ****p
    Figure Legend Snippet: SASP Factors Contribute to Vascular Remodeling in PDAC (A) Heatmap of cytokine array results from KPC mut cells following 8-day treatment with trametinib (25 nM) and/or palbociclib (500 nM). Data presented as mean of three biological replicates. (B) Cell growth analysis of 3B11 cells cultured in serum-free (basal) or conditioned media (CM) from KPC mut cells treated as in (A) (n = 3). (C) Endothelial tube formation analysis of 3B11 cells cultured in CM from (B). Quantification of total tube length is shown (n = 2; V versus T/P, p = 0.01). (D) Heatmap of cytokine array results from KPC mut cells harboring control Renilla ( Ren ) or p65 shRNAs and treated as in (A). Data presented as mean of three biological replicates. (E) IHC staining and quantification of blood vessels per field in KPC mut organoid transplant tumors harboring Ren or p65 shRNAs and treated for 2 weeks with vehicle or trametinib (1 mg/kg) and palbociclib (100 mg/kg) (n = 3; Ren T/P versus p65 T/P, p = 0.003). Arrowhead, collapsed vessel; arrow, visible lumen. (F) IHC and IF staining of tumor samples in (E). Quantification of HA staining and αSMA and VCAM-1 colocalization with blood vessels are shown (n = 2–3; Ren V versus Ren T/P, p ≤ 0.05; Ren T/P versus p65 T/P, p ≤ 0.05). (G) IHC staining and quantification of blood vessels per field in KPC mut organoid transplant tumors treated for 2 weeks with vehicle, trametinib (1 mg/kg), palbociclib (100 mg/kg), and/or a VEGFR-2 blocking antibody (DC101; 800 μg) (n = 3; T/P versus T/P+DC101, p = 0.003). (H) Flow cytometry analysis of VCAM-1 expression on endothelial cells in KPC mut cell transplant tumors treated with vehicle, trametinib (1 mg/kg), palbociclib (100 mg/kg), and/or monoclonal antibodies (mAbs) targeting CCL5 (MAB478; 50 μg), CXCL1 (MAB453; 25 μg), TGF-β (1D11; 300 μg), IL-1β (B122; 200 μg), IL-6 (MP5-20F3; 200 μg), or VEGFR-2 (DC101; 800 μg) for 2 weeks (n ≥ 4). Two-way ANOVA (B). One-way ANOVA (F and H). Error bars, mean ± SEM. ****p

    Techniques Used: Cell Culture, Immunohistochemistry, Staining, Blocking Assay, Flow Cytometry, Expressing

    13) Product Images from "Activator of thyroid and retinoid receptor increases sorafenib resistance in hepatocellular carcinoma by facilitating the Warburg effect, et al. Activator of thyroid and retinoid receptor increases sorafenib resistance in hepatocellular carcinoma by facilitating the Warburg effect"

    Article Title: Activator of thyroid and retinoid receptor increases sorafenib resistance in hepatocellular carcinoma by facilitating the Warburg effect, et al. Activator of thyroid and retinoid receptor increases sorafenib resistance in hepatocellular carcinoma by facilitating the Warburg effect

    Journal: Cancer Science

    doi: 10.1111/cas.14412

    Activator of thyroid and retinoid receptor (ACTR) enhances sorafenib resistance by affecting aerobic glycolysis in vitro. A and B, The relative viability curves of ACTR WT or KO HepG2 cells or ACTR KO HepG2 cells transiently transfected with ACTR, as well as Huh‐7 cells in DMEM with high glucose (25 mmol/L), transfected with ACTR siRNA or ACTR siRNA plus ACTR expression vector or non‐specific control for siRNA (Control siRNA); they were treated with Deoxy‐d‐glucose (2‐DG) (2.5 mmol/L) and increasing concentrations of sorafenib as indicated above. After 72 h, cell viability assays were performed using the CCK‐8. The group without treatment of sorafenib had 100% viable cells and was used as an internal control for comparison. The representative immunoblot with ACTR indicates ACTR expression levels. C and D, The relative viability curves of HepG2 cells (C) or Huh‐7 cells (D) transfected and treated as in (A) or (B) and cultured in DMEM with low glucose (5.5 mmol/L). E and F, Colony formation assays of HepG2 and Huh‐7 cells treated as in (A) and (B) with sorafenib (6 μmol/L) or not. G, Representative flow cytometry analysis of Annexin V (1:1000) and propidium iodide (1:1000) staining was carried out in HepG2 ACTR WT cells, KO cells, WT cells and KO cells treated with 2‐DG (2.5 mmol/L) and sorafenib (6 μmol/L) for 6 h. Data shown are mean ± SD of triplicate measurements that have been repeated three times with similar results. * P
    Figure Legend Snippet: Activator of thyroid and retinoid receptor (ACTR) enhances sorafenib resistance by affecting aerobic glycolysis in vitro. A and B, The relative viability curves of ACTR WT or KO HepG2 cells or ACTR KO HepG2 cells transiently transfected with ACTR, as well as Huh‐7 cells in DMEM with high glucose (25 mmol/L), transfected with ACTR siRNA or ACTR siRNA plus ACTR expression vector or non‐specific control for siRNA (Control siRNA); they were treated with Deoxy‐d‐glucose (2‐DG) (2.5 mmol/L) and increasing concentrations of sorafenib as indicated above. After 72 h, cell viability assays were performed using the CCK‐8. The group without treatment of sorafenib had 100% viable cells and was used as an internal control for comparison. The representative immunoblot with ACTR indicates ACTR expression levels. C and D, The relative viability curves of HepG2 cells (C) or Huh‐7 cells (D) transfected and treated as in (A) or (B) and cultured in DMEM with low glucose (5.5 mmol/L). E and F, Colony formation assays of HepG2 and Huh‐7 cells treated as in (A) and (B) with sorafenib (6 μmol/L) or not. G, Representative flow cytometry analysis of Annexin V (1:1000) and propidium iodide (1:1000) staining was carried out in HepG2 ACTR WT cells, KO cells, WT cells and KO cells treated with 2‐DG (2.5 mmol/L) and sorafenib (6 μmol/L) for 6 h. Data shown are mean ± SD of triplicate measurements that have been repeated three times with similar results. * P

    Techniques Used: In Vitro, Transfection, Expressing, Plasmid Preparation, CCK-8 Assay, Cell Culture, Flow Cytometry, Staining

    14) Product Images from "Cytokines Differently Define the Immunomodulation of Mesenchymal Stem Cells from the Periodontal Ligament"

    Article Title: Cytokines Differently Define the Immunomodulation of Mesenchymal Stem Cells from the Periodontal Ligament

    Journal: Cells

    doi: 10.3390/cells9051222

    Effect of IDO-1, PD-L1, and PTGS-2 inhibition in hPDLSCs on CD4 + T lymphocyte proliferation in the presence of different inflammatory stimuli (IL-1β, TNF-α, and IFN-γ). Primary hPDLSCs were stimulated with either ( a ) 50 µM PF-06840003 (IDO-1 inhibitor) or ( b ) 1 µM BMS202 (PD-1/PD-L1 inhibitor) or ( c ) 1 µM Celecoxib (PTGS-2 inhibitor) in the absence or the presence of either 5 ng/ml IL-1β or 10 ng/ml TNF-α or 100 ng/ml IFN-γ. After 48 h, hPDLSCs were applied to indirect co-culture and stimulated as described above. Allogenic CD4 + T lymphocytes were added to indirect co-culture using TC inserts. CD4 + T lymphocyte proliferation was induced by 10 µg/ml PHA-L. After five days incubation, CD4 + T lymphocyte proliferation was determined by analyzing CFSE labeled CD4 + T lymphocytes via flow cytometry. The Y-axis shows the percentage of at least once divided CD4 + T lymphocytes. Data are presented as mean value ± S.E.M. originated from five independent experiments with hPDLSCs from five different individuals. * p -value
    Figure Legend Snippet: Effect of IDO-1, PD-L1, and PTGS-2 inhibition in hPDLSCs on CD4 + T lymphocyte proliferation in the presence of different inflammatory stimuli (IL-1β, TNF-α, and IFN-γ). Primary hPDLSCs were stimulated with either ( a ) 50 µM PF-06840003 (IDO-1 inhibitor) or ( b ) 1 µM BMS202 (PD-1/PD-L1 inhibitor) or ( c ) 1 µM Celecoxib (PTGS-2 inhibitor) in the absence or the presence of either 5 ng/ml IL-1β or 10 ng/ml TNF-α or 100 ng/ml IFN-γ. After 48 h, hPDLSCs were applied to indirect co-culture and stimulated as described above. Allogenic CD4 + T lymphocytes were added to indirect co-culture using TC inserts. CD4 + T lymphocyte proliferation was induced by 10 µg/ml PHA-L. After five days incubation, CD4 + T lymphocyte proliferation was determined by analyzing CFSE labeled CD4 + T lymphocytes via flow cytometry. The Y-axis shows the percentage of at least once divided CD4 + T lymphocytes. Data are presented as mean value ± S.E.M. originated from five independent experiments with hPDLSCs from five different individuals. * p -value

    Techniques Used: Inhibition, Co-Culture Assay, Incubation, Labeling, Flow Cytometry

    Effect of pro-inflammatory stimuli (IL-1β, TNF-α, and IFN-γ) on programmed cell death 1 ligand 1 (PD-L1) and programmed cell death 1 ligand 2 (PD-L2) production in hPDLSCs. Primary hPDLSCs were treated with either 5 ng/ml IL-1β or 10 ng/ml TNF-α or 100 ng/ml IFN-γ for 48 h. Unstimulated hPDLSCs served as control. PD-L1 ( a ) and PD-L2 ( b ) gene expression levels were determined by qPCR, demonstrating the n-fold PD-L1 / PD-L2 expression compared to the control (n = 1). GAPDH served as internal reference gene. PD-L1/PD-L2 protein levels were investigated by intracellular immunostaining followed by flow cytometry analysis, determining the percentage of PD-L1 ( c ) and PD-L2 ( d ) positive cells. All data are presented as mean value ± S.E.M. received from six independent experiments with cells isolated from six different individuals. * p -value
    Figure Legend Snippet: Effect of pro-inflammatory stimuli (IL-1β, TNF-α, and IFN-γ) on programmed cell death 1 ligand 1 (PD-L1) and programmed cell death 1 ligand 2 (PD-L2) production in hPDLSCs. Primary hPDLSCs were treated with either 5 ng/ml IL-1β or 10 ng/ml TNF-α or 100 ng/ml IFN-γ for 48 h. Unstimulated hPDLSCs served as control. PD-L1 ( a ) and PD-L2 ( b ) gene expression levels were determined by qPCR, demonstrating the n-fold PD-L1 / PD-L2 expression compared to the control (n = 1). GAPDH served as internal reference gene. PD-L1/PD-L2 protein levels were investigated by intracellular immunostaining followed by flow cytometry analysis, determining the percentage of PD-L1 ( c ) and PD-L2 ( d ) positive cells. All data are presented as mean value ± S.E.M. received from six independent experiments with cells isolated from six different individuals. * p -value

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Immunostaining, Flow Cytometry, Isolation

    Effect of pro-inflammatory stimuli (IL-1β, TNF-α, and IFN-γ) on intoleamine-2,3-dioxygenase-1 (IDO-1) production and activity in hPDLSCs. Primary hPDLSCs were treated with either 5 ng/ml IL-1β or 10 ng/ml TNF-α or 100 ng/ml IFN-γ. Unstimulated hPDLSCs served as control. After 48 h treatment, IDO-1 gene expression levels ( a ) were measured using qPCR, demonstrating the n-fold IDO-1 expression compared to the control (n = 1). GAPDH served as internal reference gene. Corresponding IDO-1 protein levels were quantified by intracellular immunostaining followed by flow cytometry analysis, determining the percentage of IDO-1 positive hPDLSCs ( b ) and the corresponding mean fluorescence intensity (m.f.i.) ( c ). IDO-1 enzymatic activity was measured by quantifying L-kynurenine concentration (µM) in cell lysates ( d ) and the conditioned media ( e ) normalized to the total protein amounts in mg. All data are presented as mean value ± S.E.M. received from six independent experiments with cells isolated from six different individuals. * p -value
    Figure Legend Snippet: Effect of pro-inflammatory stimuli (IL-1β, TNF-α, and IFN-γ) on intoleamine-2,3-dioxygenase-1 (IDO-1) production and activity in hPDLSCs. Primary hPDLSCs were treated with either 5 ng/ml IL-1β or 10 ng/ml TNF-α or 100 ng/ml IFN-γ. Unstimulated hPDLSCs served as control. After 48 h treatment, IDO-1 gene expression levels ( a ) were measured using qPCR, demonstrating the n-fold IDO-1 expression compared to the control (n = 1). GAPDH served as internal reference gene. Corresponding IDO-1 protein levels were quantified by intracellular immunostaining followed by flow cytometry analysis, determining the percentage of IDO-1 positive hPDLSCs ( b ) and the corresponding mean fluorescence intensity (m.f.i.) ( c ). IDO-1 enzymatic activity was measured by quantifying L-kynurenine concentration (µM) in cell lysates ( d ) and the conditioned media ( e ) normalized to the total protein amounts in mg. All data are presented as mean value ± S.E.M. received from six independent experiments with cells isolated from six different individuals. * p -value

    Techniques Used: Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Immunostaining, Flow Cytometry, Fluorescence, Concentration Assay, Isolation

    Effect of IDO-1, PD-L1, and PTGS-2 inhibition in hPDLSCs on CD4 + T lymphocyte apoptosis in the presence of different inflammatory stimuli (IL-1β, TNF-α, and IFN-γ). Primary hPDLSCs were stimulated with either ( a ) 50 µM PF-06840003 (IDO-1 inhibitor) or ( b ) 1 µM BMS202 (PD-1/PD-L1 inhibitor) or ( c ) 1 µM Celecoxib (PTGS-2 inhibitor) in the absence or the presence of either 5 ng/ml IL-1β or 10 ng/ml TNF-α or 100 ng/ml IFN-γ. After 48 h, hPDLSCs were applied to indirect co-culture and stimulated as described above. Allogenic CD4 + T lymphocytes were added to indirect co-culture using TC inserts. After five days incubation, the percentage of apoptotic CD4 + T lymphocytes was determined by flow cytometry by labeling CD4 + T lymphocytes with Pi. The Y-axis shows the percentage of Pi positive CD4 + T lymphocytes. Data are presented as mean value ± S.E.M. received from five independent experiments with hPDLSCs from five different individuals. * p -value
    Figure Legend Snippet: Effect of IDO-1, PD-L1, and PTGS-2 inhibition in hPDLSCs on CD4 + T lymphocyte apoptosis in the presence of different inflammatory stimuli (IL-1β, TNF-α, and IFN-γ). Primary hPDLSCs were stimulated with either ( a ) 50 µM PF-06840003 (IDO-1 inhibitor) or ( b ) 1 µM BMS202 (PD-1/PD-L1 inhibitor) or ( c ) 1 µM Celecoxib (PTGS-2 inhibitor) in the absence or the presence of either 5 ng/ml IL-1β or 10 ng/ml TNF-α or 100 ng/ml IFN-γ. After 48 h, hPDLSCs were applied to indirect co-culture and stimulated as described above. Allogenic CD4 + T lymphocytes were added to indirect co-culture using TC inserts. After five days incubation, the percentage of apoptotic CD4 + T lymphocytes was determined by flow cytometry by labeling CD4 + T lymphocytes with Pi. The Y-axis shows the percentage of Pi positive CD4 + T lymphocytes. Data are presented as mean value ± S.E.M. received from five independent experiments with hPDLSCs from five different individuals. * p -value

    Techniques Used: Inhibition, Co-Culture Assay, Incubation, Flow Cytometry, Labeling

    hPDLSC mediated effect of different inflammatory stimuli (IL-1β, TNF-α, and IFN-γ) on CD4 + T lymphocyte proliferation and apoptosis. Primary hPDLSCs were stimulated with either 5 ng/ml IL-1β or 10 ng/ml TNF-α or 100 ng/ml IFN-γ. After 48 h, hPDLSCs were applied to indirect co-culture and stimulated as described above. Allogenic CD4 + T lymphocytes were added to indirect co-culture using Transwell (TC) inserts. Additionally, CD4 + T lymphocytes were cultured in the absence of hPDLSCs and stimulated with either 5 ng/ml IL-1β or 10 ng/ml TNF-α or 100 ng/ml IFN-γ. CD4 + T lymphocyte proliferation was induced by 10 µg/ml phytohemagglutinin-L (PHA-L). After five days incubation, CD4 + T lymphocyte proliferation ( a ) was determined by flow cytometry by analyzing carboxyfluorescein succinimidyl ester (CFSE) labeled CD4 + T lymphocytes. The Y-axis shows the percentage of at least once divided CD4 + T lymphocytes. Additionally, after five days incubation, the percentage of apoptotic CD4 + T lymphocytes ( b ) was determined by flow cytometry by labeling CD4 + T lymphocytes with Pi. The Y-axis shows the percentage of Pi positive CD4 + T lymphocytes. Data are presented as mean value ± S.E.M. received from five independent experiments with hPDLSCs from five different individuals. * p -value
    Figure Legend Snippet: hPDLSC mediated effect of different inflammatory stimuli (IL-1β, TNF-α, and IFN-γ) on CD4 + T lymphocyte proliferation and apoptosis. Primary hPDLSCs were stimulated with either 5 ng/ml IL-1β or 10 ng/ml TNF-α or 100 ng/ml IFN-γ. After 48 h, hPDLSCs were applied to indirect co-culture and stimulated as described above. Allogenic CD4 + T lymphocytes were added to indirect co-culture using Transwell (TC) inserts. Additionally, CD4 + T lymphocytes were cultured in the absence of hPDLSCs and stimulated with either 5 ng/ml IL-1β or 10 ng/ml TNF-α or 100 ng/ml IFN-γ. CD4 + T lymphocyte proliferation was induced by 10 µg/ml phytohemagglutinin-L (PHA-L). After five days incubation, CD4 + T lymphocyte proliferation ( a ) was determined by flow cytometry by analyzing carboxyfluorescein succinimidyl ester (CFSE) labeled CD4 + T lymphocytes. The Y-axis shows the percentage of at least once divided CD4 + T lymphocytes. Additionally, after five days incubation, the percentage of apoptotic CD4 + T lymphocytes ( b ) was determined by flow cytometry by labeling CD4 + T lymphocytes with Pi. The Y-axis shows the percentage of Pi positive CD4 + T lymphocytes. Data are presented as mean value ± S.E.M. received from five independent experiments with hPDLSCs from five different individuals. * p -value

    Techniques Used: Co-Culture Assay, Cell Culture, Incubation, Flow Cytometry, Labeling

    15) Product Images from "Molecular Profiling and Functional Analysis of Macrophage-Derived Tumor Extracellular Vesicles"

    Article Title: Molecular Profiling and Functional Analysis of Macrophage-Derived Tumor Extracellular Vesicles

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2019.05.008

    Quantification of TAM-EVs and Validation of the TAM-EV Protein Signature by IP of CD11b + Tumor-Derived EVs (A) Procedure to isolate EVs from MC38 tumors grown in LysM.Cre/ROSA mT/mG mice. (B) WB analysis of EVs isolated from cultured MC38 cells or MC38 tumors grown in LysM.Cre/ROSA mT/mG mice, after IP with anti-CD9 or control-coated magnetic beads. One representative EV preparation per condition is shown. (C and D) Flow cytometry analysis of tumor-EVs isolated from wild-type (WT) or LysM.Cre/ROSA mT/mG mice. Data show representative dot plots of GFP and tdTomato (C) and quantitative values (mean ± SD; n = 3 independent experiments; D). Statistics by two-way ANOVA, using Sidak’s multiple comparison test. (E) LC-MS/MS analysis of EVs showing quantitative values (mean ± SEM; n = 4 or 3 independent preparations) for CD11b, ALIX, and CD81, normalized to CD9. Statistics by two-way ANOVA, using Tukey’s multiple comparison test. (F) IP procedure for capturing CD11b + EVs. (G) Flow cytometry analysis of PKH67-labeled EVs bound to anti-CD9, anti-CD11b or isotype-control beads. Data show percentage values (mean ± SEM; n = 4 and 5 preparations for CD11b and CD9 IP, respectively). (H) WB analysis of tumor-derived EVs after IP. One representative EV preparation per condition is shown. (I) Venn diagrams comparing proteins enriched in IgG-EVs versus anti-CSF1R-EVs (FC > 1.7; see Table S2 ) with those detected in CD11b + IgG-EVs after IP (FC versus isotype control beads > 2; see Table S4 ). IP was performed on the same IgG-EV preparation shown in (H). (J) Radial table reporting the 62 proteins of the MC38 TAM-EV signature and their association with biological pathways. The graphical representation limits connection of each protein to two pathways (see also Table S5 ). (K) The data in (I) are shown after removing potential protein contaminants identified according to the CRAPome database. See also Figures S3 , S4 , S5 , and S6 .
    Figure Legend Snippet: Quantification of TAM-EVs and Validation of the TAM-EV Protein Signature by IP of CD11b + Tumor-Derived EVs (A) Procedure to isolate EVs from MC38 tumors grown in LysM.Cre/ROSA mT/mG mice. (B) WB analysis of EVs isolated from cultured MC38 cells or MC38 tumors grown in LysM.Cre/ROSA mT/mG mice, after IP with anti-CD9 or control-coated magnetic beads. One representative EV preparation per condition is shown. (C and D) Flow cytometry analysis of tumor-EVs isolated from wild-type (WT) or LysM.Cre/ROSA mT/mG mice. Data show representative dot plots of GFP and tdTomato (C) and quantitative values (mean ± SD; n = 3 independent experiments; D). Statistics by two-way ANOVA, using Sidak’s multiple comparison test. (E) LC-MS/MS analysis of EVs showing quantitative values (mean ± SEM; n = 4 or 3 independent preparations) for CD11b, ALIX, and CD81, normalized to CD9. Statistics by two-way ANOVA, using Tukey’s multiple comparison test. (F) IP procedure for capturing CD11b + EVs. (G) Flow cytometry analysis of PKH67-labeled EVs bound to anti-CD9, anti-CD11b or isotype-control beads. Data show percentage values (mean ± SEM; n = 4 and 5 preparations for CD11b and CD9 IP, respectively). (H) WB analysis of tumor-derived EVs after IP. One representative EV preparation per condition is shown. (I) Venn diagrams comparing proteins enriched in IgG-EVs versus anti-CSF1R-EVs (FC > 1.7; see Table S2 ) with those detected in CD11b + IgG-EVs after IP (FC versus isotype control beads > 2; see Table S4 ). IP was performed on the same IgG-EV preparation shown in (H). (J) Radial table reporting the 62 proteins of the MC38 TAM-EV signature and their association with biological pathways. The graphical representation limits connection of each protein to two pathways (see also Table S5 ). (K) The data in (I) are shown after removing potential protein contaminants identified according to the CRAPome database. See also Figures S3 , S4 , S5 , and S6 .

    Techniques Used: Derivative Assay, Mouse Assay, Western Blot, Isolation, Cell Culture, Magnetic Beads, Flow Cytometry, Liquid Chromatography with Mass Spectroscopy, Labeling

    Immunomodulatory Profile and Functions of TAM-EVs (A and B) Gene set enrichment analysis (GSEA) plots showing the correlation of TAM (TAM-Cell) and TAM-EV protein signatures with genes expressed in FACS-sorted M1-like or M2-like TAMs (A) or prognostic genes across human cancers recorded in the PRECOG database (B). (C) Flow cytometry analysis of CD8 + T cells obtained after priming OT-I splenocytes. (D) Schematic of the experiment shown in (E–G). (E and F) Flow cytometry analysis of CD8 + OT-I proliferation assessed by CellTrace dilution. (E) Representative flow profiles. (F) Quantification of the data (mean ± SEM; n = 4 cell cultures/condition). Statistics by two-way ANOVA, using Sidak’s multiple comparison test. (G) ELISA-based quantification of IFNγ in medium conditioned by OT-I CD8 + T cells. Data are shown as mean ± SEM (n = 4 cell cultures/condition). Statistics as in (F). (H) Flow cytometry analysis of CD8 + T cells purified from the spleen of C57BL/6 mice. (I) Schematic of the experiment shown in (J and K). (J) Flow cytometry analysis of CD8 + T cell proliferation assessed by CellTrace dilution (mean ± SEM; n = 4 cell cultures/condition). Statistics by one-way ANOVA, using Tukey’s multiple comparison test. (K) ELISA-based quantification of IFNγ in medium conditioned by CD8 + T cells (mean ± SEM; n = 4 cell cultures/condition). Statistics as in (J). (L) Flow cytometry analysis of CD4 + T cells purified from the spleen of BALB/c mice. (M) Schematic of the experiment shown in (N). (N) Flow cytometry analysis of CD4 + T cell proliferation assessed by CellTrace dilution (mean ± SEM; n = 4 cell cultures/condition). Statistics as in (J). (O) Schematic of the experiment shown in (P). (P) Flow cytometry analysis of activation markers (mean fluorescence intensity, MFI) in CD11b + CD11c + BMDCs (mean ± SEM; n = 4 cell cultures/condition). Statistics by two-way ANOVA, using Dunnett’s multiple comparison test (each treatment condition versus control DMSO).
    Figure Legend Snippet: Immunomodulatory Profile and Functions of TAM-EVs (A and B) Gene set enrichment analysis (GSEA) plots showing the correlation of TAM (TAM-Cell) and TAM-EV protein signatures with genes expressed in FACS-sorted M1-like or M2-like TAMs (A) or prognostic genes across human cancers recorded in the PRECOG database (B). (C) Flow cytometry analysis of CD8 + T cells obtained after priming OT-I splenocytes. (D) Schematic of the experiment shown in (E–G). (E and F) Flow cytometry analysis of CD8 + OT-I proliferation assessed by CellTrace dilution. (E) Representative flow profiles. (F) Quantification of the data (mean ± SEM; n = 4 cell cultures/condition). Statistics by two-way ANOVA, using Sidak’s multiple comparison test. (G) ELISA-based quantification of IFNγ in medium conditioned by OT-I CD8 + T cells. Data are shown as mean ± SEM (n = 4 cell cultures/condition). Statistics as in (F). (H) Flow cytometry analysis of CD8 + T cells purified from the spleen of C57BL/6 mice. (I) Schematic of the experiment shown in (J and K). (J) Flow cytometry analysis of CD8 + T cell proliferation assessed by CellTrace dilution (mean ± SEM; n = 4 cell cultures/condition). Statistics by one-way ANOVA, using Tukey’s multiple comparison test. (K) ELISA-based quantification of IFNγ in medium conditioned by CD8 + T cells (mean ± SEM; n = 4 cell cultures/condition). Statistics as in (J). (L) Flow cytometry analysis of CD4 + T cells purified from the spleen of BALB/c mice. (M) Schematic of the experiment shown in (N). (N) Flow cytometry analysis of CD4 + T cell proliferation assessed by CellTrace dilution (mean ± SEM; n = 4 cell cultures/condition). Statistics as in (J). (O) Schematic of the experiment shown in (P). (P) Flow cytometry analysis of activation markers (mean fluorescence intensity, MFI) in CD11b + CD11c + BMDCs (mean ± SEM; n = 4 cell cultures/condition). Statistics by two-way ANOVA, using Dunnett’s multiple comparison test (each treatment condition versus control DMSO).

