Structured Review

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FL118 targets both proliferative cancer cells and latent stem cell-like cancer cells: a , FL118-cisplatin combination enhances killing of <t>PANC1</t> cells. Dead cells were determined by trypan blue exclusion. b , GFPcODC-positive cells survive 50-□M cisplatin for 4 days, evaluated with trypan blue exclusion assay. c , FL118-cisplatin kills GFPcODC positive (drug-resistant) cells (arrows). Phase-contrast and GFP imaging of cells were digitally taken. d , FL118-cisplatin combination reduces PANC1 spheroid cell number. *, P
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1) Product Images from "An ABCG2 non-substrate anticancer agent FL118 targets drug-resistant cancer stem-like cells and overcomes treatment resistance of human pancreatic cancer"

Article Title: An ABCG2 non-substrate anticancer agent FL118 targets drug-resistant cancer stem-like cells and overcomes treatment resistance of human pancreatic cancer

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-018-0899-8

FL118 targets both proliferative cancer cells and latent stem cell-like cancer cells: a , FL118-cisplatin combination enhances killing of PANC1 cells. Dead cells were determined by trypan blue exclusion. b , GFPcODC-positive cells survive 50-□M cisplatin for 4 days, evaluated with trypan blue exclusion assay. c , FL118-cisplatin kills GFPcODC positive (drug-resistant) cells (arrows). Phase-contrast and GFP imaging of cells were digitally taken. d , FL118-cisplatin combination reduces PANC1 spheroid cell number. *, P
Figure Legend Snippet: FL118 targets both proliferative cancer cells and latent stem cell-like cancer cells: a , FL118-cisplatin combination enhances killing of PANC1 cells. Dead cells were determined by trypan blue exclusion. b , GFPcODC-positive cells survive 50-□M cisplatin for 4 days, evaluated with trypan blue exclusion assay. c , FL118-cisplatin kills GFPcODC positive (drug-resistant) cells (arrows). Phase-contrast and GFP imaging of cells were digitally taken. d , FL118-cisplatin combination reduces PANC1 spheroid cell number. *, P

Techniques Used: Trypan Blue Exclusion Assay, Imaging

FL118 downregulates the expression of multiple antiapoptotic proteins and upregulates certain proapoptotic proteins in pancreatic cancer cells: Subconfluent pancreatic cancer cells were treated with FL118 as shown, and the expression of antiapoptotic proteins survivin, Mcl-1, XIAP, and cIAP2 as well as proapoptotic proteins Bad, Bim and Bax was detected by western blots using corresponding antibodies for each protein. GAPDH is an internal control for protein loading. The result from PANC1 pancreatic cancer cell line is shown in a , and the result from MIA PaCa2 (Mia2) pancreatic cancer cell line is shown in ( b )
Figure Legend Snippet: FL118 downregulates the expression of multiple antiapoptotic proteins and upregulates certain proapoptotic proteins in pancreatic cancer cells: Subconfluent pancreatic cancer cells were treated with FL118 as shown, and the expression of antiapoptotic proteins survivin, Mcl-1, XIAP, and cIAP2 as well as proapoptotic proteins Bad, Bim and Bax was detected by western blots using corresponding antibodies for