    Techniques Used: FACS, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Purification, Mouse Assay, Activation Assay, Fluorescence

    Molecular and Functional Analysis of E0771 TAM-EVs (A) Schedule of subcutaneous E0771 cancer cell inoculation in C57BL/6 mice and drug administration. (B) Flow cytometry analysis of immune infiltrates in E0771 tumors. Data show percentage values (mean ± SD; n = 3 mice/condition). Statistics by unpaired two-tailed Student’s t test (left and middle) or two-way ANOVA, using Sidak’s multiple comparison test (right). (C) Yield of EVs recovered from E0771 tumors prior to and after sucrose gradient fractionation, determined by BCA (mean ± SD; n = 3 EV preparations/condition). Statistics by unpaired two-tailed Student’s t test. (D) EV concentration and size distribution by NTA in the 6 sucrose fractions (mean ± SEM; n = 3 EV preparations/condition). (E) EV protein content and EV concentration in each sucrose fraction determined by BCA and NTA, respectively (mean ± SD; n = 3 EV preparations/condition). (F) Representative TEM images of EVs. One representative EV preparation per condition is shown. Scale bars, 200 nm. (G) WB analysis of cultured E0771 cells and matched EVs. One representative EV preparation is shown. (H and I) WB analysis of E0771 tumor-EVs (H). Relative signal quantification of MRC1, COX1, and TBXAS1 is shown in (I) as mean band intensity normalized to CD9 (n = 3 EV preparations/condition). Statistics as in (C). (J) Venn diagrams comparing proteins enriched in IgG-EVs versus anti-CSF1R-EVs from E0771 tumors (FC > 1.7; see Table S7 ) with those of the MC38 TAM-EV signature (see Table S5 ). (K) LC-MS/MS analysis of TBXAS1 in E0771 tumor-EVs (mean ± SD; n = 3 EV preparations/condition). Statistics as in (C). (L) ELISA-based quantification of TXB 2 in medium conditioned by E0771 cells (mean ± SD; n = 3 EV preparations/condition). Statistics by one-way ANOVA, using Tukey’s multiple comparison test.
    Figure Legend Snippet: Molecular and Functional Analysis of E0771 TAM-EVs (A) Schedule of subcutaneous E0771 cancer cell inoculation in C57BL/6 mice and drug administration. (B) Flow cytometry analysis of immune infiltrates in E0771 tumors. Data show percentage values (mean ± SD; n = 3 mice/condition). Statistics by unpaired two-tailed Student’s t test (left and middle) or two-way ANOVA, using Sidak’s multiple comparison test (right). (C) Yield of EVs recovered from E0771 tumors prior to and after sucrose gradient fractionation, determined by BCA (mean ± SD; n = 3 EV preparations/condition). Statistics by unpaired two-tailed Student’s t test. (D) EV concentration and size distribution by NTA in the 6 sucrose fractions (mean ± SEM; n = 3 EV preparations/condition). (E) EV protein content and EV concentration in each sucrose fraction determined by BCA and NTA, respectively (mean ± SD; n = 3 EV preparations/condition). (F) Representative TEM images of EVs. One representative EV preparation per condition is shown. Scale bars, 200 nm. (G) WB analysis of cultured E0771 cells and matched EVs. One representative EV preparation is shown. (H and I) WB analysis of E0771 tumor-EVs (H). Relative signal quantification of MRC1, COX1, and TBXAS1 is shown in (I) as mean band intensity normalized to CD9 (n = 3 EV preparations/condition). Statistics as in (C). (J) Venn diagrams comparing proteins enriched in IgG-EVs versus anti-CSF1R-EVs from E0771 tumors (FC > 1.7; see Table S7 ) with those of the MC38 TAM-EV signature (see Table S5 ). (K) LC-MS/MS analysis of TBXAS1 in E0771 tumor-EVs (mean ± SD; n = 3 EV preparations/condition). Statistics as in (C). (L) ELISA-based quantification of TXB 2 in medium conditioned by E0771 cells (mean ± SD; n = 3 EV preparations/condition). Statistics by one-way ANOVA, using Tukey’s multiple comparison test.

    Techniques Used: Functional Assay, Mouse Assay, Flow Cytometry, Two Tailed Test, Fractionation, Concentration Assay, Transmission Electron Microscopy, Western Blot, Cell Culture, Liquid Chromatography with Mass Spectroscopy, Enzyme-linked Immunosorbent Assay

    EV Isolation from MC38 Tumors of IgG- and Anti-CSF1R-Treated Mice (A) Procedure to isolate EVs from IgG- and anti-CSF1R-treated tumors. (B) Flow cytometry of MC38-tumor-derived cells (day 14 post-tumor challenge; see Figure S1 A). Data show percentage values (mean ± SEM; n = 5 mice/condition). Statistics by unpaired two-tailed Student’s t test. (C) Yield of EVs prior to sucrose fractionation, determined by BCA (mean ± SEM; n = 5 independent EV preparations). Statistics as in (B). (D) Representative TEM images of EVs obtained as in (C). One representative EV preparation is shown for IgG and anti-CSF1R-treated tumors. Scale bars, 200 nm. (E) EV concentration and size distribution by NTA (mean ± SEM; n = 3 acquisitions/sample). One representative EV preparation per condition is shown. (F) Correlation between EV protein content and EV concentration, determined by BCA and NTA, respectively (mean ± SD; n = 3 serial dilutions/sample). A simple linear regression function was used. One representative EV preparation per condition is shown. (G) WB analysis of cells and matched EVs from cultured MC38 cells or MC38 tumors. One representative cell or EV preparation per condition is shown. (H) EV protein content and EV concentration in each sucrose fraction, determined by BCA and NTA, respectively (mean of 2–3 technical replicates). One representative EV preparation per condition is shown. (I) EV concentration and size distribution by NTA (mean ± SEM; n = 3 acquisitions/sample). One representative EV preparation per condition is shown. (J) Yield of EVs recovered from the third top fraction of the sucrose gradient, determined by BCA (mean ± SEM; n = 5 independent EV preparations). Statistics as in (B). (K) Representative TEM images of EVs recovered from the third top sucrose fraction. One representative EV preparation per condition is shown. Scale bars, 200 nm. (L) WB analysis of EVs after sucrose gradient fractionation. Upper panel shows a representative experiment; equal sample volumes were loaded in each lane. Lower panels show relative band intensities of MRC1 and GAPDH (mean ± SEM; n = 3 independent EV preparations, one of which is shown in the WB above). For each protein, the relative signal intensity in each fraction is indicated as percentage of the total signal from all fractions. (M) Taqman analysis of selected microRNAs (normalized to miR-16-5p; fold-change versus anti-CSF1R-EVs) in EVs after sucrose gradient fractionation (mean ± SEM; n = 3 independent EV preparations). Statistics by two-way ANOVA with Sidak’s multiple comparison test. Statistical significance of the data: ∗ p
    Figure Legend Snippet: EV Isolation from MC38 Tumors of IgG- and Anti-CSF1R-Treated Mice (A) Procedure to isolate EVs from IgG- and anti-CSF1R-treated tumors. (B) Flow cytometry of MC38-tumor-derived cells (day 14 post-tumor challenge; see Figure S1 A). Data show percentage values (mean ± SEM; n = 5 mice/condition). Statistics by unpaired two-tailed Student’s t test. (C) Yield of EVs prior to sucrose fractionation, determined by BCA (mean ± SEM; n = 5 independent EV preparations). Statistics as in (B). (D) Representative TEM images of EVs obtained as in (C). One representative EV preparation is shown for IgG and anti-CSF1R-treated tumors. Scale bars, 200 nm. (E) EV concentration and size distribution by NTA (mean ± SEM; n = 3 acquisitions/sample). One representative EV preparation per condition is shown. (F) Correlation between EV protein content and EV concentration, determined by BCA and NTA, respectively (mean ± SD; n = 3 serial dilutions/sample). A simple linear regression function was used. One representative EV preparation per condition is shown. (G) WB analysis of cells and matched EVs from cultured MC38 cells or MC38 tumors. One representative cell or EV preparation per condition is shown. (H) EV protein content and EV concentration in each sucrose fraction, determined by BCA and NTA, respectively (mean of 2–3 technical replicates). One representative EV preparation per condition is shown. (I) EV concentration and size distribution by NTA (mean ± SEM; n = 3 acquisitions/sample). One representative EV preparation per condition is shown. (J) Yield of EVs recovered from the third top fraction of the sucrose gradient, determined by BCA (mean ± SEM; n = 5 independent EV preparations). Statistics as in (B). (K) Representative TEM images of EVs recovered from the third top sucrose fraction. One representative EV preparation per condition is shown. Scale bars, 200 nm. (L) WB analysis of EVs after sucrose gradient fractionation. Upper panel shows a representative experiment; equal sample volumes were loaded in each lane. Lower panels show relative band intensities of MRC1 and GAPDH (mean ± SEM; n = 3 independent EV preparations, one of which is shown in the WB above). For each protein, the relative signal intensity in each fraction is indicated as percentage of the total signal from all fractions. (M) Taqman analysis of selected microRNAs (normalized to miR-16-5p; fold-change versus anti-CSF1R-EVs) in EVs after sucrose gradient fractionation (mean ± SEM; n = 3 independent EV preparations). Statistics by two-way ANOVA with Sidak’s multiple comparison test. Statistical significance of the data: ∗ p

    Techniques Used: Isolation, Mouse Assay, Flow Cytometry, Derivative Assay, Two Tailed Test, Fractionation, Transmission Electron Microscopy, Concentration Assay, Western Blot, Cell Culture

    Molecular and Functional Lipidomic Profile of TAM-EVs (A) Schematic illustrating AA metabolism. (B) LC-MS/MS proteome analysis of EVs showing enzymes involved in eicosanoid synthesis. Data show quantitative values (mean ± SEM; n = 4 and 3 independent preparations of MC38-tumor-derived EVs and MC38-EVs, respectively). Statistics by two-way ANOVA, using Tukey’s multiple comparison test. (C) WB analysis of cells and EVs from cultured MC38 cells or MC38 tumors. One representative EV preparation per condition is shown. (D) AA concentration (mean ± SEM; n = 4 independent EV preparations) determined by LC-MS/MS-based absolute quantification using calibration curves of internal standards. Statistics by unpaired two-tailed Student’s t test. (E) TXB 2 concentration (mean ± SEM; n = 4 independent EV preparations) determined by LC-MS/MS-based relative quantification of ion counts. Statistics as in (D). (F) Schematic illustrating putative thromboxane synthesis in TAM-EVs. (G) Quantification of eicosanoids, mostly PGs (left) and their precursor AA (right), in MC38 tumors (mean ± SEM; n = 6 mice/condition) by LC-MS/MS lipidomics. Statistics by two-way ANOVA, using Sidak’s multiple comparison test (left) or unpaired two-tailed Student’s t test (right). (H) CD8 + T cell proliferation in response to PGE 2 or PGF 2α ; the left panel shows the experimental design. The right panel shows flow cytometry analysis of CD8 + T cell proliferation assessed by CellTrace dilution (mean ± SD; n = 3 cell cultures/condition). Statistics by one-way ANOVA, using Dunnett’s multiple comparison test (each PG concentration versus control DMSO). (I) Confocal analysis of MC38 cells incubated with PKH67-labeled MC38 IgG-EVs (green). Nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). The merged panel is shown on the left. One representative experiment is shown. Scale bar, 10 μm. (J) WB analysis of MC38 cells incubated with EVs isolated from LysM.Cre/ROSA mT/mG mice. One representative EV preparation per condition is shown. (K) Schematic of the experiment shown in (L–N). (L and M) Quantification of PUFAs (L) and eicosanoids (M) in medium conditioned by MC38 cells (mean ± SEM; n = 4 cell cultures/condition) by LC-MS/MS. Statistics as in (B). (N) ELISA-based quantification of PGE 2 and TXB 2 (mean ± SEM; n = 4 cell cultures/condition) in medium conditioned by MC38 cells. Statistics by one-way ANOVA, using Tukey’s multiple comparison test. See also Figure S7 .
    Figure Legend Snippet: Molecular and Functional Lipidomic Profile of TAM-EVs (A) Schematic illustrating AA metabolism. (B) LC-MS/MS proteome analysis of EVs showing enzymes involved in eicosanoid synthesis. Data show quantitative values (mean ± SEM; n = 4 and 3 independent preparations of MC38-tumor-derived EVs and MC38-EVs, respectively). Statistics by two-way ANOVA, using Tukey’s multiple comparison test. (C) WB analysis of cells and EVs from cultured MC38 cells or MC38 tumors. One representative EV preparation per condition is shown. (D) AA concentration (mean ± SEM; n = 4 independent EV preparations) determined by LC-MS/MS-based absolute quantification using calibration curves of internal standards. Statistics by unpaired two-tailed Student’s t test. (E) TXB 2 concentration (mean ± SEM; n = 4 independent EV preparations) determined by LC-MS/MS-based relative quantification of ion counts. Statistics as in (D). (F) Schematic illustrating putative thromboxane synthesis in TAM-EVs. (G) Quantification of eicosanoids, mostly PGs (left) and their precursor AA (right), in MC38 tumors (mean ± SEM; n = 6 mice/condition) by LC-MS/MS lipidomics. Statistics by two-way ANOVA, using Sidak’s multiple comparison test (left) or unpaired two-tailed Student’s t test (right). (H) CD8 + T cell proliferation in response to PGE 2 or PGF 2α ; the left panel shows the experimental design. The right panel shows flow cytometry analysis of CD8 + T cell proliferation assessed by CellTrace dilution (mean ± SD; n = 3 cell cultures/condition). Statistics by one-way ANOVA, using Dunnett’s multiple comparison test (each PG concentration versus control DMSO). (I) Confocal analysis of MC38 cells incubated with PKH67-labeled MC38 IgG-EVs (green). Nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). The merged panel is shown on the left. One representative experiment is shown. Scale bar, 10 μm. (J) WB analysis of MC38 cells incubated with EVs isolated from LysM.Cre/ROSA mT/mG mice. One representative EV preparation per condition is shown. (K) Schematic of the experiment shown in (L–N). (L and M) Quantification of PUFAs (L) and eicosanoids (M) in medium conditioned by MC38 cells (mean ± SEM; n = 4 cell cultures/condition) by LC-MS/MS. Statistics as in (B). (N) ELISA-based quantification of PGE 2 and TXB 2 (mean ± SEM; n = 4 cell cultures/condition) in medium conditioned by MC38 cells. Statistics by one-way ANOVA, using Tukey’s multiple comparison test. See also Figure S7 .

    Techniques Used: Functional Assay, Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Western Blot, Cell Culture, Concentration Assay, Two Tailed Test, Mouse Assay, Flow Cytometry, Incubation, Labeling, Staining, Isolation, Enzyme-linked Immunosorbent Assay

    16) Product Images from "Molecular Profiling and Functional Analysis of Macrophage-Derived Tumor Extracellular Vesicles"

    Article Title: Molecular Profiling and Functional Analysis of Macrophage-Derived Tumor Extracellular Vesicles

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2019.05.008

    Quantification of TAM-EVs and Validation of the TAM-EV Protein Signature by IP of CD11b + Tumor-Derived EVs (A) Procedure to isolate EVs from MC38 tumors grown in LysM.Cre/ROSA mT/mG mice. (B) WB analysis of EVs isolated from cultured MC38 cells or MC38 tumors grown in LysM.Cre/ROSA mT/mG mice, after IP with anti-CD9 or control-coated magnetic beads. One representative EV preparation per condition is shown. (C and D) Flow cytometry analysis of tumor-EVs isolated from wild-type (WT) or LysM.Cre/ROSA mT/mG mice. Data show representative dot plots of GFP and tdTomato (C) and quantitative values (mean ± SD; n = 3 independent experiments; D). Statistics by two-way ANOVA, using Sidak’s multiple comparison test. (E) LC-MS/MS analysis of EVs showing quantitative values (mean ± SEM; n = 4 or 3 independent preparations) for CD11b, ALIX, and CD81, normalized to CD9. Statistics by two-way ANOVA, using Tukey’s multiple comparison test. (F) IP procedure for capturing CD11b + EVs. (G) Flow cytometry analysis of PKH67-labeled EVs bound to anti-CD9, anti-CD11b or isotype-control beads. Data show percentage values (mean ± SEM; n = 4 and 5 preparations for CD11b and CD9 IP, respectively). (H) WB analysis of tumor-derived EVs after IP. One representative EV preparation per condition is shown. (I) Venn diagrams comparing proteins enriched in IgG-EVs versus anti-CSF1R-EVs (FC > 1.7; see Table S2 ) with those detected in CD11b + IgG-EVs after IP (FC versus isotype control beads > 2; see Table S4 ). IP was performed on the same IgG-EV preparation shown in (H). (J) Radial table reporting the 62 proteins of the MC38 TAM-EV signature and their association with biological pathways. The graphical representation limits connection of each protein to two pathways (see also Table S5 ). (K) The data in (I) are shown after removing potential protein contaminants identified according to the CRAPome database. See also Figures S3 , S4 , S5 , and S6 .
    Figure Legend Snippet: Quantification of TAM-EVs and Validation of the TAM-EV Protein Signature by IP of CD11b + Tumor-Derived EVs (A) Procedure to isolate EVs from MC38 tumors grown in LysM.Cre/ROSA mT/mG mice. (B) WB analysis of EVs isolated from cultured MC38 cells or MC38 tumors grown in LysM.Cre/ROSA mT/mG mice, after IP with anti-CD9 or control-coated magnetic beads. One representative EV preparation per condition is shown. (C and D) Flow cytometry analysis of tumor-EVs isolated from wild-type (WT) or LysM.Cre/ROSA mT/mG mice. Data show representative dot plots of GFP and tdTomato (C) and quantitative values (mean ± SD; n = 3 independent experiments; D). Statistics by two-way ANOVA, using Sidak’s multiple comparison test. (E) LC-MS/MS analysis of EVs showing quantitative values (mean ± SEM; n = 4 or 3 independent preparations) for CD11b, ALIX, and CD81, normalized to CD9. Statistics by two-way ANOVA, using Tukey’s multiple comparison test. (F) IP procedure for capturing CD11b + EVs. (G) Flow cytometry analysis of PKH67-labeled EVs bound to anti-CD9, anti-CD11b or isotype-control beads. Data show percentage values (mean ± SEM; n = 4 and 5 preparations for CD11b and CD9 IP, respectively). (H) WB analysis of tumor-derived EVs after IP. One representative EV preparation per condition is shown. (I) Venn diagrams comparing proteins enriched in IgG-EVs versus anti-CSF1R-EVs (FC > 1.7; see Table S2 ) with those detected in CD11b + IgG-EVs after IP (FC versus isotype control beads > 2; see Table S4 ). IP was performed on the same IgG-EV preparation shown in (H). (J) Radial table reporting the 62 proteins of the MC38 TAM-EV signature and their association with biological pathways. The graphical representation limits connection of each protein to two pathways (see also Table S5 ). (K) The data in (I) are shown after removing potential protein contaminants identified according to the CRAPome database. See also Figures S3 , S4 , S5 , and S6 .

    Techniques Used: Derivative Assay, Mouse Assay, Western Blot, Isolation, Cell Culture, Magnetic Beads, Flow Cytometry, Liquid Chromatography with Mass Spectroscopy, Labeling

    Immunomodulatory Profile and Functions of TAM-EVs (A and B) Gene set enrichment analysis (GSEA) plots showing the correlation of TAM (TAM-Cell) and TAM-EV protein signatures with genes expressed in FACS-sorted M1-like or M2-like TAMs (A) or prognostic genes across human cancers recorded in the PRECOG database (B). (C) Flow cytometry analysis of CD8 + T cells obtained after priming OT-I splenocytes. (D) Schematic of the experiment shown in (E–G). (E and F) Flow cytometry analysis of CD8 + OT-I proliferation assessed by CellTrace dilution. (E) Representative flow profiles. (F) Quantification of the data (mean ± SEM; n = 4 cell cultures/condition). Statistics by two-way ANOVA, using Sidak’s multiple comparison test. (G) ELISA-based quantification of IFNγ in medium conditioned by OT-I CD8 + T cells. Data are shown as mean ± SEM (n = 4 cell cultures/condition). Statistics as in (F). (H) Flow cytometry analysis of CD8 + T cells purified from the spleen of C57BL/6 mice. (I) Schematic of the experiment shown in (J and K). (J) Flow cytometry analysis of CD8 + T cell proliferation assessed by CellTrace dilution (mean ± SEM; n = 4 cell cultures/condition). Statistics by one-way ANOVA, using Tukey’s multiple comparison test. (K) ELISA-based quantification of IFNγ in medium conditioned by CD8 + T cells (mean ± SEM; n = 4 cell cultures/condition). Statistics as in (J). (L) Flow cytometry analysis of CD4 + T cells purified from the spleen of BALB/c mice. (M) Schematic of the experiment shown in (N). (N) Flow cytometry analysis of CD4 + T cell proliferation assessed by CellTrace dilution (mean ± SEM; n = 4 cell cultures/condition). Statistics as in (J). (O) Schematic of the experiment shown in (P). (P) Flow cytometry analysis of activation markers (mean fluorescence intensity, MFI) in CD11b + CD11c + BMDCs (mean ± SEM; n = 4 cell cultures/condition). Statistics by two-way ANOVA, using Dunnett’s multiple comparison test (each treatment condition versus control DMSO).
    Figure Legend Snippet: Immunomodulatory Profile and Functions of TAM-EVs (A and B) Gene set enrichment analysis (GSEA) plots showing the correlation of TAM (TAM-Cell) and TAM-EV protein signatures with genes expressed in FACS-sorted M1-like or M2-like TAMs (A) or prognostic genes across human cancers recorded in the PRECOG database (B). (C) Flow cytometry analysis of CD8 + T cells obtained after priming OT-I splenocytes. (D) Schematic of the experiment shown in (E–G). (E and F) Flow cytometry analysis of CD8 + OT-I proliferation assessed by CellTrace dilution. (E) Representative flow profiles. (F) Quantification of the data (mean ± SEM; n = 4 cell cultures/condition). Statistics by two-way ANOVA, using Sidak’s multiple comparison test. (G) ELISA-based quantification of IFNγ in medium conditioned by OT-I CD8 + T cells. Data are shown as mean ± SEM (n = 4 cell cultures/condition). Statistics as in (F). (H) Flow cytometry analysis of CD8 + T cells purified from the spleen of C57BL/6 mice. (I) Schematic of the experiment shown in (J and K). (J) Flow cytometry analysis of CD8 + T cell proliferation assessed by CellTrace dilution (mean ± SEM; n = 4 cell cultures/condition). Statistics by one-way ANOVA, using Tukey’s multiple comparison test. (K) ELISA-based quantification of IFNγ in medium conditioned by CD8 + T cells (mean ± SEM; n = 4 cell cultures/condition). Statistics as in (J). (L) Flow cytometry analysis of CD4 + T cells purified from the spleen of BALB/c mice. (M) Schematic of the experiment shown in (N). (N) Flow cytometry analysis of CD4 + T cell proliferation assessed by CellTrace dilution (mean ± SEM; n = 4 cell cultures/condition). Statistics as in (J). (O) Schematic of the experiment shown in (P). (P) Flow cytometry analysis of activation markers (mean fluorescence intensity, MFI) in CD11b + CD11c + BMDCs (mean ± SEM; n = 4 cell cultures/condition). Statistics by two-way ANOVA, using Dunnett’s multiple comparison test (each treatment condition versus control DMSO).

    Techniques Used: FACS, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Purification, Mouse Assay, Activation Assay, Fluorescence

    Molecular and Functional Analysis of E0771 TAM-EVs (A) Schedule of subcutaneous E0771 cancer cell inoculation in C57BL/6 mice and drug administration. (B) Flow cytometry analysis of immune infiltrates in E0771 tumors. Data show percentage values (mean ± SD; n = 3 mice/condition). Statistics by unpaired two-tailed Student’s t test (left and middle) or two-way ANOVA, using Sidak’s multiple comparison test (right). (C) Yield of EVs recovered from E0771 tumors prior to and after sucrose gradient fractionation, determined by BCA (mean ± SD; n = 3 EV preparations/condition). Statistics by unpaired two-tailed Student’s t test. (D) EV concentration and size distribution by NTA in the 6 sucrose fractions (mean ± SEM; n = 3 EV preparations/condition). (E) EV protein content and EV concentration in each sucrose fraction determined by BCA and NTA, respectively (mean ± SD; n = 3 EV preparations/condition). (F) Representative TEM images of EVs. One representative EV preparation per condition is shown. Scale bars, 200 nm. (G) WB analysis of cultured E0771 cells and matched EVs. One representative EV preparation is shown. (H and I) WB analysis of E0771 tumor-EVs (H). Relative signal quantification of MRC1, COX1, and TBXAS1 is shown in (I) as mean band intensity normalized to CD9 (n = 3 EV preparations/condition). Statistics as in (C). (J) Venn diagrams comparing proteins enriched in IgG-EVs versus anti-CSF1R-EVs from E0771 tumors (FC > 1.7; see Table S7 ) with those of the MC38 TAM-EV signature (see Table S5 ). (K) LC-MS/MS analysis of TBXAS1 in E0771 tumor-EVs (mean ± SD; n = 3 EV preparations/condition). Statistics as in (C). (L) ELISA-based quantification of TXB 2 in medium conditioned by E0771 cells (mean ± SD; n = 3 EV preparations/condition). Statistics by one-way ANOVA, using Tukey’s multiple comparison test.
    Figure Legend Snippet: Molecular and Functional Analysis of E0771 TAM-EVs (A) Schedule of subcutaneous E0771 cancer cell inoculation in C57BL/6 mice and drug administration. (B) Flow cytometry analysis of immune infiltrates in E0771 tumors. Data show percentage values (mean ± SD; n = 3 mice/condition). Statistics by unpaired two-tailed Student’s t test (left and middle) or two-way ANOVA, using Sidak’s multiple comparison test (right). (C) Yield of EVs recovered from E0771 tumors prior to and after sucrose gradient fractionation, determined by BCA (mean ± SD; n = 3 EV preparations/condition). Statistics by unpaired two-tailed Student’s t test. (D) EV concentration and size distribution by NTA in the 6 sucrose fractions (mean ± SEM; n = 3 EV preparations/condition). (E) EV protein content and EV concentration in each sucrose fraction determined by BCA and NTA, respectively (mean ± SD; n = 3 EV preparations/condition). (F) Representative TEM images of EVs. One representative EV preparation per condition is shown. Scale bars, 200 nm. (G) WB analysis of cultured E0771 cells and matched EVs. One representative EV preparation is shown. (H and I) WB analysis of E0771 tumor-EVs (H). Relative signal quantification of MRC1, COX1, and TBXAS1 is shown in (I) as mean band intensity normalized to CD9 (n = 3 EV preparations/condition). Statistics as in (C). (J) Venn diagrams comparing proteins enriched in IgG-EVs versus anti-CSF1R-EVs from E0771 tumors (FC > 1.7; see Table S7 ) with those of the MC38 TAM-EV signature (see Table S5 ). (K) LC-MS/MS analysis of TBXAS1 in E0771 tumor-EVs (mean ± SD; n = 3 EV preparations/condition). Statistics as in (C). (L) ELISA-based quantification of TXB 2 in medium conditioned by E0771 cells (mean ± SD; n = 3 EV preparations/condition). Statistics by one-way ANOVA, using Tukey’s multiple comparison test.

    Techniques Used: Functional Assay, Mouse Assay, Flow Cytometry, Two Tailed Test, Fractionation, Concentration Assay, Transmission Electron Microscopy, Western Blot, Cell Culture, Liquid Chromatography with Mass Spectroscopy, Enzyme-linked Immunosorbent Assay

    EV Isolation from MC38 Tumors of IgG- and Anti-CSF1R-Treated Mice (A) Procedure to isolate EVs from IgG- and anti-CSF1R-treated tumors. (B) Flow cytometry of MC38-tumor-derived cells (day 14 post-tumor challenge; see Figure S1 A). Data show percentage values (mean ± SEM; n = 5 mice/condition). Statistics by unpaired two-tailed Student’s t test. (C) Yield of EVs prior to sucrose fractionation, determined by BCA (mean ± SEM; n = 5 independent EV preparations). Statistics as in (B). (D) Representative TEM images of EVs obtained as in (C). One representative EV preparation is shown for IgG and anti-CSF1R-treated tumors. Scale bars, 200 nm. (E) EV concentration and size distribution by NTA (mean ± SEM; n = 3 acquisitions/sample). One representative EV preparation per condition is shown. (F) Correlation between EV protein content and EV concentration, determined by BCA and NTA, respectively (mean ± SD; n = 3 serial dilutions/sample). A simple linear regression function was used. One representative EV preparation per condition is shown. (G) WB analysis of cells and matched EVs from cultured MC38 cells or MC38 tumors. One representative cell or EV preparation per condition is shown. (H) EV protein content and EV concentration in each sucrose fraction, determined by BCA and NTA, respectively (mean of 2–3 technical replicates). One representative EV preparation per condition is shown. (I) EV concentration and size distribution by NTA (mean ± SEM; n = 3 acquisitions/sample). One representative EV preparation per condition is shown. (J) Yield of EVs recovered from the third top fraction of the sucrose gradient, determined by BCA (mean ± SEM; n = 5 independent EV preparations). Statistics as in (B). (K) Representative TEM images of EVs recovered from the third top sucrose fraction. One representative EV preparation per condition is shown. Scale bars, 200 nm. (L) WB analysis of EVs after sucrose gradient fractionation. Upper panel shows a representative experiment; equal sample volumes were loaded in each lane. Lower panels show relative band intensities of MRC1 and GAPDH (mean ± SEM; n = 3 independent EV preparations, one of which is shown in the WB above). For each protein, the relative signal intensity in each fraction is indicated as percentage of the total signal from all fractions. (M) Taqman analysis of selected microRNAs (normalized to miR-16-5p; fold-change versus anti-CSF1R-EVs) in EVs after sucrose gradient fractionation (mean ± SEM; n = 3 independent EV preparations). Statistics by two-way ANOVA with Sidak’s multiple comparison test. Statistical significance of the data: ∗ p
    Figure Legend Snippet: EV Isolation from MC38 Tumors of IgG- and Anti-CSF1R-Treated Mice (A) Procedure to isolate EVs from IgG- and anti-CSF1R-treated tumors. (B) Flow cytometry of MC38-tumor-derived cells (day 14 post-tumor challenge; see Figure S1 A). Data show percentage values (mean ± SEM; n = 5 mice/condition). Statistics by unpaired two-tailed Student’s t test. (C) Yield of EVs prior to sucrose fractionation, determined by BCA (mean ± SEM; n = 5 independent EV preparations). Statistics as in (B). (D) Representative TEM images of EVs obtained as in (C). One representative EV preparation is shown for IgG and anti-CSF1R-treated tumors. Scale bars, 200 nm. (E) EV concentration and size distribution by NTA (mean ± SEM; n = 3 acquisitions/sample). One representative EV preparation per condition is shown. (F) Correlation between EV protein content and EV concentration, determined by BCA and NTA, respectively (mean ± SD; n = 3 serial dilutions/sample). A simple linear regression function was used. One representative EV preparation per condition is shown. (G) WB analysis of cells and matched EVs from cultured MC38 cells or MC38 tumors. One representative cell or EV preparation per condition is shown. (H) EV protein content and EV concentration in each sucrose fraction, determined by BCA and NTA, respectively (mean of 2–3 technical replicates). One representative EV preparation per condition is shown. (I) EV concentration and size distribution by NTA (mean ± SEM; n = 3 acquisitions/sample). One representative EV preparation per condition is shown. (J) Yield of EVs recovered from the third top fraction of the sucrose gradient, determined by BCA (mean ± SEM; n = 5 independent EV preparations). Statistics as in (B). (K) Representative TEM images of EVs recovered from the third top sucrose fraction. One representative EV preparation per condition is shown. Scale bars, 200 nm. (L) WB analysis of EVs after sucrose gradient fractionation. Upper panel shows a representative experiment; equal sample volumes were loaded in each lane. Lower panels show relative band intensities of MRC1 and GAPDH (mean ± SEM; n = 3 independent EV preparations, one of which is shown in the WB above). For each protein, the relative signal intensity in each fraction is indicated as percentage of the total signal from all fractions. (M) Taqman analysis of selected microRNAs (normalized to miR-16-5p; fold-change versus anti-CSF1R-EVs) in EVs after sucrose gradient fractionation (mean ± SEM; n = 3 independent EV preparations). Statistics by two-way ANOVA with Sidak’s multiple comparison test. Statistical significance of the data: ∗ p

    Techniques Used: Isolation, Mouse Assay, Flow Cytometry, Derivative Assay, Two Tailed Test, Fractionation, Transmission Electron Microscopy, Concentration Assay, Western Blot, Cell Culture

    Molecular and Functional Lipidomic Profile of TAM-EVs (A) Schematic illustrating AA metabolism. (B) LC-MS/MS proteome analysis of EVs showing enzymes involved in eicosanoid synthesis. Data show quantitative values (mean ± SEM; n = 4 and 3 independent preparations of MC38-tumor-derived EVs and MC38-EVs, respectively). Statistics by two-way ANOVA, using Tukey’s multiple comparison test. (C) WB analysis of cells and EVs from cultured MC38 cells or MC38 tumors. One representative EV preparation per condition is shown. (D) AA concentration (mean ± SEM; n = 4 independent EV preparations) determined by LC-MS/MS-based absolute quantification using calibration curves of internal standards. Statistics by unpaired two-tailed Student’s t test. (E) TXB 2 concentration (mean ± SEM; n = 4 independent EV preparations) determined by LC-MS/MS-based relative quantification of ion counts. Statistics as in (D). (F) Schematic illustrating putative thromboxane synthesis in TAM-EVs. (G) Quantification of eicosanoids, mostly PGs (left) and their precursor AA (right), in MC38 tumors (mean ± SEM; n = 6 mice/condition) by LC-MS/MS lipidomics. Statistics by two-way ANOVA, using Sidak’s multiple comparison test (left) or unpaired two-tailed Student’s t test (right). (H) CD8 + T cell proliferation in response to PGE 2 or PGF 2α ; the left panel shows the experimental design. The right panel shows flow cytometry analysis of CD8 + T cell proliferation assessed by CellTrace dilution (mean ± SD; n = 3 cell cultures/condition). Statistics by one-way ANOVA, using Dunnett’s multiple comparison test (each PG concentration versus control DMSO). (I) Confocal analysis of MC38 cells incubated with PKH67-labeled MC38 IgG-EVs (green). Nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). The merged panel is shown on the left. One representative experiment is shown. Scale bar, 10 μm. (J) WB analysis of MC38 cells incubated with EVs isolated from LysM.Cre/ROSA mT/mG mice. One representative EV preparation per condition is shown. (K) Schematic of the experiment shown in (L–N). (L and M) Quantification of PUFAs (L) and eicosanoids (M) in medium conditioned by MC38 cells (mean ± SEM; n = 4 cell cultures/condition) by LC-MS/MS. Statistics as in (B). (N) ELISA-based quantification of PGE 2 and TXB 2 (mean ± SEM; n = 4 cell cultures/condition) in medium conditioned by MC38 cells. Statistics by one-way ANOVA, using Tukey’s multiple comparison test. See also Figure S7 .
    Figure Legend Snippet: Molecular and Functional Lipidomic Profile of TAM-EVs (A) Schematic illustrating AA metabolism. (B) LC-MS/MS proteome analysis of EVs showing enzymes involved in eicosanoid synthesis. Data show quantitative values (mean ± SEM; n = 4 and 3 independent preparations of MC38-tumor-derived EVs and MC38-EVs, respectively). Statistics by two-way ANOVA, using Tukey’s multiple comparison test. (C) WB analysis of cells and EVs from cultured MC38 cells or MC38 tumors. One representative EV preparation per condition is shown. (D) AA concentration (mean ± SEM; n = 4 independent EV preparations) determined by LC-MS/MS-based absolute quantification using calibration curves of internal standards. Statistics by unpaired two-tailed Student’s t test. (E) TXB 2 concentration (mean ± SEM; n = 4 independent EV preparations) determined by LC-MS/MS-based relative quantification of ion counts. Statistics as in (D). (F) Schematic illustrating putative thromboxane synthesis in TAM-EVs. (G) Quantification of eicosanoids, mostly PGs (left) and their precursor AA (right), in MC38 tumors (mean ± SEM; n = 6 mice/condition) by LC-MS/MS lipidomics. Statistics by two-way ANOVA, using Sidak’s multiple comparison test (left) or unpaired two-tailed Student’s t test (right). (H) CD8 + T cell proliferation in response to PGE 2 or PGF 2α ; the left panel shows the experimental design. The right panel shows flow cytometry analysis of CD8 + T cell proliferation assessed by CellTrace dilution (mean ± SD; n = 3 cell cultures/condition). Statistics by one-way ANOVA, using Dunnett’s multiple comparison test (each PG concentration versus control DMSO). (I) Confocal analysis of MC38 cells incubated with PKH67-labeled MC38 IgG-EVs (green). Nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). The merged panel is shown on the left. One representative experiment is shown. Scale bar, 10 μm. (J) WB analysis of MC38 cells incubated with EVs isolated from LysM.Cre/ROSA mT/mG mice. One representative EV preparation per condition is shown. (K) Schematic of the experiment shown in (L–N). (L and M) Quantification of PUFAs (L) and eicosanoids (M) in medium conditioned by MC38 cells (mean ± SEM; n = 4 cell cultures/condition) by LC-MS/MS. Statistics as in (B). (N) ELISA-based quantification of PGE 2 and TXB 2 (mean ± SEM; n = 4 cell cultures/condition) in medium conditioned by MC38 cells. Statistics by one-way ANOVA, using Tukey’s multiple comparison test. See also Figure S7 .

    Techniques Used: Functional Assay, Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Western Blot, Cell Culture, Concentration Assay, Two Tailed Test, Mouse Assay, Flow Cytometry, Incubation, Labeling, Staining, Isolation, Enzyme-linked Immunosorbent Assay

    17) Product Images from "Decitabine attenuates dextran sodium sulfate-induced ulcerative colitis through regulation of immune regulatory cells and intestinal barrier"

    Article Title: Decitabine attenuates dextran sodium sulfate-induced ulcerative colitis through regulation of immune regulatory cells and intestinal barrier

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2020.4605

    Thl7/Treg-associated cytokine IL-17 expression is decreased and TGF-β and IL-10 are increased following decitabine treatment. (A) ELISA detected the secretion of IL-17, TGF-β and IL-10 cytokines in colon tissue. (B) Flow cytometry analysis of the effect of decitabine on Foxp3 in the spleen. Data are presented as the mean ± standard deviation. * P
    Figure Legend Snippet: Thl7/Treg-associated cytokine IL-17 expression is decreased and TGF-β and IL-10 are increased following decitabine treatment. (A) ELISA detected the secretion of IL-17, TGF-β and IL-10 cytokines in colon tissue. (B) Flow cytometry analysis of the effect of decitabine on Foxp3 in the spleen. Data are presented as the mean ± standard deviation. * P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Standard Deviation

    Decitabine treatment increases the proportion of CD4+ Foxp3+ T cells among CD4+ T cells in the spleen. (A) Mice spleen weights following treatments. (B) Flow cytometry analysis of the proportion of CD4+ Foxp3+ T cells in CD4+ T cells. Data are presented as the mean ± standard deviation. * P
    Figure Legend Snippet: Decitabine treatment increases the proportion of CD4+ Foxp3+ T cells among CD4+ T cells in the spleen. (A) Mice spleen weights following treatments. (B) Flow cytometry analysis of the proportion of CD4+ Foxp3+ T cells in CD4+ T cells. Data are presented as the mean ± standard deviation. * P

    Techniques Used: Mouse Assay, Flow Cytometry, Standard Deviation

    18) Product Images from "Neurons from human mesenchymal stem cells display both spontaneous and stimuli responsive activity"

    Article Title: Neurons from human mesenchymal stem cells display both spontaneous and stimuli responsive activity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0228510

    Bone marrow hMSCs from healthy donors differentiated into hMd-Neurons with phenotypical characteristics (A) Schematic representation of neuronal induction on bone marrow derived hMSCs from healthy donors. (B) Bright field images represents almost %90 of neuronal induced human bone marrow donor derived hMSCs give rise morphologically variable neuron like cells on culture at day 12 (b, d) whilst uninduced hMSCs maintains proliferation (a, d). Morphology of hMd-Neurons from human samples varies in culture as seen with neurite extension patterns (b, d). ( S2 Fig ). (C) Representative florescent images of Annexin V/Sytox green stainings showing cell viability of neuronal induced hMSCs with positive control stainings of dead cells on camptothecin (CAM) treated uninduced hMSCs. (D) Positively stained cells from Annexin V and sytox green stainings of 3 different donors were counted and the percentages of dead cells were determined. Plots indicate dead cell percentages from d2 to d12 in CAM treated, untreated (in growth medium), and neuronal induced hMSCs. (E) Graph showing time dependent cell proliferation profile of uninduced and neuronal induced hMSC during 12 days in culture by X-Celligence (Roche) real time cell analysis (RT-CA) system (F) RT-PCR demonstrating presence of βIII tubulin, Nestin, NSE, NF transcripts during neuronal differentiation (d2-d12) respectively versus an absence in uninduced hMSCs (d0). (G) Plots indicating neuronal marker expression percentages ≥%89 (Images from 3 different donors were counted to determine the neuronal marker percentages). Positively immunostained cells counted from 10 different areas of stainings and averages were calculated. (H) Representative confocal images showing neuronal protein expressions; NSE, PGP 9.5 and NeuN respectively (a, d, g) with DAPI nuclear stainings (b, e, h) of hMd-Neurons at d12 after neuronal induction. Fluorescent images of neuronal protein expressions merged with DAPI staining (c, f, i). (I) Plots represents flow cytometry results indicating 99.24% of hMd-Neurons are Nestin positive and negative for Ki-67 proliferation marker and Sox-2 expressions at day6of NI. (J) Flow cytometry analysis indicates loss of Ki-67 proliferation marker and Sox-2 expressions in Nestin positive cells. Scale bars represent 100 μm. Data are represented as mean ± S.E.M. Significance of ANOVA test *** p
    Figure Legend Snippet: Bone marrow hMSCs from healthy donors differentiated into hMd-Neurons with phenotypical characteristics (A) Schematic representation of neuronal induction on bone marrow derived hMSCs from healthy donors. (B) Bright field images represents almost %90 of neuronal induced human bone marrow donor derived hMSCs give rise morphologically variable neuron like cells on culture at day 12 (b, d) whilst uninduced hMSCs maintains proliferation (a, d). Morphology of hMd-Neurons from human samples varies in culture as seen with neurite extension patterns (b, d). ( S2 Fig ). (C) Representative florescent images of Annexin V/Sytox green stainings showing cell viability of neuronal induced hMSCs with positive control stainings of dead cells on camptothecin (CAM) treated uninduced hMSCs. (D) Positively stained cells from Annexin V and sytox green stainings of 3 different donors were counted and the percentages of dead cells were determined. Plots indicate dead cell percentages from d2 to d12 in CAM treated, untreated (in growth medium), and neuronal induced hMSCs. (E) Graph showing time dependent cell proliferation profile of uninduced and neuronal induced hMSC during 12 days in culture by X-Celligence (Roche) real time cell analysis (RT-CA) system (F) RT-PCR demonstrating presence of βIII tubulin, Nestin, NSE, NF transcripts during neuronal differentiation (d2-d12) respectively versus an absence in uninduced hMSCs (d0). (G) Plots indicating neuronal marker expression percentages ≥%89 (Images from 3 different donors were counted to determine the neuronal marker percentages). Positively immunostained cells counted from 10 different areas of stainings and averages were calculated. (H) Representative confocal images showing neuronal protein expressions; NSE, PGP 9.5 and NeuN respectively (a, d, g) with DAPI nuclear stainings (b, e, h) of hMd-Neurons at d12 after neuronal induction. Fluorescent images of neuronal protein expressions merged with DAPI staining (c, f, i). (I) Plots represents flow cytometry results indicating 99.24% of hMd-Neurons are Nestin positive and negative for Ki-67 proliferation marker and Sox-2 expressions at day6of NI. (J) Flow cytometry analysis indicates loss of Ki-67 proliferation marker and Sox-2 expressions in Nestin positive cells. Scale bars represent 100 μm. Data are represented as mean ± S.E.M. Significance of ANOVA test *** p

    Techniques Used: Derivative Assay, Positive Control, Chick Chorioallantoic Membrane Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Marker, Expressing, Flow Cytometry

    Isolated cells from bone marrow stroma of healthy donors display typical hMSC phenotype and mesodermal differentiation capacity (A, B) After isolation of human bone marrow cells from healthy donors, cells at passage 3 were analyzed by flow cytometry for phenotypic characterization. The cells showed hMSC specific marker expressions with negative expression of CD14, CD31, CD34, and CD45, while they were positive for CD29, CD44, CD73 and CD105. Representative histograms and surface antigen profiles of isolated adherent cells from 5 healthy donors are demonstrated. (C) Adipogenic differentiation was confirmed by Oil Red-O staining of lipid vacuoles (with hematoxylin counterstain); osteogenic differentiation was followed with Toluidine Blue staining of calcium deposits showing mineralization; and Alcian Blue staining of proteoglycans in chondrogenic pellet demonstrated chondrogenic differentiation.
    Figure Legend Snippet: Isolated cells from bone marrow stroma of healthy donors display typical hMSC phenotype and mesodermal differentiation capacity (A, B) After isolation of human bone marrow cells from healthy donors, cells at passage 3 were analyzed by flow cytometry for phenotypic characterization. The cells showed hMSC specific marker expressions with negative expression of CD14, CD31, CD34, and CD45, while they were positive for CD29, CD44, CD73 and CD105. Representative histograms and surface antigen profiles of isolated adherent cells from 5 healthy donors are demonstrated. (C) Adipogenic differentiation was confirmed by Oil Red-O staining of lipid vacuoles (with hematoxylin counterstain); osteogenic differentiation was followed with Toluidine Blue staining of calcium deposits showing mineralization; and Alcian Blue staining of proteoglycans in chondrogenic pellet demonstrated chondrogenic differentiation.

    Techniques Used: Isolation, Flow Cytometry, Marker, Expressing, Staining

    19) Product Images from "Endogenous TLR2 ligand embedded in the catalytic region of human cysteinyl-tRNA synthetase 1"

    Article Title: Endogenous TLR2 ligand embedded in the catalytic region of human cysteinyl-tRNA synthetase 1

    Journal: Journal for Immunotherapy of Cancer

    doi: 10.1136/jitc-2019-000277

    UNE-C1-mediated activation of APCs via TLR2/6. (A) BMDCs were incubated with different CARS1 fragments for 24 hours. Costimulatory molecules were analyzed from the gated CD11c + population. CD86 expression was evaluated by flow cytometry, and IL-6 and IL-12p70 secretion in supernatants was quantified by ELISA. (B) hTLR2 and hTLR4 HEK-Blue cells, expressing SEAP reporter gene in response to NF-Kβ activity, were treated with CARS1 or UNE-C1 in a dose-dependent manner. HEK-Blue TLR2 and TLR4 activation was evaluated by measuring SEAP secretion in culture media. (C) PMA-differentiated THP-1 cells were preincubated with the indicated amount of anti-human TLR2 or anti-human TLR4 for 1 hour and treated with CARS1 or UNE-C1 for an additional 4 hours. TNF-α from supernatants of PMA-differentiated THP-1 was measured by ELISA. (D) His-tagged CARS1 or UNE-C1 were incubated with TLR2-Flag or TLR4-flag proteins. His-ab or Mock-ab bound protein G agarose was used for immunoprecipitating his-tagged proteins. (E) Reciprocal immunoprecipitation was performed using Flag-ab or Mock-ab bound protein-G agarose. His-CARS1 or -UNE-C1 was incubated with TLR2-Flag or TLR4-flag. Interactions were determined by immunoblotting (F) BMDCs from naïve and TLR2 −/− mice were treated with CARS1 or UNE-C1 for 24 hours. IL-6 and IL-12p70 levels in supernatants were quantified. (G) CARS1 and UNE-C1 were treated on hTLR2/6 and hTLR1/2. SEAP activities were measured at OD 620 nm. Data are representative of three independent experiments. Results are presented as mean±SD, and statistical significance was analyzed with Student’s t-test (***p
    Figure Legend Snippet: UNE-C1-mediated activation of APCs via TLR2/6. (A) BMDCs were incubated with different CARS1 fragments for 24 hours. Costimulatory molecules were analyzed from the gated CD11c + population. CD86 expression was evaluated by flow cytometry, and IL-6 and IL-12p70 secretion in supernatants was quantified by ELISA. (B) hTLR2 and hTLR4 HEK-Blue cells, expressing SEAP reporter gene in response to NF-Kβ activity, were treated with CARS1 or UNE-C1 in a dose-dependent manner. HEK-Blue TLR2 and TLR4 activation was evaluated by measuring SEAP secretion in culture media. (C) PMA-differentiated THP-1 cells were preincubated with the indicated amount of anti-human TLR2 or anti-human TLR4 for 1 hour and treated with CARS1 or UNE-C1 for an additional 4 hours. TNF-α from supernatants of PMA-differentiated THP-1 was measured by ELISA. (D) His-tagged CARS1 or UNE-C1 were incubated with TLR2-Flag or TLR4-flag proteins. His-ab or Mock-ab bound protein G agarose was used for immunoprecipitating his-tagged proteins. (E) Reciprocal immunoprecipitation was performed using Flag-ab or Mock-ab bound protein-G agarose. His-CARS1 or -UNE-C1 was incubated with TLR2-Flag or TLR4-flag. Interactions were determined by immunoblotting (F) BMDCs from naïve and TLR2 −/− mice were treated with CARS1 or UNE-C1 for 24 hours. IL-6 and IL-12p70 levels in supernatants were quantified. (G) CARS1 and UNE-C1 were treated on hTLR2/6 and hTLR1/2. SEAP activities were measured at OD 620 nm. Data are representative of three independent experiments. Results are presented as mean±SD, and statistical significance was analyzed with Student’s t-test (***p

    Techniques Used: Activation Assay, Incubation, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Activity Assay, Immunoprecipitation, Mouse Assay

    UNE-C1-dependent stimulation of humoral and cellular immune responses in vivo. (A) C57BL/6 mice (n=3) were injected subcutaneously with indicated reagents. A day after, dLNs were collected, and antigen presentation on h-2k b and CD86 in a different subset of DCs was quantified. (B) After immunizing mice (n=3), pan-DCs of each group were collected from its dLNs and spleens. Collected DCs were cocultured with CD8 + T cells from OT-1 transgenic mice for 24 hours. Expression levels of CD69 on OT-1 T cells were quantified and the production of IFN-γ in the supernatants was measured by ELISA. (C) Indicated reagents were used for mice (n=5) immunization on days 0 and 7. Seven days after the last immunization, spleens and dLNs were collected from immunized mice. Percentages of OVA-specific CD8 + T cells in the spleens and dLNs were measured. (D) Mice (n=3) were immunized on days 0 and 7. On day 14, mice were injected intravenously with SIINFEKL peptide-pulsed and unpulsed splenocytes labeled with a high or low concentration of CFSE, respectively. After 24 hours, the percentage of antigen-specific killing was measured by flow cytometry. Representative flow cytometry plots showing remained pulsed and unpulsed cells from immunized mice, and bar diagram with quantitative comparison. (E) Seven days after the final immunization, the serum was collected from each group of mice (n=5), and OVA-specific total IgG, IgG1, and IgG2c were measured by ELISA. Data are representative of three independent experiments. Results are presented as mean±SEM, and statistical significance was analyzed with Student’s t-test (*p
    Figure Legend Snippet: UNE-C1-dependent stimulation of humoral and cellular immune responses in vivo. (A) C57BL/6 mice (n=3) were injected subcutaneously with indicated reagents. A day after, dLNs were collected, and antigen presentation on h-2k b and CD86 in a different subset of DCs was quantified. (B) After immunizing mice (n=3), pan-DCs of each group were collected from its dLNs and spleens. Collected DCs were cocultured with CD8 + T cells from OT-1 transgenic mice for 24 hours. Expression levels of CD69 on OT-1 T cells were quantified and the production of IFN-γ in the supernatants was measured by ELISA. (C) Indicated reagents were used for mice (n=5) immunization on days 0 and 7. Seven days after the last immunization, spleens and dLNs were collected from immunized mice. Percentages of OVA-specific CD8 + T cells in the spleens and dLNs were measured. (D) Mice (n=3) were immunized on days 0 and 7. On day 14, mice were injected intravenously with SIINFEKL peptide-pulsed and unpulsed splenocytes labeled with a high or low concentration of CFSE, respectively. After 24 hours, the percentage of antigen-specific killing was measured by flow cytometry. Representative flow cytometry plots showing remained pulsed and unpulsed cells from immunized mice, and bar diagram with quantitative comparison. (E) Seven days after the final immunization, the serum was collected from each group of mice (n=5), and OVA-specific total IgG, IgG1, and IgG2c were measured by ELISA. Data are representative of three independent experiments. Results are presented as mean±SEM, and statistical significance was analyzed with Student’s t-test (*p

    Techniques Used: In Vivo, Mouse Assay, Injection, Transgenic Assay, Expressing, Enzyme-linked Immunosorbent Assay, Labeling, Concentration Assay, Flow Cytometry

    Effect of secreted CARS1 on TNF-α secretion from macrophages (A) CARS1 secretion was tested by incubating HCT116 cells under different conditions, including SF, TNF-α (10 ng/mL), tunicamycin (1 µg/mL), arsenite (12.5 µM), CoCl 2 (100 µM), Wnt3a (200 ng/mL), IL-2 (10 ng/mL), IL-4 (10 ng/mL), VEGF (20 ng/mL), BMP4 (50 ng/mL), PDGF (100 ng/mL), EGF (100 ng/mL) FGF-2 (50 ng/mL), FGF-4 (50 ng/mL), IGF (50 ng/mL), and glutamine-free conditions for 24 hours. The amount of CARS1 secreted in the medium and WCL was detected. (B) HCT116 cells were treated with tunicamycin (Tunica) in dose-dependent and time-dependent manners. Proteins in the medium were precipitated and detected by an antibody against CARS1. (C) HCT116 cells were treated with TNF-α in dose-dependent and time-dependent manners to detect secreted CARS1. (D, E) CARS1 and BSA were conjugated with Alexa-Fluor 647 and treated for 30 min to different cell types. CARS1-bound cells were visualized and analyzed by confocal microscopy (D) and flow cytometry (E), respectively. (F) RAW264.7 cells were treated with either 100 nM of CARS1 or KARS1 for 4 hour. Boiled CARS1 and KARS1 were used for negative controls. (G) The production of TNF-α was measured by ELISA after treating 100 nM of CARS1 on PMA-differentiated THP-1 cells. CARS1 was preincubated with proteinase K (50 µg/mL) or boiled for 1 hour. Before adding CARS1, some cells were preincubated with polymyxin B (10 µg/mL) for 1 hour. Data are representative of three independent experiments. Results are presented as mean±SD, and statistical significance was analyzed with Student’s t-test (***p
    Figure Legend Snippet: Effect of secreted CARS1 on TNF-α secretion from macrophages (A) CARS1 secretion was tested by incubating HCT116 cells under different conditions, including SF, TNF-α (10 ng/mL), tunicamycin (1 µg/mL), arsenite (12.5 µM), CoCl 2 (100 µM), Wnt3a (200 ng/mL), IL-2 (10 ng/mL), IL-4 (10 ng/mL), VEGF (20 ng/mL), BMP4 (50 ng/mL), PDGF (100 ng/mL), EGF (100 ng/mL) FGF-2 (50 ng/mL), FGF-4 (50 ng/mL), IGF (50 ng/mL), and glutamine-free conditions for 24 hours. The amount of CARS1 secreted in the medium and WCL was detected. (B) HCT116 cells were treated with tunicamycin (Tunica) in dose-dependent and time-dependent manners. Proteins in the medium were precipitated and detected by an antibody against CARS1. (C) HCT116 cells were treated with TNF-α in dose-dependent and time-dependent manners to detect secreted CARS1. (D, E) CARS1 and BSA were conjugated with Alexa-Fluor 647 and treated for 30 min to different cell types. CARS1-bound cells were visualized and analyzed by confocal microscopy (D) and flow cytometry (E), respectively. (F) RAW264.7 cells were treated with either 100 nM of CARS1 or KARS1 for 4 hour. Boiled CARS1 and KARS1 were used for negative controls. (G) The production of TNF-α was measured by ELISA after treating 100 nM of CARS1 on PMA-differentiated THP-1 cells. CARS1 was preincubated with proteinase K (50 µg/mL) or boiled for 1 hour. Before adding CARS1, some cells were preincubated with polymyxin B (10 µg/mL) for 1 hour. Data are representative of three independent experiments. Results are presented as mean±SD, and statistical significance was analyzed with Student’s t-test (***p

    Techniques Used: Confocal Microscopy, Flow Cytometry, Enzyme-linked Immunosorbent Assay

    20) Product Images from "Design, development and characterization of ACT017, a humanized Fab that blocks platelet's glycoprotein VI function without causing bleeding risks"

    Article Title: Design, development and characterization of ACT017, a humanized Fab that blocks platelet's glycoprotein VI function without causing bleeding risks

    Journal: mAbs

    doi: 10.1080/19420862.2017.1336592

    Functional characterization of ACT017 in vitro. (A) Isotherm binding curve of ACT017 to immobilized GPVI-Fc. (B) Inhibition of GPVI-Fc binding to immobilized collagen by increasing concentrations of ACT017. (C) In vitro binding of ACT017 conjugated to Alexa488 to human platelets analyzed by flow cytometry (red: whole blood; black: PRP). (D) Typical residual platelet aggregation as the ratio of the response in the presence of ACT017 to the response without ACT017 as a function of ACT017 concentration (Black: residual intensity, Gray: residual velocity). The insert shows a typical aggregation curve obtained after pre-incubation of human PRP with increasing concentrations of ACT017 (0–10 μg/mL).
    Figure Legend Snippet: Functional characterization of ACT017 in vitro. (A) Isotherm binding curve of ACT017 to immobilized GPVI-Fc. (B) Inhibition of GPVI-Fc binding to immobilized collagen by increasing concentrations of ACT017. (C) In vitro binding of ACT017 conjugated to Alexa488 to human platelets analyzed by flow cytometry (red: whole blood; black: PRP). (D) Typical residual platelet aggregation as the ratio of the response in the presence of ACT017 to the response without ACT017 as a function of ACT017 concentration (Black: residual intensity, Gray: residual velocity). The insert shows a typical aggregation curve obtained after pre-incubation of human PRP with increasing concentrations of ACT017 (0–10 μg/mL).

    Techniques Used: Functional Assay, In Vitro, Binding Assay, Inhibition, Flow Cytometry, Cytometry, Concentration Assay, Incubation

    21) Product Images from "The MEK inhibitor selumetinib complements CTLA-4 blockade by reprogramming the tumor immune microenvironment"

    Article Title: The MEK inhibitor selumetinib complements CTLA-4 blockade by reprogramming the tumor immune microenvironment

    Journal: Journal for Immunotherapy of Cancer

    doi: 10.1186/s40425-017-0268-8

    Selumetinib alters the phenotype of antigen presenting cells and suppresses T-cell activation in vitro. a Human PBMCs stimulated with SEA, anti-CD3 antibody and either 30 μg/ml of tremelimumab or isotype control, were incubated with increasing concentrations of selumetinib for 72 h. Levels of IL-2 in supernatants were determined by immuno-assay. Data presented as mean (± SEM) of triplicates. b Flow cytometry analysis of mouse CT26 tumor cells following 48 h treatment with selumetinib or DMSO vehicle control and stained for H2-Kd and PD-L1. c Flow cytometry analysis of human monocyte-derived dendritic cells after 8 days in culture with GM-CSF and IL-4. Cells were either untreated or activated with CD40L and treated with selumetinib or DMSO vehicle control for the last 48 h of culture. Histograms for staining with specific antibodies for mDCs activated with CD40L and treated with DMSO vehicle (solid line); or mDCs left untreated (dashed line); and isotype control staining of untreated mDCs (filled). Percentage of gated cells are shown in histograms for the CD40L-activated + DMSO condition. d The percentages of CD80 + , CD83 + , CD86 high and HLA-DR high cells of total live cells, and frequency of live cells out of total cells are shown. Plotted data are single measurements
    Figure Legend Snippet: Selumetinib alters the phenotype of antigen presenting cells and suppresses T-cell activation in vitro. a Human PBMCs stimulated with SEA, anti-CD3 antibody and either 30 μg/ml of tremelimumab or isotype control, were incubated with increasing concentrations of selumetinib for 72 h. Levels of IL-2 in supernatants were determined by immuno-assay. Data presented as mean (± SEM) of triplicates. b Flow cytometry analysis of mouse CT26 tumor cells following 48 h treatment with selumetinib or DMSO vehicle control and stained for H2-Kd and PD-L1. c Flow cytometry analysis of human monocyte-derived dendritic cells after 8 days in culture with GM-CSF and IL-4. Cells were either untreated or activated with CD40L and treated with selumetinib or DMSO vehicle control for the last 48 h of culture. Histograms for staining with specific antibodies for mDCs activated with CD40L and treated with DMSO vehicle (solid line); or mDCs left untreated (dashed line); and isotype control staining of untreated mDCs (filled). Percentage of gated cells are shown in histograms for the CD40L-activated + DMSO condition. d The percentages of CD80 + , CD83 + , CD86 high and HLA-DR high cells of total live cells, and frequency of live cells out of total cells are shown. Plotted data are single measurements

    Techniques Used: Activation Assay, In Vitro, Incubation, IA, Flow Cytometry, Cytometry, Staining, Derivative Assay

    Selumetinib in combination with anti-CTLA-4 alters the composition of innate cells within tumors. On day 8 after initiation of anti-CTLA-4, selumetinib or combination treatment, cells were isolated from tumors and analysed by flow cytometry to identify and characterise myeloid cells and plasmacytoid DCs (pDC). a Gating strategy used to identify CD11b + Ly6G + Ly6C Int (I), CD11b + Ly6G − Ly6C + MHCII lo/− (II), CD11b + Ly6G − Ly6C + MHCII + (III), CD11b + Ly6G − Ly6C − MHCII lo/− (IV), CD11b + Ly6G − Ly6C − MHCII + (V) cells and CD11b − B220 + PDCA1 + pDCs. Phenotypic analysis of CD11c, PD-L1 and CD86 expression on cells are shown as histograms with matched isotype staining (black line) and target antigen staining (grey filled). b Frequencies of myeloid cells (I-V) and pDCs (VI) out of total CD45 + cells. Plotted are mean ± SD. Each group contained 8 mice. * P
    Figure Legend Snippet: Selumetinib in combination with anti-CTLA-4 alters the composition of innate cells within tumors. On day 8 after initiation of anti-CTLA-4, selumetinib or combination treatment, cells were isolated from tumors and analysed by flow cytometry to identify and characterise myeloid cells and plasmacytoid DCs (pDC). a Gating strategy used to identify CD11b + Ly6G + Ly6C Int (I), CD11b + Ly6G − Ly6C + MHCII lo/− (II), CD11b + Ly6G − Ly6C + MHCII + (III), CD11b + Ly6G − Ly6C − MHCII lo/− (IV), CD11b + Ly6G − Ly6C − MHCII + (V) cells and CD11b − B220 + PDCA1 + pDCs. Phenotypic analysis of CD11c, PD-L1 and CD86 expression on cells are shown as histograms with matched isotype staining (black line) and target antigen staining (grey filled). b Frequencies of myeloid cells (I-V) and pDCs (VI) out of total CD45 + cells. Plotted are mean ± SD. Each group contained 8 mice. * P

    Techniques Used: Isolation, Flow Cytometry, Cytometry, Expressing, Staining, Mouse Assay

    Frequency and effector function of T-cells following selumetinib, anti-CTLA-4 and combination treatment in vivo. a Schema showing treatment schedule. b Immunohistochemical analysis of tumors for p-ERK 1 h following the last dose with 25 mg/kg selumetinib bid (3 doses in total, over 24 h) or vehicle control. Following 8 days of anti-CTLA-4, selumetinib or combination treatment, cells isolated from spleens and tumors were analysed by flow cytometry analysis. Spleens from non-tumor bearing BALB/c mice were also included in the analysis. c CD8 + T-cell and ( d ) CD4 + T-cell populations are presented as percentages of CD45 + cells. e Frequency of Foxp3 + CD4 + regulatory T-cells (Tregs) of total CD4 + T-cells. f Ratio of CD8 + T-cells to Tregs. Effects of treatment on the frequency of Ki67-positive cells ( g ) of total CD8 + T-cells, ( h ) CD4 + T-cells or ( i ) Tregs. Data points in scatter plots represent individual animals, treatment groups each contained 8 mice. Plotted are means ± SD. * P
    Figure Legend Snippet: Frequency and effector function of T-cells following selumetinib, anti-CTLA-4 and combination treatment in vivo. a Schema showing treatment schedule. b Immunohistochemical analysis of tumors for p-ERK 1 h following the last dose with 25 mg/kg selumetinib bid (3 doses in total, over 24 h) or vehicle control. Following 8 days of anti-CTLA-4, selumetinib or combination treatment, cells isolated from spleens and tumors were analysed by flow cytometry analysis. Spleens from non-tumor bearing BALB/c mice were also included in the analysis. c CD8 + T-cell and ( d ) CD4 + T-cell populations are presented as percentages of CD45 + cells. e Frequency of Foxp3 + CD4 + regulatory T-cells (Tregs) of total CD4 + T-cells. f Ratio of CD8 + T-cells to Tregs. Effects of treatment on the frequency of Ki67-positive cells ( g ) of total CD8 + T-cells, ( h ) CD4 + T-cells or ( i ) Tregs. Data points in scatter plots represent individual animals, treatment groups each contained 8 mice. Plotted are means ± SD. * P

    Techniques Used: In Vivo, Immunohistochemistry, Isolation, Flow Cytometry, Cytometry, Mouse Assay

    22) Product Images from "Immunoregulatory factors in multiple sclerosis patients during and after pregnancy: relevance of natural killer cells"

    Article Title: Immunoregulatory factors in multiple sclerosis patients during and after pregnancy: relevance of natural killer cells

    Journal:

    doi: 10.1111/j.1365-2249.2007.03555.x

    Representative staining for flow cytometry experiments. (a) Shown is a representative forward-scatter/side-scatter plot. R2 shows the monocyte gate (of CD14-positive monocytes). The lower square shows the lymphocyte gate. (b) Shown is a dot plot of the
    Figure Legend Snippet: Representative staining for flow cytometry experiments. (a) Shown is a representative forward-scatter/side-scatter plot. R2 shows the monocyte gate (of CD14-positive monocytes). The lower square shows the lymphocyte gate. (b) Shown is a dot plot of the

    Techniques Used: Staining, Flow Cytometry, Cytometry

    23) Product Images from "Natural Killer Cells Exhibit a Peculiar Phenotypic Profile in Systemic Sclerosis and Are Potent Inducers of Endothelial Microparticles Release"

    Article Title: Natural Killer Cells Exhibit a Peculiar Phenotypic Profile in Systemic Sclerosis and Are Potent Inducers of Endothelial Microparticles Release

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01665

    Activation receptors and markers in natural killer (NK) cells of systemic sclerosis (SSc) patients. NKG2D (A) , DNAM-1 (B) , CD69 (C) , and CD16 (D) expression levels were evaluated with flow cytometry analysis of NK cells among peripheral blood mononuclear cells from SSc patients and healthy controls (HCtl). Results were expressed as median fluorescence intensity (MFI) and median ± interquartile range. Statistical difference was established using Mann–Whitney U test. * p
    Figure Legend Snippet: Activation receptors and markers in natural killer (NK) cells of systemic sclerosis (SSc) patients. NKG2D (A) , DNAM-1 (B) , CD69 (C) , and CD16 (D) expression levels were evaluated with flow cytometry analysis of NK cells among peripheral blood mononuclear cells from SSc patients and healthy controls (HCtl). Results were expressed as median fluorescence intensity (MFI) and median ± interquartile range. Statistical difference was established using Mann–Whitney U test. * p

    Techniques Used: Activation Assay, Expressing, Flow Cytometry, Cytometry, Fluorescence, MANN-WHITNEY

    CX3CR1 expression in circulating cytotoxic immune cells from systemic sclerosis (SSc) patients. Percentages of CX3CR1 expressing immune cells were evaluated with flow cytometry analysis on the surface of CD8 T cells (A) ; γδ T cells (B) ; Vδ1 (C) ; and Vδ2 T cells (D) from SSc patients ( n = 15) in comparison with healthy controls (HCtl) ( n = 15). Results were expressed as median percentages and median ± interquartile range.
    Figure Legend Snippet: CX3CR1 expression in circulating cytotoxic immune cells from systemic sclerosis (SSc) patients. Percentages of CX3CR1 expressing immune cells were evaluated with flow cytometry analysis on the surface of CD8 T cells (A) ; γδ T cells (B) ; Vδ1 (C) ; and Vδ2 T cells (D) from SSc patients ( n = 15) in comparison with healthy controls (HCtl) ( n = 15). Results were expressed as median percentages and median ± interquartile range.

    Techniques Used: Expressing, Flow Cytometry, Cytometry

    Chemokine receptors expression in natural killer (NK) cells in systemic sclerosis (SSc) patients. CX3CR1 (A,B) and CXCR4 expression levels and percentages (C,D) were assessed with flow cytometry analysis in whole NK cell from SSc patients ( n = 15) in comparison with healthy controls (HCtl) ( n = 15). Results were expressed as percentages of positive cells among NK cells (A,C) and median fluorescence intensity (B,D) . Results were depicted as median ± interquartile range. Statistical difference was established using Mann–Whitney U test. ** p
    Figure Legend Snippet: Chemokine receptors expression in natural killer (NK) cells in systemic sclerosis (SSc) patients. CX3CR1 (A,B) and CXCR4 expression levels and percentages (C,D) were assessed with flow cytometry analysis in whole NK cell from SSc patients ( n = 15) in comparison with healthy controls (HCtl) ( n = 15). Results were expressed as percentages of positive cells among NK cells (A,C) and median fluorescence intensity (B,D) . Results were depicted as median ± interquartile range. Statistical difference was established using Mann–Whitney U test. ** p

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Fluorescence, MANN-WHITNEY

    Natural killer (NK) cells degranulation of systemic sclerosis (SSc) patients toward microvascular endothelial cell (EC) target. Cytolytic degranulation of NK cells was assessed by flow cytometry analysis of CD107a/b expression of NK cells among peripheral blood mononuclear cells (PBMCs) after a 4-h coculture with human microvascular dermal ECs (HMVEC-d) with an effector/target ratio of 2/1. (A) PBMCs from patients with SSc ( n = 6) and healthy controls (HCtl) ( n = 6) were cultured with HMVEC-d with or without thymoglobulin (ATG). (B) PBMCs from SSc patients ( n = 6) and HCtl ( n = 6) were cultured with HMVEC-d and either SSc serum or HCtl serum and conversely. Cumulative data from five independent experiments. Results were expressed as median percentages ± interquartile range. Statistical significance was established using the non-parametric paired Wilcoxon U -test. * p
    Figure Legend Snippet: Natural killer (NK) cells degranulation of systemic sclerosis (SSc) patients toward microvascular endothelial cell (EC) target. Cytolytic degranulation of NK cells was assessed by flow cytometry analysis of CD107a/b expression of NK cells among peripheral blood mononuclear cells (PBMCs) after a 4-h coculture with human microvascular dermal ECs (HMVEC-d) with an effector/target ratio of 2/1. (A) PBMCs from patients with SSc ( n = 6) and healthy controls (HCtl) ( n = 6) were cultured with HMVEC-d with or without thymoglobulin (ATG). (B) PBMCs from SSc patients ( n = 6) and HCtl ( n = 6) were cultured with HMVEC-d and either SSc serum or HCtl serum and conversely. Cumulative data from five independent experiments. Results were expressed as median percentages ± interquartile range. Statistical significance was established using the non-parametric paired Wilcoxon U -test. * p

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Cell Culture

    Number of circulating cytotoxic immune cells in systemic sclerosis (SSc) patients. Percentages of circulating immune cells were assessed in the peripheral blood of SSc patients ( n = 15) with flow cytometry and compared with healthy controls (HCtl) ( n = 14). Absolute numbers of circulating cells including CD8 T cells (A) ; natural killer (NK) cells (B) ; NK cells CD56 dim (C) ; γδ T cells (D) ; Vδ1 (E) ; and Vδ2 T cells (F) were obtained based on the white blood lymphocyte count and expressed as number of cells per mm 3 . Results were expressed as median ± interquartile range. Statistical difference was established using Mann–Whitney U test. * p
    Figure Legend Snippet: Number of circulating cytotoxic immune cells in systemic sclerosis (SSc) patients. Percentages of circulating immune cells were assessed in the peripheral blood of SSc patients ( n = 15) with flow cytometry and compared with healthy controls (HCtl) ( n = 14). Absolute numbers of circulating cells including CD8 T cells (A) ; natural killer (NK) cells (B) ; NK cells CD56 dim (C) ; γδ T cells (D) ; Vδ1 (E) ; and Vδ2 T cells (F) were obtained based on the white blood lymphocyte count and expressed as number of cells per mm 3 . Results were expressed as median ± interquartile range. Statistical difference was established using Mann–Whitney U test. * p

    Techniques Used: Flow Cytometry, Cytometry, MANN-WHITNEY

    24) Product Images from "Myeloma cells inhibit osteogenic differentiation of mesenchymal stem cells and kill osteoblasts via TRAIL-induced apoptosis"

    Article Title: Myeloma cells inhibit osteogenic differentiation of mesenchymal stem cells and kill osteoblasts via TRAIL-induced apoptosis

    Journal: Archives of Medical Science : AMS

    doi: 10.5114/aoms.2010.14459

    The OBs induced from BMMSCs or human osteoblast cell line HFOB1.19 grew well in medium containing rhTRAIL and were not sensitive to rhTRAIL induced apoptosis in vitr o as MTT assays or flow cytometry using the annexin V/PI binding assay (A and B). The percentage of apoptosis increased significantly whether the OBs or HFOB1.19 were precultured or co-cultured with XG7/RPMI 8266 MM cells, but the proportion of apoptosis decreased significantly when anti-TRAIL-R2 mab were added to the cultured (C)
    Figure Legend Snippet: The OBs induced from BMMSCs or human osteoblast cell line HFOB1.19 grew well in medium containing rhTRAIL and were not sensitive to rhTRAIL induced apoptosis in vitr o as MTT assays or flow cytometry using the annexin V/PI binding assay (A and B). The percentage of apoptosis increased significantly whether the OBs or HFOB1.19 were precultured or co-cultured with XG7/RPMI 8266 MM cells, but the proportion of apoptosis decreased significantly when anti-TRAIL-R2 mab were added to the cultured (C)

    Techniques Used: MTT Assay, Flow Cytometry, Cytometry, Binding Assay, Cell Culture

    25) Product Images from "Membrane raft microdomains mediate lateral assemblies required for HIV-1 infection"

    Article Title: Membrane raft microdomains mediate lateral assemblies required for HIV-1 infection

    Journal: EMBO Reports

    doi: 10.1093/embo-reports/kvd025

    Fig. 4. Cholesterol depletion inhibits env -mediated fusion. ( A ) Untreated (control) or CD-treated MT-2 cells were incubated alone (gray area) or with gp120 (dotted line) and binding monitored in flow cytometry. ( B ) Untreated or CD-treated MT-2 or HEK-293, as a CD4– cell, were incubated with NL4-3 and, after removal of unbound virus, cell lysates prepared. Total lysates were resolved by SDS–PAGE and sequentially blotted with anti-gp120 and anti-tubulin antibodies, as indicated. ( C ) Untreated or CD-treated MT-2 cells were exposed to NL4-3 as in Figure 3, and after 6 h culture, total DNA was amplified by PCR with primers specific for HIV gag . Either 100 or 10 ng of total DNA from the PCR products were hybridized in solution with an internal 32 P-labeled gag probe, resolved by SDS–PAGE and autoradiographed. Serial dilutions of proviral 8E5 DNA were run in parallel for standardization. HLA-DQα amplification is shown as a loading control. ( D ) Untreated, CD-treated and cholesterol-replenished (CD+Cho) HeLa-CD4 cells were mixed with BSC40-env cells and cell fusion events measured. *Statistically significant difference, p
    Figure Legend Snippet: Fig. 4. Cholesterol depletion inhibits env -mediated fusion. ( A ) Untreated (control) or CD-treated MT-2 cells were incubated alone (gray area) or with gp120 (dotted line) and binding monitored in flow cytometry. ( B ) Untreated or CD-treated MT-2 or HEK-293, as a CD4– cell, were incubated with NL4-3 and, after removal of unbound virus, cell lysates prepared. Total lysates were resolved by SDS–PAGE and sequentially blotted with anti-gp120 and anti-tubulin antibodies, as indicated. ( C ) Untreated or CD-treated MT-2 cells were exposed to NL4-3 as in Figure 3, and after 6 h culture, total DNA was amplified by PCR with primers specific for HIV gag . Either 100 or 10 ng of total DNA from the PCR products were hybridized in solution with an internal 32 P-labeled gag probe, resolved by SDS–PAGE and autoradiographed. Serial dilutions of proviral 8E5 DNA were run in parallel for standardization. HLA-DQα amplification is shown as a loading control. ( D ) Untreated, CD-treated and cholesterol-replenished (CD+Cho) HeLa-CD4 cells were mixed with BSC40-env cells and cell fusion events measured. *Statistically significant difference, p

    Techniques Used: Incubation, Binding Assay, Flow Cytometry, Cytometry, SDS Page, Amplification, Polymerase Chain Reaction, Labeling

    26) Product Images from "Fluorescent CXCR4 targeting peptide as alternative for antibody staining in Ewing sarcoma"

    Article Title: Fluorescent CXCR4 targeting peptide as alternative for antibody staining in Ewing sarcoma

    Journal: BMC Cancer

    doi: 10.1186/s12885-017-3352-z

    Semi-quantitative detection of MSAP-Ac-TZ14011 in EWS cell lines correlated significantly with CXCR4 RNA expression levels. a Cells of the EWS cell lines A673, IARC-EW7, L1062, 6647 and TC32 were harvested and stained without ( upper graphs ) or with ( lower graphs ) MSAP-Ac-TZ14011. Fluorescence was detected at 710–40 nm. The calculated percentage of positive cells is indicated in each panel. b The baseline corrected geometric mean cytometry fluorescence intensities (GFI) detected after MSAP-Ac-TZ14011 staining of the in ( a ) described EWS cell lines were correlated to the previous determined CXCR4 RNA expression levels (x-axis). Linear regression analysis demonstrated a significant correlation between the by MSAP-Ac-TZ14011 detected CXCR4 levels and RNA expression levels ( P -value and 95% certainty borders are displaced). Both figures are representatives ( n = 3)
    Figure Legend Snippet: Semi-quantitative detection of MSAP-Ac-TZ14011 in EWS cell lines correlated significantly with CXCR4 RNA expression levels. a Cells of the EWS cell lines A673, IARC-EW7, L1062, 6647 and TC32 were harvested and stained without ( upper graphs ) or with ( lower graphs ) MSAP-Ac-TZ14011. Fluorescence was detected at 710–40 nm. The calculated percentage of positive cells is indicated in each panel. b The baseline corrected geometric mean cytometry fluorescence intensities (GFI) detected after MSAP-Ac-TZ14011 staining of the in ( a ) described EWS cell lines were correlated to the previous determined CXCR4 RNA expression levels (x-axis). Linear regression analysis demonstrated a significant correlation between the by MSAP-Ac-TZ14011 detected CXCR4 levels and RNA expression levels ( P -value and 95% certainty borders are displaced). Both figures are representatives ( n = 3)

    Techniques Used: RNA Expression, Staining, Fluorescence, Cytometry

    27) Product Images from "The preRC protein ORCA organizes heterochromatin by assembling histone H3 lysine 9 methyltransferases on chromatin"

    Article Title: The preRC protein ORCA organizes heterochromatin by assembling histone H3 lysine 9 methyltransferases on chromatin

    Journal: eLife

    doi: 10.7554/eLife.06496

    BrdU incorporation preferentially at perinucleolar regions in cells lacking ORCA. ( A ) IP of DD-T7-ORCA siRNA NTV from U2OS cells using T7 Ab. DD-T7-ORCA siRNA NTV and endogenous Orc were analyzed by IB. ( B ) Patterns of BrdU incorporation in control and ORCA depleted cells in late S phase. Scale bar, 10µm. ( C ) H3K9me3 and HP1α immunofluorescence in control and ORCA depleted cells. Scale bar, 10µm. ( D ) Flow cytometry of control and ORCA knockdown cells at 0, 4, 8 and 12h post release from Aphidicolin block. DOI: http://dx.doi.org/10.7554/eLife.06496.017
    Figure Legend Snippet: BrdU incorporation preferentially at perinucleolar regions in cells lacking ORCA. ( A ) IP of DD-T7-ORCA siRNA NTV from U2OS cells using T7 Ab. DD-T7-ORCA siRNA NTV and endogenous Orc were analyzed by IB. ( B ) Patterns of BrdU incorporation in control and ORCA depleted cells in late S phase. Scale bar, 10µm. ( C ) H3K9me3 and HP1α immunofluorescence in control and ORCA depleted cells. Scale bar, 10µm. ( D ) Flow cytometry of control and ORCA knockdown cells at 0, 4, 8 and 12h post release from Aphidicolin block. DOI: http://dx.doi.org/10.7554/eLife.06496.017

    Techniques Used: BrdU Incorporation Assay, Immunofluorescence, Flow Cytometry, Cytometry, Blocking Assay

    28) Product Images from "Cellular and cytokine-dependent immunosuppressive mechanisms of grm1-transgenic murine melanoma"

    Article Title: Cellular and cytokine-dependent immunosuppressive mechanisms of grm1-transgenic murine melanoma

    Journal: Cancer Immunology, Immunotherapy

    doi: 10.1007/s00262-012-1290-9

    Frequency of regulatory T cells ex vivo (T regs ; CD4 + CD25 + FoxP3 + ). a Representative flow cytometry analysis of gated CD4 + T cells in tdLN and ndLN of tumor-bearing LLA-TG-3 mice. Numbers in top right quadrants indicate CD25 + FoxP3 + cells; b the frequency of T regs was analyzed as in a in spleens and LN of wt and LLA-TG-3 mice ex vivo; n = 13 for wt mice; n = 10 for no tumor and low tumor burden LLA-TG-3 mice; n = 11 for high tumor burden LLA-TG-3 mice. Depicted are mean values + SEM. *** p
    Figure Legend Snippet: Frequency of regulatory T cells ex vivo (T regs ; CD4 + CD25 + FoxP3 + ). a Representative flow cytometry analysis of gated CD4 + T cells in tdLN and ndLN of tumor-bearing LLA-TG-3 mice. Numbers in top right quadrants indicate CD25 + FoxP3 + cells; b the frequency of T regs was analyzed as in a in spleens and LN of wt and LLA-TG-3 mice ex vivo; n = 13 for wt mice; n = 10 for no tumor and low tumor burden LLA-TG-3 mice; n = 11 for high tumor burden LLA-TG-3 mice. Depicted are mean values + SEM. *** p

    Techniques Used: Ex Vivo, Flow Cytometry, Cytometry, Mouse Assay

    Gating strategy; CD4 + and CD8 + T-cell subsets ex vivo. a Gating strategy used for all flow cytometry data shown; b – d frequency of activated CD4 + T-cell subsets in spleen and lymph nodes of wild-type (wt) and LLA-TG-3 mice; e – g frequency of activated CD8 + T-cell subsets in spleen and lymph nodes of wt and LLA-TG-3 mice; n = 14 for wt mice; n = 10 for no tumor and low tumor burden LLA-TG-3 mice; n = 13 for high tumor burden LLA-TG-3 mice. Depicted are mean values + SEM. * p
    Figure Legend Snippet: Gating strategy; CD4 + and CD8 + T-cell subsets ex vivo. a Gating strategy used for all flow cytometry data shown; b – d frequency of activated CD4 + T-cell subsets in spleen and lymph nodes of wild-type (wt) and LLA-TG-3 mice; e – g frequency of activated CD8 + T-cell subsets in spleen and lymph nodes of wt and LLA-TG-3 mice; n = 14 for wt mice; n = 10 for no tumor and low tumor burden LLA-TG-3 mice; n = 13 for high tumor burden LLA-TG-3 mice. Depicted are mean values + SEM. * p

    Techniques Used: Ex Vivo, Flow Cytometry, Cytometry, Mouse Assay

    Frequency of CD4 + and CD8 + T cells upon IL-2 and PMA stimulation. a Representative dot plots of CD4 and CD8 stainings in spleen, tumor-draining (tdLN), and non-draining (ndLN) lymph nodes of a high tumor burden LLA-TG-3 mouse upon IL-2/PMA stimulation; b representative flow cytometry analysis of gated CD8 + T cells in tumor-draining LN of low and high tumor burden LLA-TG-3 mice upon stimulation with IL-2 (500 IU/ml) or IL-2 and PMA (200 nM). Numbers in top right quadrants indicate CD69 + CD25 + cells; c the frequency of CD69 + /CD25 + CD8 + T cells in spleen, tdLN, and ndLN of wt and LLA-TG-3 mice was analyzed as in a ; n = 10 for wt mice; n = 6 for no tumor and low tumor burden LLA-TG-3 mice; n = 8 for high tumor burden LLA-TG-3 mice. Depicted are mean values + SEM. ** p
    Figure Legend Snippet: Frequency of CD4 + and CD8 + T cells upon IL-2 and PMA stimulation. a Representative dot plots of CD4 and CD8 stainings in spleen, tumor-draining (tdLN), and non-draining (ndLN) lymph nodes of a high tumor burden LLA-TG-3 mouse upon IL-2/PMA stimulation; b representative flow cytometry analysis of gated CD8 + T cells in tumor-draining LN of low and high tumor burden LLA-TG-3 mice upon stimulation with IL-2 (500 IU/ml) or IL-2 and PMA (200 nM). Numbers in top right quadrants indicate CD69 + CD25 + cells; c the frequency of CD69 + /CD25 + CD8 + T cells in spleen, tdLN, and ndLN of wt and LLA-TG-3 mice was analyzed as in a ; n = 10 for wt mice; n = 6 for no tumor and low tumor burden LLA-TG-3 mice; n = 8 for high tumor burden LLA-TG-3 mice. Depicted are mean values + SEM. ** p

    Techniques Used: Flow Cytometry, Cytometry, Mouse Assay

    29) Product Images from "Downregulation of Microparticle Release and Pro-Inflammatory Properties of Activated Human Polymorphonuclear Neutrophils by LMW Fucoidan"

    Article Title: Downregulation of Microparticle Release and Pro-Inflammatory Properties of Activated Human Polymorphonuclear Neutrophils by LMW Fucoidan

    Journal: Journal of Innate Immunity

    doi: 10.1159/000494220

    Microparticle production induced in an endotoxemia model is inhibited by low-molecular-weight fucoidan (LMW-Fuc). Mice were subjected to one of the following treatments: sham (intraperitoneal injection [IP] 15 min of saline 0.9% + intravenous injection [IV] 15 min of saline 0.9% + IV 30 min of saline 0.9%), LMW-Fuc (IP 15 min of saline 0.9% + IV 15 min of LMW-Fuc 25 µg/g + IV 30 min of saline 0.9%), LPS/fMLP (IP 15 min of LPS 0.2 µg/g + IV 15 min of saline 0.9% + IV 30 min of fMLP 100 nM) and LPS/LMW-Fuc/fMLP (IP 15 min of LPS 0.2 µg/g + IV 15 min of LMW-Fuc 25 µg/g + IV 30 min of fMLP 100 nM). After treatment, mice were euthanized and the collected plasma was submitted to a centrifugation to remove cells and debris, and the supernatant was subjected to ultracentrifugation at 100,000 g for 4 h. Microparticles in the pellet were quantified by flow cytometry for annexin V + ( a ) or Ly6G + ( b ) events, using annexin V and anti-mouse Ly6G-PE (Biolegend, cat No. 127607). Results are representative of five independent experiments. Data are displayed as means ± SDM. * p
    Figure Legend Snippet: Microparticle production induced in an endotoxemia model is inhibited by low-molecular-weight fucoidan (LMW-Fuc). Mice were subjected to one of the following treatments: sham (intraperitoneal injection [IP] 15 min of saline 0.9% + intravenous injection [IV] 15 min of saline 0.9% + IV 30 min of saline 0.9%), LMW-Fuc (IP 15 min of saline 0.9% + IV 15 min of LMW-Fuc 25 µg/g + IV 30 min of saline 0.9%), LPS/fMLP (IP 15 min of LPS 0.2 µg/g + IV 15 min of saline 0.9% + IV 30 min of fMLP 100 nM) and LPS/LMW-Fuc/fMLP (IP 15 min of LPS 0.2 µg/g + IV 15 min of LMW-Fuc 25 µg/g + IV 30 min of fMLP 100 nM). After treatment, mice were euthanized and the collected plasma was submitted to a centrifugation to remove cells and debris, and the supernatant was subjected to ultracentrifugation at 100,000 g for 4 h. Microparticles in the pellet were quantified by flow cytometry for annexin V + ( a ) or Ly6G + ( b ) events, using annexin V and anti-mouse Ly6G-PE (Biolegend, cat No. 127607). Results are representative of five independent experiments. Data are displayed as means ± SDM. * p

    Techniques Used: Molecular Weight, Mouse Assay, Injection, Centrifugation, Flow Cytometry, Cytometry

    Low-molecular-weight fucoidan (LMW-Fuc) inhibits protection from apoptosis in activated polymorphonuclear neutrophils (PMNs). PMNs were left untreated or were incubated with 10 µg/mL LMW-Fuc for 5 min before one of the following treatments: 1 µg/mL lipopolysaccharide (LPS) for 20 h; 100 nM N-formyl-methionine-leucine-phenylalanine (fMLP) for 20 h; or LPS priming for 5 min followed by stimulation with fMLP for 20 h (LPS/fMLP). Furthermore, PMNs were primed with LPS for 5 min before treatment with LMW-Fuc for 5 min, and then stimulated with fMLP for 20 h (LPS/LMW-Fuc/fMLP group). All treatments were performed at 37°C (in a 5% CO 2 atmosphere). a PMNs were stained with Diff-Quik TM (Dade Behring, Dudingen, Switzerland), and apoptotic cells (i.e., those with piknotic nuclei and decreased volume) were counted by direct light microscopy observation. The results are representative of three to five different experiments. b PMNs were fixed and labeled with annexin V and propidium iodide (PI) and then analyzed by flow cytometry, to identify apoptotic cells (annexin V + /PI − and annexin V + /PI + ). c PMNs were fixed and marked with the JC1 probe to detect apoptosis-related loss of mitochondrial membrane potential (i.e., decrease in the ratio of red/green JC1 + cells) by flow cytometry. Results are representative of three independent experiments. Data are displayed as means ± SDM. * p
    Figure Legend Snippet: Low-molecular-weight fucoidan (LMW-Fuc) inhibits protection from apoptosis in activated polymorphonuclear neutrophils (PMNs). PMNs were left untreated or were incubated with 10 µg/mL LMW-Fuc for 5 min before one of the following treatments: 1 µg/mL lipopolysaccharide (LPS) for 20 h; 100 nM N-formyl-methionine-leucine-phenylalanine (fMLP) for 20 h; or LPS priming for 5 min followed by stimulation with fMLP for 20 h (LPS/fMLP). Furthermore, PMNs were primed with LPS for 5 min before treatment with LMW-Fuc for 5 min, and then stimulated with fMLP for 20 h (LPS/LMW-Fuc/fMLP group). All treatments were performed at 37°C (in a 5% CO 2 atmosphere). a PMNs were stained with Diff-Quik TM (Dade Behring, Dudingen, Switzerland), and apoptotic cells (i.e., those with piknotic nuclei and decreased volume) were counted by direct light microscopy observation. The results are representative of three to five different experiments. b PMNs were fixed and labeled with annexin V and propidium iodide (PI) and then analyzed by flow cytometry, to identify apoptotic cells (annexin V + /PI − and annexin V + /PI + ). c PMNs were fixed and marked with the JC1 probe to detect apoptosis-related loss of mitochondrial membrane potential (i.e., decrease in the ratio of red/green JC1 + cells) by flow cytometry. Results are representative of three independent experiments. Data are displayed as means ± SDM. * p

    Techniques Used: Molecular Weight, Incubation, Staining, Diff-Quik, Light Microscopy, Labeling, Flow Cytometry, Cytometry

    Microparticle (MP) production by activated polymorphonuclear neutrophils (PMNs) is inhibited by low-molecular-weight fucoidan (LMW-Fuc). Human PMNs remained untreated or were treated with 10 µg/mL LMW-Fuc (LMW-Fuc group), 1 µg/mL lipopolysaccharide (LPS) for 5 min (priming) followed by stimulation with 100 nM N-formyl-methionine-leucine-phenylalanine (fMLP) for 1 h ( a and d ) or 30 min ( b and c ) (LPS/fMLP group), or LPS primed for 5 min, treated with LMW-Fuc for 5 min, and then fMLP-stimulated (LPS/LMW-Fuc/fMLP group) for 30 min ( a and b ) or 1 h ( c and d ). All treatments were performed at 37°C (in a 5% CO 2 atmosphere). After treatment, PMNs were visualized by scanning electron microscopy ( a ) showing membrane blebs ( a , b , and d ) and membrane ruffles ( c ). Membrane ruffles are indicated by white arrows. Scale bar: 2 µm. b Whole cell lysates of treated PMNs were subjected to SDS-PAGE and immunoblotting for the detection of myosin light chain (MLC) and phospho-MLC (pMLC). Densitometry of pMLC was performed using ImageJ, and results were normalized to MLC levels (loading control). Intact cells were removed by centrifugation, and supernatants were subjected to ultracentrifugation at 100,000 g for 4 h, to produce a pellet containing MP. c MP released by treated PMNs were quantified using flow cytometry after labeling with annexin V. d Purified MP (1 µg/lane) were subjected to SDS-PAGE and Western blotting for the detection of p47 phox , a cytosolic subunit of NADPH oxidase-2 (NOX-2). Actin is shown as a loading control. Results are representative of four independent experiments. b , c Data are displayed as means ± SDM. * p
    Figure Legend Snippet: Microparticle (MP) production by activated polymorphonuclear neutrophils (PMNs) is inhibited by low-molecular-weight fucoidan (LMW-Fuc). Human PMNs remained untreated or were treated with 10 µg/mL LMW-Fuc (LMW-Fuc group), 1 µg/mL lipopolysaccharide (LPS) for 5 min (priming) followed by stimulation with 100 nM N-formyl-methionine-leucine-phenylalanine (fMLP) for 1 h ( a and d ) or 30 min ( b and c ) (LPS/fMLP group), or LPS primed for 5 min, treated with LMW-Fuc for 5 min, and then fMLP-stimulated (LPS/LMW-Fuc/fMLP group) for 30 min ( a and b ) or 1 h ( c and d ). All treatments were performed at 37°C (in a 5% CO 2 atmosphere). After treatment, PMNs were visualized by scanning electron microscopy ( a ) showing membrane blebs ( a , b , and d ) and membrane ruffles ( c ). Membrane ruffles are indicated by white arrows. Scale bar: 2 µm. b Whole cell lysates of treated PMNs were subjected to SDS-PAGE and immunoblotting for the detection of myosin light chain (MLC) and phospho-MLC (pMLC). Densitometry of pMLC was performed using ImageJ, and results were normalized to MLC levels (loading control). Intact cells were removed by centrifugation, and supernatants were subjected to ultracentrifugation at 100,000 g for 4 h, to produce a pellet containing MP. c MP released by treated PMNs were quantified using flow cytometry after labeling with annexin V. d Purified MP (1 µg/lane) were subjected to SDS-PAGE and Western blotting for the detection of p47 phox , a cytosolic subunit of NADPH oxidase-2 (NOX-2). Actin is shown as a loading control. Results are representative of four independent experiments. b , c Data are displayed as means ± SDM. * p

    Techniques Used: Molecular Weight, Electron Microscopy, SDS Page, Centrifugation, Flow Cytometry, Cytometry, Labeling, Purification, Western Blot

    30) Product Images from "Effect of colorectal cancer-derived extracellular vesicles on the immunophenotype and cytokine secretion profile of monocytes and macrophages"

    Article Title: Effect of colorectal cancer-derived extracellular vesicles on the immunophenotype and cytokine secretion profile of monocytes and macrophages

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-018-0229-y

    Effect of dynasore hydrate on the SW480 and SW620 EV-induced changes on the expression of the surface marker CD14 and on the gene expression of CXCL10 and IL-10 in M0 macrophages. a Flow cytometry analysis showing the percentage of CD14-positive M0 macrophages. The graphs represent mean ± SD (n = 2). Statistical analysis was carried out with t-test. * p ≤ 0.05, ** p ≤ 0.01 b qPCR analysis showing changes in CXCL10 and IL-10 gene expression (n = 3).
    Figure Legend Snippet: Effect of dynasore hydrate on the SW480 and SW620 EV-induced changes on the expression of the surface marker CD14 and on the gene expression of CXCL10 and IL-10 in M0 macrophages. a Flow cytometry analysis showing the percentage of CD14-positive M0 macrophages. The graphs represent mean ± SD (n = 2). Statistical analysis was carried out with t-test. * p ≤ 0.05, ** p ≤ 0.01 b qPCR analysis showing changes in CXCL10 and IL-10 gene expression (n = 3).

    Techniques Used: Expressing, Marker, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction

    SW480 and SW620-derived EV uptake in THP-1 monocytes and M0 macrophages. a Flow cytometry analysis showing concentration-dependent uptake of Syto RNA select labelled EVs by THP-1 monocytes. The graphs show the percentage of Syto RNA select-positive THP-1 monocytes in relation to EV concentration (left) and Syto RNA Select-positive THP-1 monocytes (M, middle) and M0 macrophages (M0, right) following incubation with Syto RNA Select labelled EV at final concentration 10 μg/mL. Data are shown as mean ± SD ( n = 5). Statistical analysis was carried out with one-way ANOVA test. b Representative flow cytometry histograms showing Syto RNA Select labelled SW480 and SW620 EV uptake in THP-1 monocytes and M0 macrophages ( n ≥ 4). Grey lines represent untreated cells; black lines represent SW480 or SW620 EV (10 μg/mL) treated monocytes (M) or macrophages (M0). Histogram bar shows the percentage of Syto RNA select positive cells in the respective analysis. c Representative fluorescence microscopy images showing Syto RNA select labelled SW480 EV and SW620 EV uptake in THP-1 monocytes (n = 3). THP-1 monocytes were incubated with Syto RNA select labelled SW480 or SW620 EVs (10 μg/mL) for 1 h (green). The cytoskeleton was labelled with F-actin probe ActinRed 555 (red). The nuclei were stained with Hoechst 33,342 (blue). Scale bar is 10 μm
    Figure Legend Snippet: SW480 and SW620-derived EV uptake in THP-1 monocytes and M0 macrophages. a Flow cytometry analysis showing concentration-dependent uptake of Syto RNA select labelled EVs by THP-1 monocytes. The graphs show the percentage of Syto RNA select-positive THP-1 monocytes in relation to EV concentration (left) and Syto RNA Select-positive THP-1 monocytes (M, middle) and M0 macrophages (M0, right) following incubation with Syto RNA Select labelled EV at final concentration 10 μg/mL. Data are shown as mean ± SD ( n = 5). Statistical analysis was carried out with one-way ANOVA test. b Representative flow cytometry histograms showing Syto RNA Select labelled SW480 and SW620 EV uptake in THP-1 monocytes and M0 macrophages ( n ≥ 4). Grey lines represent untreated cells; black lines represent SW480 or SW620 EV (10 μg/mL) treated monocytes (M) or macrophages (M0). Histogram bar shows the percentage of Syto RNA select positive cells in the respective analysis. c Representative fluorescence microscopy images showing Syto RNA select labelled SW480 EV and SW620 EV uptake in THP-1 monocytes (n = 3). THP-1 monocytes were incubated with Syto RNA select labelled SW480 or SW620 EVs (10 μg/mL) for 1 h (green). The cytoskeleton was labelled with F-actin probe ActinRed 555 (red). The nuclei were stained with Hoechst 33,342 (blue). Scale bar is 10 μm

    Techniques Used: Derivative Assay, Flow Cytometry, Cytometry, Concentration Assay, Incubation, Fluorescence, Microscopy, Staining

    SW480 and SW620-derived EV effect on immunophenotype and cytokine secretion of THP-1 monocytes (M), inactive macrophages (M0) and polarized macrophages (M1 and M2). a, b, c Flow cytometry analysis showing the percentage of CD14, HLA-DR and CD206 positive cells (mean with range, n = 4) at M, M0, M1 and M2 stages following incubation with 10 μg/mL of SW480 EVs or SW620 EVs. Statistical analysis was carried out with two-way ANOVA test. * p ≤ 0.05; ** p ≤ 0.01 vs. untreated controls of the respective monocyte-macrophage cell subset. d Cytokine and chemokine secretion pattern in M, M0, M1 and M2 stages following incubation with 10 μg/mL of SW480 EVs or SW620 EVs analysed by Luminex assay (mean with range, n = 3). Statistical analysis was carried out with multiple t-tests using Holm-Sidak method for multiple comparison correction. * p ≤ 0.05 vs. untreated controls of the respective monocyte-macrophage cell subtype
    Figure Legend Snippet: SW480 and SW620-derived EV effect on immunophenotype and cytokine secretion of THP-1 monocytes (M), inactive macrophages (M0) and polarized macrophages (M1 and M2). a, b, c Flow cytometry analysis showing the percentage of CD14, HLA-DR and CD206 positive cells (mean with range, n = 4) at M, M0, M1 and M2 stages following incubation with 10 μg/mL of SW480 EVs or SW620 EVs. Statistical analysis was carried out with two-way ANOVA test. * p ≤ 0.05; ** p ≤ 0.01 vs. untreated controls of the respective monocyte-macrophage cell subset. d Cytokine and chemokine secretion pattern in M, M0, M1 and M2 stages following incubation with 10 μg/mL of SW480 EVs or SW620 EVs analysed by Luminex assay (mean with range, n = 3). Statistical analysis was carried out with multiple t-tests using Holm-Sidak method for multiple comparison correction. * p ≤ 0.05 vs. untreated controls of the respective monocyte-macrophage cell subtype

    Techniques Used: Derivative Assay, Flow Cytometry, Cytometry, Incubation, Luminex

    SW480 and SW620-derived EV uptake pathway studies in THP-1 monocytes and M0 macrophages by flow cytometry analysis. a Fluorescence intensity of THP-1 monocytes following incubation with Syto RNA select labelled EV (10 μg/mL) in the presence of uptake inhibitors: 5 μM 5-ethyl-N-isopropyl amiloride (EIPA), 80 μM dynasore hydrate, 10 μM chlorpromazine, 20 μM nystatin and 20 μM cytochalasin D. Untreated cells were used as negative control and EV-treated cells served as positive control. The graph represents mean ± SD (n = 3). b, c Flow cytometry histograms showing fluorescence intensity of THP-1 monocytes following incubation with 10 μg/mL of Syto RNA Select labeled SW480 EVs (b) or SW620 EVs (c) in the presence or absence of the uptake inhibitors. Images are representative of 3 biological replicates. d Flow cytometry analysis of M0 macrophages following incubation with Syto RNA select labelled SW480 and SW620 EVs (10 μg/mL) in the presence of uptake inhibitors: 5 μM 5-ethyl-N-isopropyl amiloride (EIPA), 80 μM dynasore hydrate, 10 μM chlorpromazine, 20 μM nystatin and 20 μM cytochalasin D. Untreated M0 cells were used as a negative control and EV-treated M0 cells served as positive control. The graph represents mean ± SD ( n = 2). e, f Flow cytometry histograms showing fluorescence intensity of M0 macrophages following incubation with 10 μg/mL of Syto RNA Select labeled SW480 EVs (e) or SW620 EVs (f) in the presence or absence of uptake inhibitors. Statistical analysis carried out with a two-way ANOVA test followed by Sidak’s post-test. * p ≤ 0.05 vs. EV-treated cells of the respective monocyte-macrophage cell subset
    Figure Legend Snippet: SW480 and SW620-derived EV uptake pathway studies in THP-1 monocytes and M0 macrophages by flow cytometry analysis. a Fluorescence intensity of THP-1 monocytes following incubation with Syto RNA select labelled EV (10 μg/mL) in the presence of uptake inhibitors: 5 μM 5-ethyl-N-isopropyl amiloride (EIPA), 80 μM dynasore hydrate, 10 μM chlorpromazine, 20 μM nystatin and 20 μM cytochalasin D. Untreated cells were used as negative control and EV-treated cells served as positive control. The graph represents mean ± SD (n = 3). b, c Flow cytometry histograms showing fluorescence intensity of THP-1 monocytes following incubation with 10 μg/mL of Syto RNA Select labeled SW480 EVs (b) or SW620 EVs (c) in the presence or absence of the uptake inhibitors. Images are representative of 3 biological replicates. d Flow cytometry analysis of M0 macrophages following incubation with Syto RNA select labelled SW480 and SW620 EVs (10 μg/mL) in the presence of uptake inhibitors: 5 μM 5-ethyl-N-isopropyl amiloride (EIPA), 80 μM dynasore hydrate, 10 μM chlorpromazine, 20 μM nystatin and 20 μM cytochalasin D. Untreated M0 cells were used as a negative control and EV-treated M0 cells served as positive control. The graph represents mean ± SD ( n = 2). e, f Flow cytometry histograms showing fluorescence intensity of M0 macrophages following incubation with 10 μg/mL of Syto RNA Select labeled SW480 EVs (e) or SW620 EVs (f) in the presence or absence of uptake inhibitors. Statistical analysis carried out with a two-way ANOVA test followed by Sidak’s post-test. * p ≤ 0.05 vs. EV-treated cells of the respective monocyte-macrophage cell subset

    Techniques Used: Derivative Assay, Flow Cytometry, Cytometry, Fluorescence, Incubation, Negative Control, Positive Control, Labeling

    31) Product Images from "Proteomic Identification of Heat Shock-Induced Danger Signals in a Melanoma Cell Lysate Used in Dendritic Cell-Based Cancer Immunotherapy"

    Article Title: Proteomic Identification of Heat Shock-Induced Danger Signals in a Melanoma Cell Lysate Used in Dendritic Cell-Based Cancer Immunotherapy

    Journal: Journal of Immunology Research

    doi: 10.1155/2018/3982942

    The HS conditioning of TRIMEL melanoma cells contributes to its in vitro DC maturation capacity. Representative density plots (a) and statistical quantification (b) of the DC-associated marker expression MHCI, MHCII, and CD80 in primary human cytokine-activated monocytes stimulated with TRIMEL (HS), or with the same lysate generated without heat shock conditioning (no-HS) (100 μ g/mL) or without lysate (unstimulated (Unst)). (b) The quantification of the maturation marker expression considered the % positive cells, the geometric mean fluorescence intensity (gMFI) of the positive cells, and the integrated MFI (iMFI: % positive cells × gMFI of positive cells/100). The expression of surface markers was assessed by flow cytometry (CD11c + cells were gated). Data represent three independent experiments with PBMC derived from three different stage IV MM patients. (c) Bars indicate the average fold induction and standard deviation (SD) of the iMFI of DC markers relative to monocytes stimulated with no-HS lysate. ∗ p
    Figure Legend Snippet: The HS conditioning of TRIMEL melanoma cells contributes to its in vitro DC maturation capacity. Representative density plots (a) and statistical quantification (b) of the DC-associated marker expression MHCI, MHCII, and CD80 in primary human cytokine-activated monocytes stimulated with TRIMEL (HS), or with the same lysate generated without heat shock conditioning (no-HS) (100 μ g/mL) or without lysate (unstimulated (Unst)). (b) The quantification of the maturation marker expression considered the % positive cells, the geometric mean fluorescence intensity (gMFI) of the positive cells, and the integrated MFI (iMFI: % positive cells × gMFI of positive cells/100). The expression of surface markers was assessed by flow cytometry (CD11c + cells were gated). Data represent three independent experiments with PBMC derived from three different stage IV MM patients. (c) Bars indicate the average fold induction and standard deviation (SD) of the iMFI of DC markers relative to monocytes stimulated with no-HS lysate. ∗ p

    Techniques Used: In Vitro, Marker, Expressing, Generated, Fluorescence, Flow Cytometry, Cytometry, Derivative Assay, Standard Deviation

    32) Product Images from "The AP2 binding site of synaptotagmin 1 is not an internalization signal but a regulator of endocytosis"

    Article Title: The AP2 binding site of synaptotagmin 1 is not an internalization signal but a regulator of endocytosis

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200103040

    Comparison of synaptotagmin 1 internalization in CHO and PC12 cells. (a) wtPC12 or (b) CHO stably transfected with synaptotagmin 1 (CHOsyn1) were labeled at 4°C with the 604.1 antibody and then moved to 37°C for the indicated periods. Cells were cooled to 4°C and antibody remaining at the surface after the 37°C incubation was detected with a fluorescein-conjugated secondary antibody. The intensity of fluorescence was determined by flow cytometry. Data were expressed as the percentage of the initial value at t = 0. (c) The expression level of synaptotagmin 1 in different CHOsyn1 clones was determined by flow cytometry after permeabilization of the cells and staining with 604-1 antibody. These values are expressed along the x-axis. The same clones were then analyzed for internalization of synaptotagmin 1 using the same assay as in panels a and b. The values obtained after 10 min at 37°C correspond to the y-axis. The same measurements were done in parallel on PC12 cells. (d) wt PC12, CHOsyn1, and HEK cells stably expressing synaptotagmin 1 (HEKsyn1) were examined for internalization of synaptotagmin 1 using 125 I -604.1 antibody. Cells were labeled at 4°C and shifted to 37°C for different time points. The internalized antibody was determined by surface acid stripping and expressed as a fraction of total cell associated counts. Each time point was done in triplicate. In this and subsequent figures, when standard deviations are not apparent, they were too small to be represented graphically.
    Figure Legend Snippet: Comparison of synaptotagmin 1 internalization in CHO and PC12 cells. (a) wtPC12 or (b) CHO stably transfected with synaptotagmin 1 (CHOsyn1) were labeled at 4°C with the 604.1 antibody and then moved to 37°C for the indicated periods. Cells were cooled to 4°C and antibody remaining at the surface after the 37°C incubation was detected with a fluorescein-conjugated secondary antibody. The intensity of fluorescence was determined by flow cytometry. Data were expressed as the percentage of the initial value at t = 0. (c) The expression level of synaptotagmin 1 in different CHOsyn1 clones was determined by flow cytometry after permeabilization of the cells and staining with 604-1 antibody. These values are expressed along the x-axis. The same clones were then analyzed for internalization of synaptotagmin 1 using the same assay as in panels a and b. The values obtained after 10 min at 37°C correspond to the y-axis. The same measurements were done in parallel on PC12 cells. (d) wt PC12, CHOsyn1, and HEK cells stably expressing synaptotagmin 1 (HEKsyn1) were examined for internalization of synaptotagmin 1 using 125 I -604.1 antibody. Cells were labeled at 4°C and shifted to 37°C for different time points. The internalized antibody was determined by surface acid stripping and expressed as a fraction of total cell associated counts. Each time point was done in triplicate. In this and subsequent figures, when standard deviations are not apparent, they were too small to be represented graphically.

    Techniques Used: Stable Transfection, Transfection, Labeling, Incubation, Fluorescence, Flow Cytometry, Cytometry, Expressing, Clone Assay, Staining, Stripping Membranes

    33) Product Images from "Mesenchymal stem cells-derived exosomes are more immunosuppressive than microparticles in inflammatory arthritis"

    Article Title: Mesenchymal stem cells-derived exosomes are more immunosuppressive than microparticles in inflammatory arthritis

    Journal: Theranostics

    doi: 10.7150/thno.21072

    Isolation and characterization of exosomes and microparticles isolated from murine MSCs. (A) Experimental protocol for isolation of microparticles (MPs) and exosomes (Exos) from MSCs-conditioned medium using differential ultracentrifugation. (B) Quantification of EVs produced by 10 6 MSCs and expressed as equivalent protein. MSCs were naïve or primed with 20 ng/mL IFN-γ (n=5 biological replicates). (C) Representative pictures of MPs (18K) and Exos (100 k) by transmission electron microscopy. Bars represent 200 nm in large pictures and inserts for MPs; for Exos, bars are 200 nm for large pictures and 100 nm for inserts. (D) Number and size of Exos (left) and MPs (right) detected in 1 mL (corresponding to 1 µg EVs equivalent protein) by Nano Tracking Analysis. Letters (A to E) indicate various population peaks (n=3 biological replicates). (E) Quantification of Exos and MPs particle numbers related to the quantity of protein (n=3 biological replicates). (F) Mean size of Exos (left) and MPs (right) in the fractions represented in (D) (n=3 biological replicates). (G) Percentage of MPs in each fraction (A to E) related to total MPs (n=3 biological replicates). (H) Expression of MSCs membrane markers (Sca-1, CD44, CD29) and of exosomal markers (CD9, CD81) on Exos (top) and MPs (bottom) isolated from naïve MSCs analyzed by flow cytometry.
    Figure Legend Snippet: Isolation and characterization of exosomes and microparticles isolated from murine MSCs. (A) Experimental protocol for isolation of microparticles (MPs) and exosomes (Exos) from MSCs-conditioned medium using differential ultracentrifugation. (B) Quantification of EVs produced by 10 6 MSCs and expressed as equivalent protein. MSCs were naïve or primed with 20 ng/mL IFN-γ (n=5 biological replicates). (C) Representative pictures of MPs (18K) and Exos (100 k) by transmission electron microscopy. Bars represent 200 nm in large pictures and inserts for MPs; for Exos, bars are 200 nm for large pictures and 100 nm for inserts. (D) Number and size of Exos (left) and MPs (right) detected in 1 mL (corresponding to 1 µg EVs equivalent protein) by Nano Tracking Analysis. Letters (A to E) indicate various population peaks (n=3 biological replicates). (E) Quantification of Exos and MPs particle numbers related to the quantity of protein (n=3 biological replicates). (F) Mean size of Exos (left) and MPs (right) in the fractions represented in (D) (n=3 biological replicates). (G) Percentage of MPs in each fraction (A to E) related to total MPs (n=3 biological replicates). (H) Expression of MSCs membrane markers (Sca-1, CD44, CD29) and of exosomal markers (CD9, CD81) on Exos (top) and MPs (bottom) isolated from naïve MSCs analyzed by flow cytometry.

    Techniques Used: Isolation, Produced, Transmission Assay, Electron Microscopy, Expressing, Flow Cytometry, Cytometry

    MPs and Exos exert immunosuppressive functions on B lymphocytes. (A) Percentage of CD138 + plasmablasts obtained after activation (Ctrl) or culturing with naïve MSCs or 50 ng Exos or MPs from naïve or IFN-γ primed MSCs (n=5 biological replicates). Representative flow cytometry pictures are shown below. (B) Concentration of total IgG, TNFα, IL6, IL10 in supernatants from plasmablasts in (A) as expressed in arbitrary unit (a.u.) or pg/mL (n=5 biological replicates). (C) Amounts of TGF-β1, PGE2, IL-6, IL1-RA in 1 µg of Exos or MPs as evaluated by ELISA (n=5 biological replicates). Statistical analysis used a non-parametric Kruskal-Wallis test with Dunn's multiple comparison post-test. *: p
    Figure Legend Snippet: MPs and Exos exert immunosuppressive functions on B lymphocytes. (A) Percentage of CD138 + plasmablasts obtained after activation (Ctrl) or culturing with naïve MSCs or 50 ng Exos or MPs from naïve or IFN-γ primed MSCs (n=5 biological replicates). Representative flow cytometry pictures are shown below. (B) Concentration of total IgG, TNFα, IL6, IL10 in supernatants from plasmablasts in (A) as expressed in arbitrary unit (a.u.) or pg/mL (n=5 biological replicates). (C) Amounts of TGF-β1, PGE2, IL-6, IL1-RA in 1 µg of Exos or MPs as evaluated by ELISA (n=5 biological replicates). Statistical analysis used a non-parametric Kruskal-Wallis test with Dunn's multiple comparison post-test. *: p

    Techniques Used: Activation Assay, Flow Cytometry, Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Freshly isolated extracellular vesicles from murine MSCs exert immunosuppressive functions. (A) Experimental protocol for isolation of total extracellular vesicles (EVs) using differential ultracentrifugation. (B) Proliferation of Concanavalin A-activated murine splenocytes cultured alone for 3 days (Ctrl) or incubated with naïve or IFN-γ (20 ng/mL)-primed MSCs (n=3 biological replicates). (C) Proliferation of Concanavalin A-activated murine splenocytes cultured alone for 3 days (C) or incubated with ultracentrifuged production medium (UM) or naïve MSCs or MSCs-conditioned medium (CM) pre (Pr)- or post (Po)-100,000 × g centrifugation according to (A). CM was depleted in cells and debris (by 300 × g and 2500 × g centrifugation steps). MSCs were naïve or primed with 20 ng/mL IFN-γ (n=3 biological replicates). (D) Proliferation of Concanavalin A-activated murine splenocytes cultured alone for 3 days (Ctrl) or incubated with increasing amounts of EVs (n=4 biological replicates). (E) Proliferation of Concanavalin A-activated murine splenocytes cultured alone for 3 days (Ctrl) or incubated with 50 ng of freshly isolated or freeze-thawed EVs (n=4 biological replicates). (F) TEM analysis of freshly isolated (4°C) or freeze-thawed EVs (-80°C). Bar is 100 nm. (G) Expression of MSCs membrane markers (Sca-1, CD44, CD29) and of CD81 exosomal marker on freshly isolated (4°C) or freeze-thawed EVs (-80°C) analyzed by flow cytometry. (H) Number and median size of freshly isolated (4°C) or freeze-thawed EVs (-80°C) by Nano Tracking Analysis. Statistical analysis used a non-parametric Kruskal-Wallis test with Dunn's multiple comparison post-test (B, C, D, E) or a Mann-Whitney test (H). *: p
    Figure Legend Snippet: Freshly isolated extracellular vesicles from murine MSCs exert immunosuppressive functions. (A) Experimental protocol for isolation of total extracellular vesicles (EVs) using differential ultracentrifugation. (B) Proliferation of Concanavalin A-activated murine splenocytes cultured alone for 3 days (Ctrl) or incubated with naïve or IFN-γ (20 ng/mL)-primed MSCs (n=3 biological replicates). (C) Proliferation of Concanavalin A-activated murine splenocytes cultured alone for 3 days (C) or incubated with ultracentrifuged production medium (UM) or naïve MSCs or MSCs-conditioned medium (CM) pre (Pr)- or post (Po)-100,000 × g centrifugation according to (A). CM was depleted in cells and debris (by 300 × g and 2500 × g centrifugation steps). MSCs were naïve or primed with 20 ng/mL IFN-γ (n=3 biological replicates). (D) Proliferation of Concanavalin A-activated murine splenocytes cultured alone for 3 days (Ctrl) or incubated with increasing amounts of EVs (n=4 biological replicates). (E) Proliferation of Concanavalin A-activated murine splenocytes cultured alone for 3 days (Ctrl) or incubated with 50 ng of freshly isolated or freeze-thawed EVs (n=4 biological replicates). (F) TEM analysis of freshly isolated (4°C) or freeze-thawed EVs (-80°C). Bar is 100 nm. (G) Expression of MSCs membrane markers (Sca-1, CD44, CD29) and of CD81 exosomal marker on freshly isolated (4°C) or freeze-thawed EVs (-80°C) analyzed by flow cytometry. (H) Number and median size of freshly isolated (4°C) or freeze-thawed EVs (-80°C) by Nano Tracking Analysis. Statistical analysis used a non-parametric Kruskal-Wallis test with Dunn's multiple comparison post-test (B, C, D, E) or a Mann-Whitney test (H). *: p

    Techniques Used: Isolation, Cell Culture, Incubation, Centrifugation, Transmission Electron Microscopy, Expressing, Marker, Flow Cytometry, Cytometry, MANN-WHITNEY

    34) Product Images from "Methanol fixed fibroblasts serve as feeder cells to maintain stem cells in the pluripotent state in vitro"

    Article Title: Methanol fixed fibroblasts serve as feeder cells to maintain stem cells in the pluripotent state in vitro

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26238-2

    Human and porcine iPS cells were maintained on feeders derived from methanol fixed fibroblasts. ( A ) Human iPS cells were cultured on MT-MEF (a), MT-3T3 (b), MT-hMSC (c), and MMC-MEF (d) in KSR medium, and on MT-MEF (e) and Matrigel (f) in mTeSR medium. ( B ) Immunofluorescence analysis of OCT4 and SOX2 expressions in human iPS cells. ( C ) Flow cytometry analysis of SSEA-4 in human iPS cells. ( D ) Porcine iPS cells were cultured on MT-MEF, MT-3T3, and MMC-MEF. ( E ) RT-PCR analysis of OCT4 , SOX2 , NANOG , and ESRRB expressions in porcine iPS cells. Scale bar, 200 μm.
    Figure Legend Snippet: Human and porcine iPS cells were maintained on feeders derived from methanol fixed fibroblasts. ( A ) Human iPS cells were cultured on MT-MEF (a), MT-3T3 (b), MT-hMSC (c), and MMC-MEF (d) in KSR medium, and on MT-MEF (e) and Matrigel (f) in mTeSR medium. ( B ) Immunofluorescence analysis of OCT4 and SOX2 expressions in human iPS cells. ( C ) Flow cytometry analysis of SSEA-4 in human iPS cells. ( D ) Porcine iPS cells were cultured on MT-MEF, MT-3T3, and MMC-MEF. ( E ) RT-PCR analysis of OCT4 , SOX2 , NANOG , and ESRRB expressions in porcine iPS cells. Scale bar, 200 μm.

    Techniques Used: Derivative Assay, Cell Culture, Immunofluorescence, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction

    Maintenance of self-renewal and pluripotency of J1 mES on methanol fixed fibroblasts. J1 mES cells were cultured on methanol fixed MEF (MT-MEF) and NIH3T3 (MT-3T3) cells, and Mitomycin C treated MEF (MMC-MEF). ( A ) Morphology and AP staining of J1 mES cells. ( B ) Growth curve of J1 mES. ( C ) qRT-PCR analysis of pluripotent genes in J1 mES. ( D , E ) Immunofluorescence ( D ) and flow cytometry analysis ( E ) of pluripotent markers OCT4 and SSEA-1 in J1 mES. Nuclei were stained by Hoechst 33342 (Hoe). ( F ) Teratoma formation of J1 mES. Arrows indicate tissues from the three germ layers. Scale bar, 400 μm for A, 200 μm for F, and 100 μm for D.
    Figure Legend Snippet: Maintenance of self-renewal and pluripotency of J1 mES on methanol fixed fibroblasts. J1 mES cells were cultured on methanol fixed MEF (MT-MEF) and NIH3T3 (MT-3T3) cells, and Mitomycin C treated MEF (MMC-MEF). ( A ) Morphology and AP staining of J1 mES cells. ( B ) Growth curve of J1 mES. ( C ) qRT-PCR analysis of pluripotent genes in J1 mES. ( D , E ) Immunofluorescence ( D ) and flow cytometry analysis ( E ) of pluripotent markers OCT4 and SSEA-1 in J1 mES. Nuclei were stained by Hoechst 33342 (Hoe). ( F ) Teratoma formation of J1 mES. Arrows indicate tissues from the three germ layers. Scale bar, 400 μm for A, 200 μm for F, and 100 μm for D.

    Techniques Used: Cell Culture, Staining, Quantitative RT-PCR, Immunofluorescence, Flow Cytometry, Cytometry

    Application of methanol fixed fibroblasts for drug screening and repeated usage. ( A ) Diagram of drug screening. ( B ) The stably transfected cell lines derived from J1 cells transfected by miR70/EGFP and piPS cells transfected by METTL3-EGFP and miR370/EGFP, respectively, which were cultured on MT-MEF and MT-3T3. ( C , D ) Repeated usage of MT-MEF ( C ) and MT-3T3 ( D ) for four times (R0-R3). ( E , F ) Flow cytometry analysis of SSEA-1 expression in J1 mES cultured on MT-MEF ( E ) and MT-3T3 ( F ), which were used repeatedly for four times (R0-R3). Scale bar, 100 μm for B, 200 μm for C and D.
    Figure Legend Snippet: Application of methanol fixed fibroblasts for drug screening and repeated usage. ( A ) Diagram of drug screening. ( B ) The stably transfected cell lines derived from J1 cells transfected by miR70/EGFP and piPS cells transfected by METTL3-EGFP and miR370/EGFP, respectively, which were cultured on MT-MEF and MT-3T3. ( C , D ) Repeated usage of MT-MEF ( C ) and MT-3T3 ( D ) for four times (R0-R3). ( E , F ) Flow cytometry analysis of SSEA-1 expression in J1 mES cultured on MT-MEF ( E ) and MT-3T3 ( F ), which were used repeatedly for four times (R0-R3). Scale bar, 100 μm for B, 200 μm for C and D.

    Techniques Used: Stable Transfection, Transfection, Derivative Assay, Cell Culture, Flow Cytometry, Cytometry, Expressing

    Culture of mouse ES on fibroblasts fixed by methanol. J1 mES cells were cultured on MEF cells fixed by MT (MT-MEF) and GA (GA-MEF), respectively. ( A ) Morphology and AP staining of J1 mES cells. ( B ) Growth curve of J1 cells. ( C ) qRT-PCR analysis of Oct4 , Nanog , and Sox2 expressions in J1 cells. ( D ) Flow cytometry analysis of SSEA-1 expression in J1 cells. ( E ) Scanning electron microscope analysis of MT-MEF, GA-MEF, and MMC-MEF. ( F ) J1 cells were cultured on MT fixed C2C12, PEF, and PK-15 cells. Phase 1, MT fixed cells; Phase 2, morphology of J1 cells cultured on MT fixed cells. Scale bar, 200 μm. Data indicate mean ± SD, *P
    Figure Legend Snippet: Culture of mouse ES on fibroblasts fixed by methanol. J1 mES cells were cultured on MEF cells fixed by MT (MT-MEF) and GA (GA-MEF), respectively. ( A ) Morphology and AP staining of J1 mES cells. ( B ) Growth curve of J1 cells. ( C ) qRT-PCR analysis of Oct4 , Nanog , and Sox2 expressions in J1 cells. ( D ) Flow cytometry analysis of SSEA-1 expression in J1 cells. ( E ) Scanning electron microscope analysis of MT-MEF, GA-MEF, and MMC-MEF. ( F ) J1 cells were cultured on MT fixed C2C12, PEF, and PK-15 cells. Phase 1, MT fixed cells; Phase 2, morphology of J1 cells cultured on MT fixed cells. Scale bar, 200 μm. Data indicate mean ± SD, *P

    Techniques Used: Cell Culture, Staining, Quantitative RT-PCR, Flow Cytometry, Cytometry, Expressing, Microscopy

    35) Product Images from "S1P1 receptor overrides regulatory T cell-mediated immune suppression through Akt-mTOR"

    Article Title: S1P1 receptor overrides regulatory T cell-mediated immune suppression through Akt-mTOR

    Journal: Nature immunology

    doi: 10.1038/ni.1743

    S1P 1 -Tg mice show disrupted immune homeostasis and develop age-related autoimmunity due to defects in the T reg compartment ( a - e ) Analysis of WT and S1P 1 -Tg mice. ( a ) Flow cytometry of T cell activation markers from peripheral lymphoid organs of aged mice (10 months). MLN, mesenteric lymph nodes. Data are representative of 6 independent experiments. ( b ) Proliferative response to TCR stimulation of T conv cells from WT and S1P 1 -Tg mice (2 months). Data are representative of 6 independent experiments. ( c ) Titers of anti-nuclear antigen and anti-ds DNA antibodies of aged mice (10 months). Data are the mean (±s.d.) of > 10 mice of each genotype and are representative of 4 independent experiments. ( d ) Effector cytokine production of activated T cells from WT and S1P 1 -Tg mice (5-6 months). Data are representative of 2 independent experiments. ( e ) Serum titers of IgG1 and IgG2a (5-6 months). Data are the mean of 5 mice of each genotype and are representative of 3 independent experiments. ( f - h ) Analysis of WT and S1P 1 -Tg T cells in the mixed BM chimeras (6-9 months after reconstitution), including expression of activation markers ( f ), proliferation ( g ), and effector cytokine production ( h ). Data are representative of 3 independent experiments. *, P
    Figure Legend Snippet: S1P 1 -Tg mice show disrupted immune homeostasis and develop age-related autoimmunity due to defects in the T reg compartment ( a - e ) Analysis of WT and S1P 1 -Tg mice. ( a ) Flow cytometry of T cell activation markers from peripheral lymphoid organs of aged mice (10 months). MLN, mesenteric lymph nodes. Data are representative of 6 independent experiments. ( b ) Proliferative response to TCR stimulation of T conv cells from WT and S1P 1 -Tg mice (2 months). Data are representative of 6 independent experiments. ( c ) Titers of anti-nuclear antigen and anti-ds DNA antibodies of aged mice (10 months). Data are the mean (±s.d.) of > 10 mice of each genotype and are representative of 4 independent experiments. ( d ) Effector cytokine production of activated T cells from WT and S1P 1 -Tg mice (5-6 months). Data are representative of 2 independent experiments. ( e ) Serum titers of IgG1 and IgG2a (5-6 months). Data are the mean of 5 mice of each genotype and are representative of 3 independent experiments. ( f - h ) Analysis of WT and S1P 1 -Tg T cells in the mixed BM chimeras (6-9 months after reconstitution), including expression of activation markers ( f ), proliferation ( g ), and effector cytokine production ( h ). Data are representative of 3 independent experiments. *, P

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Activation Assay, Expressing

    S1P 1 is necessary for Akt activation in T reg cells ( a , b ) IL-2 activated signaling pathways in thymic T reg precursors ( a ) and Foxp3 + reg T reg cells ( b ) from WT and S1P 1 -KO mice. Purified CD4 + CD25 + Foxp3 − cells ( a ) or CD4 + Foxp3 + cells ( b ) were stimulated with medium alone or IL-2, and activation of Akt, STAT5 and Erk were examined by flow cytometry using phospho-specific antibodies. Data are representative of 3 independent experiments. ( c ) Suppressive activity of T reg cells transduced with Cre-GFP retrovirus alone or in combination with constitutively active Akt. Foxp3 + T reg cells from the periphery of S1pr1 +/+ and S1pr1 fl/fl mice were transduced with Cre-expressing retrovirus (Cre-GFP) alone or in combination with empty control (MiT) or constitutively active Akt (ca-Akt) retroviruses, and transduced cells were sorted and used in the T-cell suppression assays with different T conv and T reg ratios. Data are representative of 2 independent experiments. *, P
    Figure Legend Snippet: S1P 1 is necessary for Akt activation in T reg cells ( a , b ) IL-2 activated signaling pathways in thymic T reg precursors ( a ) and Foxp3 + reg T reg cells ( b ) from WT and S1P 1 -KO mice. Purified CD4 + CD25 + Foxp3 − cells ( a ) or CD4 + Foxp3 + cells ( b ) were stimulated with medium alone or IL-2, and activation of Akt, STAT5 and Erk were examined by flow cytometry using phospho-specific antibodies. Data are representative of 3 independent experiments. ( c ) Suppressive activity of T reg cells transduced with Cre-GFP retrovirus alone or in combination with constitutively active Akt. Foxp3 + T reg cells from the periphery of S1pr1 +/+ and S1pr1 fl/fl mice were transduced with Cre-expressing retrovirus (Cre-GFP) alone or in combination with empty control (MiT) or constitutively active Akt (ca-Akt) retroviruses, and transduced cells were sorted and used in the T-cell suppression assays with different T conv and T reg ratios. Data are representative of 2 independent experiments. *, P

    Techniques Used: Activation Assay, Mouse Assay, Purification, Flow Cytometry, Cytometry, Activity Assay, Transduction, Expressing

    S1P 1 blocks thymic differentiation of T reg cells ( a ) Flow cytometry of total and gated CD4SP thymocytes isolated from WT control, S1P 1 -KO and S1P 1 -Tg FTOC. Panels on the right show the proportions and absolute numbers of Foxp3 + CD4SP T reg cells with the mean (+s.d.) calculated from ≥8 mice of each genotype. ( b ) Flow cytometry of gated CD4SP thymocytes from WT control, S1P 1 -KO and S1P 1 -Tg mice. Panels on the right show the proportions and absolute numbers of the CD4 + CD25 + Foxp3 − precursor population, with the mean (+s.d.) calculated from ≥8 mice of each genotype. ( c ) Induction of Foxp3 expression in the CD4 + CD25 + Foxp3 − population in vitro . CD4 + CD25 + Foxp3 − cells were purified and stimulated with medium alone, IL-2 or IL-15 for 20 h, and induction of Foxp3 expression was measured by flow cytometry. The lower panel shows an IL-2 dependent dose response curve. Data are representative of 5 independent experiments. *, P
    Figure Legend Snippet: S1P 1 blocks thymic differentiation of T reg cells ( a ) Flow cytometry of total and gated CD4SP thymocytes isolated from WT control, S1P 1 -KO and S1P 1 -Tg FTOC. Panels on the right show the proportions and absolute numbers of Foxp3 + CD4SP T reg cells with the mean (+s.d.) calculated from ≥8 mice of each genotype. ( b ) Flow cytometry of gated CD4SP thymocytes from WT control, S1P 1 -KO and S1P 1 -Tg mice. Panels on the right show the proportions and absolute numbers of the CD4 + CD25 + Foxp3 − precursor population, with the mean (+s.d.) calculated from ≥8 mice of each genotype. ( c ) Induction of Foxp3 expression in the CD4 + CD25 + Foxp3 − population in vitro . CD4 + CD25 + Foxp3 − cells were purified and stimulated with medium alone, IL-2 or IL-15 for 20 h, and induction of Foxp3 expression was measured by flow cytometry. The lower panel shows an IL-2 dependent dose response curve. Data are representative of 5 independent experiments. *, P

    Techniques Used: Flow Cytometry, Cytometry, Isolation, Mouse Assay, Expressing, In Vitro, Purification

    Enhanced peripheral population and suppressive activity of S1P 1 -KO T reg cells ( a ) Flow cytometry of gated CD4 T cells from the spleen and peripheral lymph nodes (PLN) of WT and S1P 1 -KO mice. The panel on the right shows the proportions of Foxp3 + T reg cells among total CD4 + T cell population, with the mean (+s.d.) calculated from 4 mice of each genotype. ( b ) Flow cytometry analysis of T reg markers (Foxp3, CD25, GITR and CTLA4) in PLN of WT and S1P 1 -KO mice. Data are representative of 2 independent experiments. Similar findings were observed in other peripheral lymphoid organs (not shown). ( c ) In vitro T-cell suppression assays using Foxp3 + CD4SP cells from WT and S1P 1 -KO mice. The left panel shows a representative proliferative assay of 4 independent experiments, the middle panel is the percentage of suppression with the mean (±s.d.) calculated from 4 experiments, and the right panel shows a representative of 2 independent experiments measuring IL-2 production. ( d ) In vitro T-cell suppression assays using S1P 1 -deleted peripheral T reg cells. Foxp3 + T reg cells from the periphery of S1pr1 +/+ and S1pr1 fl/fl mice were transduced with Cre-expressing retrovirus (Cre-GFP), and sorted GFP + T reg cells were used in the T-cell suppression assays with different T conv and T reg ratios; freshly isolated T reg cells were used as a comparison. The left panel is a representative of 3 independent experiments, and the right panel shows the percentage of suppression with the mean (+s.d.) calculated from 3 experiments. *, P
    Figure Legend Snippet: Enhanced peripheral population and suppressive activity of S1P 1 -KO T reg cells ( a ) Flow cytometry of gated CD4 T cells from the spleen and peripheral lymph nodes (PLN) of WT and S1P 1 -KO mice. The panel on the right shows the proportions of Foxp3 + T reg cells among total CD4 + T cell population, with the mean (+s.d.) calculated from 4 mice of each genotype. ( b ) Flow cytometry analysis of T reg markers (Foxp3, CD25, GITR and CTLA4) in PLN of WT and S1P 1 -KO mice. Data are representative of 2 independent experiments. Similar findings were observed in other peripheral lymphoid organs (not shown). ( c ) In vitro T-cell suppression assays using Foxp3 + CD4SP cells from WT and S1P 1 -KO mice. The left panel shows a representative proliferative assay of 4 independent experiments, the middle panel is the percentage of suppression with the mean (±s.d.) calculated from 4 experiments, and the right panel shows a representative of 2 independent experiments measuring IL-2 production. ( d ) In vitro T-cell suppression assays using S1P 1 -deleted peripheral T reg cells. Foxp3 + T reg cells from the periphery of S1pr1 +/+ and S1pr1 fl/fl mice were transduced with Cre-expressing retrovirus (Cre-GFP), and sorted GFP + T reg cells were used in the T-cell suppression assays with different T conv and T reg ratios; freshly isolated T reg cells were used as a comparison. The left panel is a representative of 3 independent experiments, and the right panel shows the percentage of suppression with the mean (+s.d.) calculated from 3 experiments. *, P

    Techniques Used: Activity Assay, Flow Cytometry, Cytometry, Mouse Assay, In Vitro, Transduction, Expressing, Isolation

    S1P 1 induces activation of Akt-mTOR to inhibit T reg development and function ( a ) IL-2 activated signaling pathways in thymic T reg precursors from WT and S1P 1 -Tg mice. CD4 + CD25 + Foxp3 − cells were purified and stimulated with medium alone or IL-2, and activation of Akt, STAT5, Erk and S6 ribosomal protein (S6) were examined by flow cytometry using phospho-specific antibodies. Data are representative of 4 independent experiments. ( b ) Effects of drug treatments on IL-2 induced Foxp3 expression in T reg precursors. CD4 + CD25 + Foxp3 − cells were treated with U0126, LY294002 and Rapamycin for 30 minutes, followed by IL-2 stimulation. Data are representative of 3 independent experiments. ( c ) IL-2 activated signaling pathways in peripheral T reg cells from WT and S1P 1 -Tg mice. T reg cells were stimulated with medium alone or IL-2, and activation of Akt, STAT5, Erk and S6 ribosomal protein (S6) were examined by flow cytometry using phospho-specific antibodies. Data are representative of 5 independent experiments. ( d ) Suppressive activity of T reg cells transduced with dn-Akt retrovirus. WT and S1P 1 -Tg T reg cells were transduced with control (MiT) and dn-Akt expressing (dn-Akt) retroviruses (non-transduced cells are shown on the right as a comparison), and transduced cells were sorted and used in the T-cell suppression assays with different T conv and T reg ratios. Data are representative of 3 independent experiments. *, P
    Figure Legend Snippet: S1P 1 induces activation of Akt-mTOR to inhibit T reg development and function ( a ) IL-2 activated signaling pathways in thymic T reg precursors from WT and S1P 1 -Tg mice. CD4 + CD25 + Foxp3 − cells were purified and stimulated with medium alone or IL-2, and activation of Akt, STAT5, Erk and S6 ribosomal protein (S6) were examined by flow cytometry using phospho-specific antibodies. Data are representative of 4 independent experiments. ( b ) Effects of drug treatments on IL-2 induced Foxp3 expression in T reg precursors. CD4 + CD25 + Foxp3 − cells were treated with U0126, LY294002 and Rapamycin for 30 minutes, followed by IL-2 stimulation. Data are representative of 3 independent experiments. ( c ) IL-2 activated signaling pathways in peripheral T reg cells from WT and S1P 1 -Tg mice. T reg cells were stimulated with medium alone or IL-2, and activation of Akt, STAT5, Erk and S6 ribosomal protein (S6) were examined by flow cytometry using phospho-specific antibodies. Data are representative of 5 independent experiments. ( d ) Suppressive activity of T reg cells transduced with dn-Akt retrovirus. WT and S1P 1 -Tg T reg cells were transduced with control (MiT) and dn-Akt expressing (dn-Akt) retroviruses (non-transduced cells are shown on the right as a comparison), and transduced cells were sorted and used in the T-cell suppression assays with different T conv and T reg ratios. Data are representative of 3 independent experiments. *, P

    Techniques Used: Activation Assay, Mouse Assay, Purification, Flow Cytometry, Cytometry, Expressing, Activity Assay, Transduction

    S1P 1 negatively regulates thymic Foxp3 + T reg population ( a , b ) Flow cytometry of total and gated CD4SP thymocytes isolated from wild-type (WT) control, S1P 1 -KO ( a ) and S1P 1 -Tg mice ( b ). Panels on the right show the proportions and absolute numbers of Foxp3 + CD4SP T reg cells. Data are the mean (+s.d.) of 8-14 mice of each genotype from 7 experiments. ( c ) Foxp3 expression in bone marrow chimeras following retroviral transduction of S1P 1 . Bone marrow stem cells from WT mice were transduced with retrovirus expressing S1P 1 (S1P 1 -GFP) or empty vector (GFP), and transferred into sublethally irradiated Rag1 -/- mice. At 6-8 weeks after reconstitution, Foxp3 expression was analyzed in gated CD4SP thymocytes. Data are representative of 2 independent experiments. ( d ) Expression of Foxp3 in mixed bone marrow chimeras. Bone marrow stem cells from WT (CD45.1 + ) and S1P 1 -KO or S1P 1 -Tg mice (CD45.2 + ) were mixed at 1:1, and transferred into Rag1 -/- mice to generate mixed bone marrow chimeras. At 6-8 weeks after reconstitution, Foxp3 expression was analyzed in CD4SP thymocytes, and cells from different donors were distinguished by their CD45.1 and CD45.2 expression. Data are representative of 3 independent experiments. *, P
    Figure Legend Snippet: S1P 1 negatively regulates thymic Foxp3 + T reg population ( a , b ) Flow cytometry of total and gated CD4SP thymocytes isolated from wild-type (WT) control, S1P 1 -KO ( a ) and S1P 1 -Tg mice ( b ). Panels on the right show the proportions and absolute numbers of Foxp3 + CD4SP T reg cells. Data are the mean (+s.d.) of 8-14 mice of each genotype from 7 experiments. ( c ) Foxp3 expression in bone marrow chimeras following retroviral transduction of S1P 1 . Bone marrow stem cells from WT mice were transduced with retrovirus expressing S1P 1 (S1P 1 -GFP) or empty vector (GFP), and transferred into sublethally irradiated Rag1 -/- mice. At 6-8 weeks after reconstitution, Foxp3 expression was analyzed in gated CD4SP thymocytes. Data are representative of 2 independent experiments. ( d ) Expression of Foxp3 in mixed bone marrow chimeras. Bone marrow stem cells from WT (CD45.1 + ) and S1P 1 -KO or S1P 1 -Tg mice (CD45.2 + ) were mixed at 1:1, and transferred into Rag1 -/- mice to generate mixed bone marrow chimeras. At 6-8 weeks after reconstitution, Foxp3 expression was analyzed in CD4SP thymocytes, and cells from different donors were distinguished by their CD45.1 and CD45.2 expression. Data are representative of 3 independent experiments. *, P

    Techniques Used: Flow Cytometry, Cytometry, Isolation, Mouse Assay, Expressing, Transduction, Plasmid Preparation, Irradiation

    36) Product Images from "Dissection of the Transformation of Primary Human Hematopoietic Cells by the Oncogene NUP98-HOXA9"

    Article Title: Dissection of the Transformation of Primary Human Hematopoietic Cells by the Oncogene NUP98-HOXA9

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006719

    Flow cytometry shows homeodomain-independent disruption of human primary CD34+ cell differentiation by NUP98-HOXA9. (A) Flow cytometry for erythroid differentiation: Cells from the CFC plates (see Fig. 7 above) were harvested and stained with antibodies to CD45 and CD235a. The CD235a+ gate was plotted on a histogram (lower panels) to show the expression of CD235a relative to control cells. (B) Flow cytometry for myeloid differentiation: Cells from the CFC plates (see Fig. 7 above) were harvested and stained with CD45, CD33 and CD11b; the CD33+ gate was plotted on a histogram to show CD11b expression compared to control (lower panels). The percentages of cells falling within each gate are shown.
    Figure Legend Snippet: Flow cytometry shows homeodomain-independent disruption of human primary CD34+ cell differentiation by NUP98-HOXA9. (A) Flow cytometry for erythroid differentiation: Cells from the CFC plates (see Fig. 7 above) were harvested and stained with antibodies to CD45 and CD235a. The CD235a+ gate was plotted on a histogram (lower panels) to show the expression of CD235a relative to control cells. (B) Flow cytometry for myeloid differentiation: Cells from the CFC plates (see Fig. 7 above) were harvested and stained with CD45, CD33 and CD11b; the CD33+ gate was plotted on a histogram to show CD11b expression compared to control (lower panels). The percentages of cells falling within each gate are shown.

    Techniques Used: Flow Cytometry, Cytometry, Cell Differentiation, Staining, Expressing

    37) Product Images from "Targeting Human-Cytomegalovirus-Infected Cells by Redirecting T Cells Using an Anti-CD3/Anti-Glycoprotein B Bispecific Antibody"

    Article Title: Targeting Human-Cytomegalovirus-Infected Cells by Redirecting T Cells Using an Anti-CD3/Anti-Glycoprotein B Bispecific Antibody

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01719-17

    The bispecific antibody activates T cells, as shown by the proliferation and secretion of Th1-type cytokines. (A) Dual specificity of the bispecific antibody. Biotinylated gB protein was incubated with CD4 or CD8 T cells pretreated with the bispecific (red) or hu272.7 (green) antibody. PE-conjugated streptavidin was used to detect the binding signal as an indication of dual specificity. (B) Antigen-specific proliferation of T cells mediated by the bispecific antibody. T cells were prestained with CFSE and were then incubated with the bispecific antibody in culture plates coated with recombinant gB protein (left) or BSA (right) for 2 days. The proliferation of T cells in response to the antibody treatment was then determined by measuring the CFSE signal by flow cytometry. (C) Activation of T cells as shown by cytokine production. T cells were incubated with HCMV-infected (circles) or uninfected (squares) ARPE-19 cells in the presence of the bispecific antibody in titration. The supernatants were collected 2 days later for the measurement of TNF-α and IFN-γ production. Statistical significance was determined by two-way ANOVA. (D to F) Two days after the incubation of HCMV-infected (red) or uninfected (green) ARPE-19 cells with the BsAb, T cells were analyzed by flow cytometry for activation markers CD25 (D) and CD69 (E) and for degranulation marker CD107a (F).
    Figure Legend Snippet: The bispecific antibody activates T cells, as shown by the proliferation and secretion of Th1-type cytokines. (A) Dual specificity of the bispecific antibody. Biotinylated gB protein was incubated with CD4 or CD8 T cells pretreated with the bispecific (red) or hu272.7 (green) antibody. PE-conjugated streptavidin was used to detect the binding signal as an indication of dual specificity. (B) Antigen-specific proliferation of T cells mediated by the bispecific antibody. T cells were prestained with CFSE and were then incubated with the bispecific antibody in culture plates coated with recombinant gB protein (left) or BSA (right) for 2 days. The proliferation of T cells in response to the antibody treatment was then determined by measuring the CFSE signal by flow cytometry. (C) Activation of T cells as shown by cytokine production. T cells were incubated with HCMV-infected (circles) or uninfected (squares) ARPE-19 cells in the presence of the bispecific antibody in titration. The supernatants were collected 2 days later for the measurement of TNF-α and IFN-γ production. Statistical significance was determined by two-way ANOVA. (D to F) Two days after the incubation of HCMV-infected (red) or uninfected (green) ARPE-19 cells with the BsAb, T cells were analyzed by flow cytometry for activation markers CD25 (D) and CD69 (E) and for degranulation marker CD107a (F).

    Techniques Used: Incubation, Binding Assay, Recombinant, Flow Cytometry, Cytometry, Activation Assay, Infection, Titration, Marker

    Humanization of a rabbit HCMV gB-specific antibody and detection of gB expression on the surfaces of HCMV-infected cells. (A) Sequence alignment of the closest human germ lines (IGHV3-53*04), rabbit antibody 272.7, and the humanized antibody (hu272.7). The combined CDRs determined are boxed. Antibody humanization was performed by CDR grafting. (B) The humanized antibody maintained affinity and specificity for gB. The rabbit 272.7 and hu272.7 antibodies in titration were tested for binding to gB protein by ELISA. EC 50 s were deduced from four-parameter curve fitting. The statistical significance of differences between the rabbit 272.7 and hu272.7 antibodies was analyzed by two-way ANOVA. n.s., not significant ( P > 0.05). (C) Detection of gB expression on the surfaces of HCMV-infected ARPE-19 cells by a flow cytometry assay. The mean fluorescence intensities ± SD of gB-specific signals from triplicate samples are shown. The data are representative results from two independent experiments. Statistical significance was determined by the unpaired two-tailed t test. **, P
    Figure Legend Snippet: Humanization of a rabbit HCMV gB-specific antibody and detection of gB expression on the surfaces of HCMV-infected cells. (A) Sequence alignment of the closest human germ lines (IGHV3-53*04), rabbit antibody 272.7, and the humanized antibody (hu272.7). The combined CDRs determined are boxed. Antibody humanization was performed by CDR grafting. (B) The humanized antibody maintained affinity and specificity for gB. The rabbit 272.7 and hu272.7 antibodies in titration were tested for binding to gB protein by ELISA. EC 50 s were deduced from four-parameter curve fitting. The statistical significance of differences between the rabbit 272.7 and hu272.7 antibodies was analyzed by two-way ANOVA. n.s., not significant ( P > 0.05). (C) Detection of gB expression on the surfaces of HCMV-infected ARPE-19 cells by a flow cytometry assay. The mean fluorescence intensities ± SD of gB-specific signals from triplicate samples are shown. The data are representative results from two independent experiments. Statistical significance was determined by the unpaired two-tailed t test. **, P

    Techniques Used: Expressing, Infection, Sequencing, Titration, Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Fluorescence, Two Tailed Test

    Design and characterization of the bispecific antibody (BsAb). (A) Schematic depiction of the bispecific antibody with gB and CD3 specificities. (Gly 4 Ser) 3 linkers were constructed between the V H and V L domains of their respective scFvs. (B) Bispecific-antibody expression in HEK293F cells. Single-chain controls and the bispecific antibody were expressed by transfection of the scFv-K-Fc or scFv-H-Fc construct, or both, and were affinity purified with protein A. The constructs were then evaluated by SDS-PAGE under either reducing conditions (lanes 1 to 3) or nonreducing conditions (lanes 4 to 6). Lanes 1 and 4, single-chain hu272.7 scFv-K proteins; lanes 2 and 5, OKT3 scFv-H; lanes 3 and 6, the bispecific antibody. K, knob monomer; H, hole monomer; K/H, knob or hole mixed monomer; KK, knob homodimer; HH, hole homodimer; KH, knob-into-hole heterodimer. The densitometry values of the bands were analyzed by ImageJ software and are shown below the bands in lanes 4 to 6. The percentages of dimers were calculated and are shown below the gel. (C) Specificity of the BsAb for immobilized gB as determined by ELISA. Statistical significance was determined by two-way ANOVA. (D) Testing of the specificities of the BsAb (red) and the hu272.7 antibody (green) for T lymphocytes by flow cytometry. The binding signal is detected by a PE-conjugated anti-human Fc antibody.
    Figure Legend Snippet: Design and characterization of the bispecific antibody (BsAb). (A) Schematic depiction of the bispecific antibody with gB and CD3 specificities. (Gly 4 Ser) 3 linkers were constructed between the V H and V L domains of their respective scFvs. (B) Bispecific-antibody expression in HEK293F cells. Single-chain controls and the bispecific antibody were expressed by transfection of the scFv-K-Fc or scFv-H-Fc construct, or both, and were affinity purified with protein A. The constructs were then evaluated by SDS-PAGE under either reducing conditions (lanes 1 to 3) or nonreducing conditions (lanes 4 to 6). Lanes 1 and 4, single-chain hu272.7 scFv-K proteins; lanes 2 and 5, OKT3 scFv-H; lanes 3 and 6, the bispecific antibody. K, knob monomer; H, hole monomer; K/H, knob or hole mixed monomer; KK, knob homodimer; HH, hole homodimer; KH, knob-into-hole heterodimer. The densitometry values of the bands were analyzed by ImageJ software and are shown below the bands in lanes 4 to 6. The percentages of dimers were calculated and are shown below the gel. (C) Specificity of the BsAb for immobilized gB as determined by ELISA. Statistical significance was determined by two-way ANOVA. (D) Testing of the specificities of the BsAb (red) and the hu272.7 antibody (green) for T lymphocytes by flow cytometry. The binding signal is detected by a PE-conjugated anti-human Fc antibody.

    Techniques Used: Construct, Expressing, Transfection, Affinity Purification, SDS Page, Software, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Binding Assay

    38) Product Images from "Immune protection against reinfection with nonprimate hepacivirus"

    Article Title: Immune protection against reinfection with nonprimate hepacivirus

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1619380114

    Immune cell frequencies as determined by flow cytometry. PBMCs were stained with antibodies directed against CD8, CD4, CD3, PanB, CD13, CD1w2, and Mac387 to detect T lymphocytes (CD3 + CD4 + and CD3 + CD8 + ), B lymphocytes (PanB + ), CD13 + cells (expressed on
    Figure Legend Snippet: Immune cell frequencies as determined by flow cytometry. PBMCs were stained with antibodies directed against CD8, CD4, CD3, PanB, CD13, CD1w2, and Mac387 to detect T lymphocytes (CD3 + CD4 + and CD3 + CD8 + ), B lymphocytes (PanB + ), CD13 + cells (expressed on

    Techniques Used: Flow Cytometry, Cytometry, Staining

    Development of a flow cytometry-based method to measure immune cell frequencies and intracellular IFN-γ expression. PBMCs were isolated on a weekly basis postinoculation by centrifugation on a Ficoll-Hypaque density gradient and stored at −150
    Figure Legend Snippet: Development of a flow cytometry-based method to measure immune cell frequencies and intracellular IFN-γ expression. PBMCs were isolated on a weekly basis postinoculation by centrifugation on a Ficoll-Hypaque density gradient and stored at −150

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Isolation, Centrifugation

    39) Product Images from "Experimental abdominal aortic aneurysm formation is mediated by IL-17 and attenuated by mesenchymal stem cell treatment"

    Article Title: Experimental abdominal aortic aneurysm formation is mediated by IL-17 and attenuated by mesenchymal stem cell treatment

    Journal: Circulation

    doi: 10.1161/CIRCULATIONAHA.111.083451

    CD4+ T cell infiltration and IL-17 production is increased in aortas from elastase-perfused WT mice. Flow cytometry analysis of cell counts was performed on aortic tissue from control or elastase-perfused WT mice on days 3, 7 and 14. Results are shown as percentage of cell numbers compared to WT controls; n=4–5 mice/group; *p
    Figure Legend Snippet: CD4+ T cell infiltration and IL-17 production is increased in aortas from elastase-perfused WT mice. Flow cytometry analysis of cell counts was performed on aortic tissue from control or elastase-perfused WT mice on days 3, 7 and 14. Results are shown as percentage of cell numbers compared to WT controls; n=4–5 mice/group; *p

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry

    40) Product Images from "Efficient Delivery of Human Cytomegalovirus T Cell Antigens by Attenuated Sendai Virus Vectors"

    Article Title: Efficient Delivery of Human Cytomegalovirus T Cell Antigens by Attenuated Sendai Virus Vectors

    Journal: Journal of Virology

    doi: 10.1128/JVI.00569-18

    SeV efficiently infects moDCs with rcSeV, eliciting higher transgene expression than rdSeV. MoDCs from 3 different HCMV seronegative blood donors were infected at the indicated MOIs, and the intracellular presence of the transgenes GFP, IE-1, and pp65 was quantified via flow cytometry after 24 (A and B) and 48 h (C and D) (nd, not determined). (A and C) Results are presented as the percentage of cells positive for a given antigen, with connected lines indicating values that were obtained using cells from an individual donor. (B and D) Median fluorescence intensity (MFI) values were normalized to the signals obtained from uninfected cells, with bars representing the means and standard deviations of values from all donors.
    Figure Legend Snippet: SeV efficiently infects moDCs with rcSeV, eliciting higher transgene expression than rdSeV. MoDCs from 3 different HCMV seronegative blood donors were infected at the indicated MOIs, and the intracellular presence of the transgenes GFP, IE-1, and pp65 was quantified via flow cytometry after 24 (A and B) and 48 h (C and D) (nd, not determined). (A and C) Results are presented as the percentage of cells positive for a given antigen, with connected lines indicating values that were obtained using cells from an individual donor. (B and D) Median fluorescence intensity (MFI) values were normalized to the signals obtained from uninfected cells, with bars representing the means and standard deviations of values from all donors.

    Techniques Used: Expressing, Infection, Flow Cytometry, Cytometry, Fluorescence

    Infection with both Sendai vectors leads to efficient restimulation of T cells by infected moDCs. (A) Direct presentation assay. moDCs from 3 different HCMV seronegative blood donors were infected at the indicated MOIs with IE-1- or pp65-expressing vectors. At 24 hpi, antigen-specific T cell clones were added at an effector/target ratio of 1:1. After 6 h of cocultivation in the presence of brefeldin A (BFA), cells were stained for CD8 and intracellular IFN-γ and analyzed via flow cytometry. (B) Removal of extracellular virions before cross presentation: HeLa cells were transduced at an MOI of 10 with rdSeV-pp65. At 24 hpi, the supernatant from the overnight culture (lane 0) as well as 4 subsequent washing steps with cell culture medium (lanes 1 to 4) was collected and added to moDCs. Twenty-four h later, a pp65-specific, HLA-matched T cell clone was added to moDCs at an effector/target ratio of 1:1. After 6 h in the presence of BFA, CD8/IFN-γ staining and flow cytometry analysis were performed. (C) Cross-presentation assay. HeLa cells were transduced at the indicated MOIs with rdSeV-pp65. At 24 hpi, cells were washed 4 times and added to moDCs from 3 individual donors at a 1:1 ratio. After 24 h of cocultivation, an antigen-specific T cell clone was added for a HeLa/DC/T cell ratio of 1:1:1. T cell restimulation was measured after 6 h as described for panel A. Bars represent the means and standard deviations of values from all donors (A and C) or 3 independent experiments (B) (nd, not determined).
    Figure Legend Snippet: Infection with both Sendai vectors leads to efficient restimulation of T cells by infected moDCs. (A) Direct presentation assay. moDCs from 3 different HCMV seronegative blood donors were infected at the indicated MOIs with IE-1- or pp65-expressing vectors. At 24 hpi, antigen-specific T cell clones were added at an effector/target ratio of 1:1. After 6 h of cocultivation in the presence of brefeldin A (BFA), cells were stained for CD8 and intracellular IFN-γ and analyzed via flow cytometry. (B) Removal of extracellular virions before cross presentation: HeLa cells were transduced at an MOI of 10 with rdSeV-pp65. At 24 hpi, the supernatant from the overnight culture (lane 0) as well as 4 subsequent washing steps with cell culture medium (lanes 1 to 4) was collected and added to moDCs. Twenty-four h later, a pp65-specific, HLA-matched T cell clone was added to moDCs at an effector/target ratio of 1:1. After 6 h in the presence of BFA, CD8/IFN-γ staining and flow cytometry analysis were performed. (C) Cross-presentation assay. HeLa cells were transduced at the indicated MOIs with rdSeV-pp65. At 24 hpi, cells were washed 4 times and added to moDCs from 3 individual donors at a 1:1 ratio. After 24 h of cocultivation, an antigen-specific T cell clone was added for a HeLa/DC/T cell ratio of 1:1:1. T cell restimulation was measured after 6 h as described for panel A. Bars represent the means and standard deviations of values from all donors (A and C) or 3 independent experiments (B) (nd, not determined).

    Techniques Used: Infection, Expressing, Clone Assay, Staining, Flow Cytometry, Cytometry, Cell Culture

    Attenuated rdSeV is less cytotoxic than rcSeV. moDCs from 3 different HCMV seronegative blood donors were infected at the indicated MOIs with IE-1- or pp65-expressing vectors. After 24 or 48 h, samples were costained with annexin V/7-AAD, and cells positive for one or both markers were quantified by flow cytometry. Bars represent the means and standard deviations (SD) of values from all donors (nd, not determined).
    Figure Legend Snippet: Attenuated rdSeV is less cytotoxic than rcSeV. moDCs from 3 different HCMV seronegative blood donors were infected at the indicated MOIs with IE-1- or pp65-expressing vectors. After 24 or 48 h, samples were costained with annexin V/7-AAD, and cells positive for one or both markers were quantified by flow cytometry. Bars represent the means and standard deviations (SD) of values from all donors (nd, not determined).

    Techniques Used: Infection, Expressing, Flow Cytometry, Cytometry

    SeV is capable of infecting T cells, NK cells, and monocytes. (A) Gating strategy for discriminating different leukocyte populations. PBMCs from 3 different HCMV seronegative blood donors were infected at an MOI of 1 or 10 with rcSeV-GFP. (B) At 24 hpi, the amount of GFP-positive cells in the indicated populations was determined by flow cytometry. Bars represent the means and standard deviations (SD) of values from all donors.
    Figure Legend Snippet: SeV is capable of infecting T cells, NK cells, and monocytes. (A) Gating strategy for discriminating different leukocyte populations. PBMCs from 3 different HCMV seronegative blood donors were infected at an MOI of 1 or 10 with rcSeV-GFP. (B) At 24 hpi, the amount of GFP-positive cells in the indicated populations was determined by flow cytometry. Bars represent the means and standard deviations (SD) of values from all donors.

    Techniques Used: Infection, Flow Cytometry, Cytometry

    SeV induces maturation of dendritic cells. moDCs from 3 different HCMV seronegative blood donors were infected at the indicated MOIs with IE-1- or pp65-expressing vectors. After 24 or 48 h, samples were stained for CD80, CD83, CD86, or HLA-DR and analyzed via flow cytometry. Obtained median fluorescence intensity (MFI) values were normalized to those of uninfected cells. Log 2 values which represent the means (± standard deviations) from all donors are displayed in a heatmap indicating upregulation (blue) or downregulation (red) of a given marker (nd, not determined).
    Figure Legend Snippet: SeV induces maturation of dendritic cells. moDCs from 3 different HCMV seronegative blood donors were infected at the indicated MOIs with IE-1- or pp65-expressing vectors. After 24 or 48 h, samples were stained for CD80, CD83, CD86, or HLA-DR and analyzed via flow cytometry. Obtained median fluorescence intensity (MFI) values were normalized to those of uninfected cells. Log 2 values which represent the means (± standard deviations) from all donors are displayed in a heatmap indicating upregulation (blue) or downregulation (red) of a given marker (nd, not determined).

    Techniques Used: Infection, Expressing, Staining, Flow Cytometry, Cytometry, Fluorescence, Marker

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    Flow Cytometry:

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    Fluorescence:

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    Centrifugation:

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    Cell Culture:

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    Cytometry:

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    Microarray:

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    Incubation:

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    Expressing:

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    FACS:

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    Staining:

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    Thermo Fisher click it edu flow cytometry cell proliferation assay
    Loss of USP22 in cancer cells results in G1-phase cell cycle arrest. H1299 human lung cancer cells depleted of USP22 via infection with an shRNA-encoding lentivirus (or a luciferase shRNA as a control). After selection, cells counted and plated at 80,000 cells/mL on day 2 postinfection. ( A ) Cell number determined by direct counting of triplicate wells in a six-well plate via hemocytometer at the indicated time points. ( B ) Colony growth assessed via fixation and methylene blue staining of foci at day 7 postinfection. ( C ) Efficient knockdown of USP22 confirmed by both qRT-PCR and immunoblot (IB). ( D ) Cell viability quantified via flow <t>cytometry.</t> ( E ) Quantification of three experimental replicates representing average and SD of percent cell death, based on permeability. ( F ) Apoptosis measured by Annexin V-PE and 7AAD DNA staining, quantified by flow cytometry. ( G ) Quantification of three experimental replicates representing average and SD of percent apoptosis based on population of Annexin V-PE + cells. NS, not significant. ( H ) PARP and CASPASE-3 (CAS-3) cleavage demonstrated by IB. The black arrowhead indicates cleaved species. ( I ) Progression through the cell cycle determined by <t>EdU</t> incorporation and PI staining followed by flow cytometry; the percent of cells in G1 phase is represented in blue. ( J ) Quantification of three experimental replicates of cell cycle phase distribution. Error bars indicate SD based on three independent experiments. * P
    Click It Edu Flow Cytometry Cell Proliferation Assay, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mir 218 expression analysis
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    Thermo Fisher apoptosis analysis
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    Thermo Fisher flow cytometric analysis
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    Loss of USP22 in cancer cells results in G1-phase cell cycle arrest. H1299 human lung cancer cells depleted of USP22 via infection with an shRNA-encoding lentivirus (or a luciferase shRNA as a control). After selection, cells counted and plated at 80,000 cells/mL on day 2 postinfection. ( A ) Cell number determined by direct counting of triplicate wells in a six-well plate via hemocytometer at the indicated time points. ( B ) Colony growth assessed via fixation and methylene blue staining of foci at day 7 postinfection. ( C ) Efficient knockdown of USP22 confirmed by both qRT-PCR and immunoblot (IB). ( D ) Cell viability quantified via flow cytometry. ( E ) Quantification of three experimental replicates representing average and SD of percent cell death, based on permeability. ( F ) Apoptosis measured by Annexin V-PE and 7AAD DNA staining, quantified by flow cytometry. ( G ) Quantification of three experimental replicates representing average and SD of percent apoptosis based on population of Annexin V-PE + cells. NS, not significant. ( H ) PARP and CASPASE-3 (CAS-3) cleavage demonstrated by IB. The black arrowhead indicates cleaved species. ( I ) Progression through the cell cycle determined by EdU incorporation and PI staining followed by flow cytometry; the percent of cells in G1 phase is represented in blue. ( J ) Quantification of three experimental replicates of cell cycle phase distribution. Error bars indicate SD based on three independent experiments. * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Control of CCND1 ubiquitylation by the catalytic SAGA subunit USP22 is essential for cell cycle progression through G1 in cancer cells

    doi: 10.1073/pnas.1807704115

    Figure Lengend Snippet: Loss of USP22 in cancer cells results in G1-phase cell cycle arrest. H1299 human lung cancer cells depleted of USP22 via infection with an shRNA-encoding lentivirus (or a luciferase shRNA as a control). After selection, cells counted and plated at 80,000 cells/mL on day 2 postinfection. ( A ) Cell number determined by direct counting of triplicate wells in a six-well plate via hemocytometer at the indicated time points. ( B ) Colony growth assessed via fixation and methylene blue staining of foci at day 7 postinfection. ( C ) Efficient knockdown of USP22 confirmed by both qRT-PCR and immunoblot (IB). ( D ) Cell viability quantified via flow cytometry. ( E ) Quantification of three experimental replicates representing average and SD of percent cell death, based on permeability. ( F ) Apoptosis measured by Annexin V-PE and 7AAD DNA staining, quantified by flow cytometry. ( G ) Quantification of three experimental replicates representing average and SD of percent apoptosis based on population of Annexin V-PE + cells. NS, not significant. ( H ) PARP and CASPASE-3 (CAS-3) cleavage demonstrated by IB. The black arrowhead indicates cleaved species. ( I ) Progression through the cell cycle determined by EdU incorporation and PI staining followed by flow cytometry; the percent of cells in G1 phase is represented in blue. ( J ) Quantification of three experimental replicates of cell cycle phase distribution. Error bars indicate SD based on three independent experiments. * P

    Article Snippet: Cell cycle analysis was conducted using the Click-iT EdU flow cytometry cell proliferation assay where cells were labeled with EdU for 2 h, harvested, and stained according to the manufacterer’s instructions (Thermo Fisher).

    Techniques: Infection, shRNA, Luciferase, Selection, Staining, Quantitative RT-PCR, Flow Cytometry, Cytometry, Permeability

    Expressions of miR-218 and Runx2 in PTC tissues. Notes: qRT-PCR was performed to detect the expressions of ( A ) miR-218 and ( B ) Runx2 mRNA in PTC tissues and matched adjacent normal tissues. ( C ) Correlation analysis between miR-218 and Runx2 mRNA expressions in PTC tissues. Abbreviations: PTC, papillary thyroid cancer; qRT-PCR, quantitative real-time PCR.

    Journal: OncoTargets and therapy

    Article Title: miR-218 overexpression suppresses tumorigenesis of papillary thyroid cancer via inactivation of PTEN/PI3K/AKT pathway by targeting Runx2

    doi: 10.2147/OTT.S172152

    Figure Lengend Snippet: Expressions of miR-218 and Runx2 in PTC tissues. Notes: qRT-PCR was performed to detect the expressions of ( A ) miR-218 and ( B ) Runx2 mRNA in PTC tissues and matched adjacent normal tissues. ( C ) Correlation analysis between miR-218 and Runx2 mRNA expressions in PTC tissues. Abbreviations: PTC, papillary thyroid cancer; qRT-PCR, quantitative real-time PCR.

    Article Snippet: For miR-218 expression analysis, miRNAs were reversely transcribed using the TaqMan MiRNA Reverse Transcript Kit (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    miR-218 inhibits EMT of PTC cells. Notes: Western blot was conducted to determine the expression levels of E-cadherin, N-cadherin, and vimentin in TPC-1 cells transfected with miR-control or miR-218 mimic. * P

    Journal: OncoTargets and therapy

    Article Title: miR-218 overexpression suppresses tumorigenesis of papillary thyroid cancer via inactivation of PTEN/PI3K/AKT pathway by targeting Runx2

    doi: 10.2147/OTT.S172152

    Figure Lengend Snippet: miR-218 inhibits EMT of PTC cells. Notes: Western blot was conducted to determine the expression levels of E-cadherin, N-cadherin, and vimentin in TPC-1 cells transfected with miR-control or miR-218 mimic. * P

    Article Snippet: For miR-218 expression analysis, miRNAs were reversely transcribed using the TaqMan MiRNA Reverse Transcript Kit (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Western Blot, Expressing, Transfection

    miR-218 directly targets Runx2 and regulates its expression in PTC cells. Notes: ( A ) Diagram of the binding sites between miR-218 and Runx2 3′UTR. ( B ) The relative luciferase activity was measured in K-1 and TPC-1 cells after transfection with miR-218 or miR-control and luciferase reporter vectors with wild-type (WT) or mutant (MUT) Runx2 3′UTR. ( C ) Western blot analysis of Runx2 expression level in K-1 and TPC-1 cells transfected with miR-218 mimic, miR-218 inhibitor, or miR-control. * P

    Journal: OncoTargets and therapy

    Article Title: miR-218 overexpression suppresses tumorigenesis of papillary thyroid cancer via inactivation of PTEN/PI3K/AKT pathway by targeting Runx2

    doi: 10.2147/OTT.S172152

    Figure Lengend Snippet: miR-218 directly targets Runx2 and regulates its expression in PTC cells. Notes: ( A ) Diagram of the binding sites between miR-218 and Runx2 3′UTR. ( B ) The relative luciferase activity was measured in K-1 and TPC-1 cells after transfection with miR-218 or miR-control and luciferase reporter vectors with wild-type (WT) or mutant (MUT) Runx2 3′UTR. ( C ) Western blot analysis of Runx2 expression level in K-1 and TPC-1 cells transfected with miR-218 mimic, miR-218 inhibitor, or miR-control. * P

    Article Snippet: For miR-218 expression analysis, miRNAs were reversely transcribed using the TaqMan MiRNA Reverse Transcript Kit (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Expressing, Binding Assay, Luciferase, Activity Assay, Transfection, Mutagenesis, Western Blot

    miR-218 exerts anticancer effects in PTC via inactivation of PTEN/PI3K/AKT pathway by targeting Runx2 in vitro. Notes: After TPC-1 cells were transfected with miR-218 alone or in combination with pcDNA-Runx2, cell viability, invasion, and apoptosis were examined by ( A ) CCK-8 assay, ( B ) Transwell invasion assay, and ( C ) flow cytometry analysis, respectively. ( D and E ) The protein levels of phosphorylated AKT (p-AKT) (Ser473), total AKT (t-AKT), and PTEN were determined by Western blot in TPC-1 cells transfected with si-Runx2, miR-218 , miR-218 + pcDNA-Runx2, or respective controls blot. * P

    Journal: OncoTargets and therapy

    Article Title: miR-218 overexpression suppresses tumorigenesis of papillary thyroid cancer via inactivation of PTEN/PI3K/AKT pathway by targeting Runx2

    doi: 10.2147/OTT.S172152

    Figure Lengend Snippet: miR-218 exerts anticancer effects in PTC via inactivation of PTEN/PI3K/AKT pathway by targeting Runx2 in vitro. Notes: After TPC-1 cells were transfected with miR-218 alone or in combination with pcDNA-Runx2, cell viability, invasion, and apoptosis were examined by ( A ) CCK-8 assay, ( B ) Transwell invasion assay, and ( C ) flow cytometry analysis, respectively. ( D and E ) The protein levels of phosphorylated AKT (p-AKT) (Ser473), total AKT (t-AKT), and PTEN were determined by Western blot in TPC-1 cells transfected with si-Runx2, miR-218 , miR-218 + pcDNA-Runx2, or respective controls blot. * P

    Article Snippet: For miR-218 expression analysis, miRNAs were reversely transcribed using the TaqMan MiRNA Reverse Transcript Kit (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: In Vitro, Transfection, CCK-8 Assay, Transwell Invasion Assay, Flow Cytometry, Cytometry, Western Blot

    A schematic model of the mechanism of miR-218 in PTC. Abbreviation: PTC, papillary thyroid cancer.

    Journal: OncoTargets and therapy

    Article Title: miR-218 overexpression suppresses tumorigenesis of papillary thyroid cancer via inactivation of PTEN/PI3K/AKT pathway by targeting Runx2

    doi: 10.2147/OTT.S172152

    Figure Lengend Snippet: A schematic model of the mechanism of miR-218 in PTC. Abbreviation: PTC, papillary thyroid cancer.

    Article Snippet: For miR-218 expression analysis, miRNAs were reversely transcribed using the TaqMan MiRNA Reverse Transcript Kit (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques:

    Expressions of miR-218 and Runx2 in PTC cells. Notes: ( A ) The expressions of miR-218 in PTC cell lines (TPC-1 and K-1) and human thyroid epithelial cell line Nthy-ori3-1 were evaluated by qRT-PCR. The expression levels of Runx2 at ( B ) mRNA and ( C ) protein levels in TPC-1, K-1, and Nthy-ori3-1 cells were assessed by qRT-PCR and Western blot. * P

    Journal: OncoTargets and therapy

    Article Title: miR-218 overexpression suppresses tumorigenesis of papillary thyroid cancer via inactivation of PTEN/PI3K/AKT pathway by targeting Runx2

    doi: 10.2147/OTT.S172152

    Figure Lengend Snippet: Expressions of miR-218 and Runx2 in PTC cells. Notes: ( A ) The expressions of miR-218 in PTC cell lines (TPC-1 and K-1) and human thyroid epithelial cell line Nthy-ori3-1 were evaluated by qRT-PCR. The expression levels of Runx2 at ( B ) mRNA and ( C ) protein levels in TPC-1, K-1, and Nthy-ori3-1 cells were assessed by qRT-PCR and Western blot. * P

    Article Snippet: For miR-218 expression analysis, miRNAs were reversely transcribed using the TaqMan MiRNA Reverse Transcript Kit (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Quantitative RT-PCR, Expressing, Western Blot

    miR-218 overexpression inhibits PTC tumor growth in vivo. Notes: TPC-1 cells transfected with lentiviral vectors containing miR-218 or miR-control were injected into nude mice. The mice ( A ) body weights and ( B ) tumor volumes were monitored every 5 days. ( C ) The mice were killed after 35 days, and the tumor weights were measured. The expression of Runx2 at mRNA and protein levels in removed tumors was assessed by ( D ) qRT-PCR and ( E ) Western blot. ( F ) The protein levels of phosphorylated AKT (p-AKT) (Ser473), PTEN, and total AKT (t-AKT) in excised tumors were determined by Western blot. ( G ) The protein levels of p-mTOR, t-mTOR, p-GSK-3β, and t-GSK-3β in excised tumors were examined by Western blot. * P

    Journal: OncoTargets and therapy

    Article Title: miR-218 overexpression suppresses tumorigenesis of papillary thyroid cancer via inactivation of PTEN/PI3K/AKT pathway by targeting Runx2

    doi: 10.2147/OTT.S172152

    Figure Lengend Snippet: miR-218 overexpression inhibits PTC tumor growth in vivo. Notes: TPC-1 cells transfected with lentiviral vectors containing miR-218 or miR-control were injected into nude mice. The mice ( A ) body weights and ( B ) tumor volumes were monitored every 5 days. ( C ) The mice were killed after 35 days, and the tumor weights were measured. The expression of Runx2 at mRNA and protein levels in removed tumors was assessed by ( D ) qRT-PCR and ( E ) Western blot. ( F ) The protein levels of phosphorylated AKT (p-AKT) (Ser473), PTEN, and total AKT (t-AKT) in excised tumors were determined by Western blot. ( G ) The protein levels of p-mTOR, t-mTOR, p-GSK-3β, and t-GSK-3β in excised tumors were examined by Western blot. * P

    Article Snippet: For miR-218 expression analysis, miRNAs were reversely transcribed using the TaqMan MiRNA Reverse Transcript Kit (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Over Expression, In Vivo, Transfection, Injection, Mouse Assay, Expressing, Quantitative RT-PCR, Western Blot

    Effects of miR-218 overexpression on tumorigenesis of PTC in vitro. Notes: K-1 and TPC-1 cells were transfected with miR-218 or miR-control, and further experiments were performed at 24 or 48 hours post-transfection. ( A ) qRT-PCR analysis of miR-218 expression in transfected K-1 and TPC-1 cells. ( B ) CCK-8 assay was conducted in transfected K-1 and TPC-1 cells to measure the cell viability. Cell invasive ability was examined by Transwell invasion assay in transfected ( C ) K-1 and ( D ) TPC-1 cells (magnification ×200). Flow cytometry analysis of apoptosis in transfected ( E ) K-1 and ( F ) TPC-1 cells. * P

    Journal: OncoTargets and therapy

    Article Title: miR-218 overexpression suppresses tumorigenesis of papillary thyroid cancer via inactivation of PTEN/PI3K/AKT pathway by targeting Runx2

    doi: 10.2147/OTT.S172152

    Figure Lengend Snippet: Effects of miR-218 overexpression on tumorigenesis of PTC in vitro. Notes: K-1 and TPC-1 cells were transfected with miR-218 or miR-control, and further experiments were performed at 24 or 48 hours post-transfection. ( A ) qRT-PCR analysis of miR-218 expression in transfected K-1 and TPC-1 cells. ( B ) CCK-8 assay was conducted in transfected K-1 and TPC-1 cells to measure the cell viability. Cell invasive ability was examined by Transwell invasion assay in transfected ( C ) K-1 and ( D ) TPC-1 cells (magnification ×200). Flow cytometry analysis of apoptosis in transfected ( E ) K-1 and ( F ) TPC-1 cells. * P

    Article Snippet: For miR-218 expression analysis, miRNAs were reversely transcribed using the TaqMan MiRNA Reverse Transcript Kit (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Over Expression, In Vitro, Transfection, Quantitative RT-PCR, Expressing, CCK-8 Assay, Transwell Invasion Assay, Flow Cytometry, Cytometry

    Effects of lincRNA-p21 on lung cancer cell proliferation, apoptosis and migration. (A) A549 and PC9 cells were transfected with lincRNA-p21 overexpression plasmid or lincRNA-p21 siRNA for 72 h, and the cell proliferation was detected using a Cell Counting Kit-8 assay. (B) The cells were transfected with lincRNA-p21 overexpression plasmid or lincRNA-p21 siRNA for 72 h, and cell apoptosis was detected by flow cytometric analysis. (C) The transfected cells were subjected to Transwell analysis for 24 h (magnification ×200). *P

    Journal: Oncology Reports

    Article Title: lincRNA-p21 inhibits the progression of non-small cell lung cancer via targeting miR-17-5p

    doi: 10.3892/or.2018.6900

    Figure Lengend Snippet: Effects of lincRNA-p21 on lung cancer cell proliferation, apoptosis and migration. (A) A549 and PC9 cells were transfected with lincRNA-p21 overexpression plasmid or lincRNA-p21 siRNA for 72 h, and the cell proliferation was detected using a Cell Counting Kit-8 assay. (B) The cells were transfected with lincRNA-p21 overexpression plasmid or lincRNA-p21 siRNA for 72 h, and cell apoptosis was detected by flow cytometric analysis. (C) The transfected cells were subjected to Transwell analysis for 24 h (magnification ×200). *P

    Article Snippet: Apoptosis analysis An Annexin V-FITC and propidium iodide (PI) staining kit (Dead Cell Apoptosis kit with Annexin V Alexa Fluor™ 488 & PI; Thermo Fisher Scientific, Inc.) was used to detect the cell apoptosis according to the manufacturer's protocol.

    Techniques: Migration, Transfection, Over Expression, Plasmid Preparation, Cell Counting, Flow Cytometry

    Effects of miR-17-5p on lung cancer cell proliferation, apoptosis and migration. (A) lincRNA-p21 overexpression plasmid- or lincRNA-p21 siRNA-transfected lung cancer cells were treated miR-17-5p mimics or inhibitor for 72 h, and cell proliferation was detected using a Cell Counting Kit-8 assay. (B) lincRNA-p21 overexpression plasmid- or lincRNA-p21 siRNA-transfected lung cancer cells were treated miR-17-5p mimics or inhibitor for 72 h, and the cells apoptosis were detected by flow cytometric analysis. (C) lincRNA-p21 overexpression plasmid- or lincRNA-p21 siRNA-transfected lung cancer cells were treated miR-17-5p mimics or inhibitor for 24 h, and the cells were subjected to Transwell analysis (magnification, ×200). *P

    Journal: Oncology Reports

    Article Title: lincRNA-p21 inhibits the progression of non-small cell lung cancer via targeting miR-17-5p

    doi: 10.3892/or.2018.6900

    Figure Lengend Snippet: Effects of miR-17-5p on lung cancer cell proliferation, apoptosis and migration. (A) lincRNA-p21 overexpression plasmid- or lincRNA-p21 siRNA-transfected lung cancer cells were treated miR-17-5p mimics or inhibitor for 72 h, and cell proliferation was detected using a Cell Counting Kit-8 assay. (B) lincRNA-p21 overexpression plasmid- or lincRNA-p21 siRNA-transfected lung cancer cells were treated miR-17-5p mimics or inhibitor for 72 h, and the cells apoptosis were detected by flow cytometric analysis. (C) lincRNA-p21 overexpression plasmid- or lincRNA-p21 siRNA-transfected lung cancer cells were treated miR-17-5p mimics or inhibitor for 24 h, and the cells were subjected to Transwell analysis (magnification, ×200). *P

    Article Snippet: Apoptosis analysis An Annexin V-FITC and propidium iodide (PI) staining kit (Dead Cell Apoptosis kit with Annexin V Alexa Fluor™ 488 & PI; Thermo Fisher Scientific, Inc.) was used to detect the cell apoptosis according to the manufacturer's protocol.

    Techniques: Migration, Over Expression, Plasmid Preparation, Transfection, Cell Counting, Flow Cytometry

    CART transfection outperforms electroporation and causes minimal reconfiguration of NK cell phenotype. NK cells (500,000 cells per well) were transfected with GFP-encoding mRNA via L2000 (31 ng mRNA/well), electroporation (31 ng/well (Elec) or 10,0000 ng mRNA/well (Elec high)), or CART ( 2 ) (31 ng mRNA/well). Flow cytometric analysis of NK cell viability (A) and transfection efficacy (B) 18 hours post-transfection with GFP-encoding mRNA. C) UMAP dimensionality reduction of mass cytometry data faceted by treatment condition. D) Heatmap of mean marker expression, with samples and markers hierarchically clustered.

    Journal: bioRxiv

    Article Title: Charge-Altering Releasable Transporters Enable Specific Phenotypic Manipulation of Resting Primary Natural Killer Cells

    doi: 10.1101/2020.02.28.970491

    Figure Lengend Snippet: CART transfection outperforms electroporation and causes minimal reconfiguration of NK cell phenotype. NK cells (500,000 cells per well) were transfected with GFP-encoding mRNA via L2000 (31 ng mRNA/well), electroporation (31 ng/well (Elec) or 10,0000 ng mRNA/well (Elec high)), or CART ( 2 ) (31 ng mRNA/well). Flow cytometric analysis of NK cell viability (A) and transfection efficacy (B) 18 hours post-transfection with GFP-encoding mRNA. C) UMAP dimensionality reduction of mass cytometry data faceted by treatment condition. D) Heatmap of mean marker expression, with samples and markers hierarchically clustered.

    Article Snippet: Flow cytometric analysis of transfection efficacy 6 hours post-treatment with CART/mRNA polyplexes, NK cells were stained with LIVE/DEAD™ Fixable Yellow Staining Kit (Thermo Fisher Scientific) for 20 minutes at room temperature, then stained with anti-CD3-PE (BioLegend, Clone UCHT1), anti-CD14-APC (BioLegend, Clone HCD14), anti-CD16-PerCP/Cy5.5 (BioLegend, Clone 3G8), and anti-CD56-PE-Cy7 (BioLegend, Clone HCD56) for 20 minutes at room temperature.

    Techniques: Transfection, Electroporation, Mass Cytometry, Marker, Expressing

    CART-mediated transfection of anti-CD19 CAR generates cytotoxic human CAR NK cells. Isolated primary resting human NK cells were transfected with an mRNA encoding a anti-human CD19-41BB-CD3ζ CAR (hCAR). The cells were incubated for a total of 18 hours before being co-cultured with Raji (■), wild-type Nalm6 (●), or CD19KO Nalm6 (○) target cells for 6 hours followed by flow cytometric analysis. A) anti-human CD19 CAR (hCAR) expression by NK cells in the absence of target cells, as detected by FITC-conjugated soluble human CD19. B) Percentage of dead Raji cells after E:T 10:1 co-culture with transfected NK cells. C) Representative flow cytometry plots of CD107a, IFNγ, and TNFα between BDK − and BDK + populations of CART-transfected NK cells co-cultured with Raji cells at E:T 1:3. This analysis is quantified across all n = 11 donors in (D) . E) Percentage of dead wild-type or CD19KO Nalm6 cells after E:T 20:1 co-culture with transfected NK cells. F) CD107a, IFNγ, and TNFα expression after E:T 1:3 co-culture with Nalm6 cells. For E) and F), n = 8. For all panels, *, p

    Journal: bioRxiv

    Article Title: Charge-Altering Releasable Transporters Enable Specific Phenotypic Manipulation of Resting Primary Natural Killer Cells

    doi: 10.1101/2020.02.28.970491

    Figure Lengend Snippet: CART-mediated transfection of anti-CD19 CAR generates cytotoxic human CAR NK cells. Isolated primary resting human NK cells were transfected with an mRNA encoding a anti-human CD19-41BB-CD3ζ CAR (hCAR). The cells were incubated for a total of 18 hours before being co-cultured with Raji (■), wild-type Nalm6 (●), or CD19KO Nalm6 (○) target cells for 6 hours followed by flow cytometric analysis. A) anti-human CD19 CAR (hCAR) expression by NK cells in the absence of target cells, as detected by FITC-conjugated soluble human CD19. B) Percentage of dead Raji cells after E:T 10:1 co-culture with transfected NK cells. C) Representative flow cytometry plots of CD107a, IFNγ, and TNFα between BDK − and BDK + populations of CART-transfected NK cells co-cultured with Raji cells at E:T 1:3. This analysis is quantified across all n = 11 donors in (D) . E) Percentage of dead wild-type or CD19KO Nalm6 cells after E:T 20:1 co-culture with transfected NK cells. F) CD107a, IFNγ, and TNFα expression after E:T 1:3 co-culture with Nalm6 cells. For E) and F), n = 8. For all panels, *, p

    Article Snippet: Flow cytometric analysis of transfection efficacy 6 hours post-treatment with CART/mRNA polyplexes, NK cells were stained with LIVE/DEAD™ Fixable Yellow Staining Kit (Thermo Fisher Scientific) for 20 minutes at room temperature, then stained with anti-CD3-PE (BioLegend, Clone UCHT1), anti-CD14-APC (BioLegend, Clone HCD14), anti-CD16-PerCP/Cy5.5 (BioLegend, Clone 3G8), and anti-CD56-PE-Cy7 (BioLegend, Clone HCD56) for 20 minutes at room temperature.

    Techniques: Transfection, Isolation, Incubation, Cell Culture, Expressing, Co-Culture Assay, Flow Cytometry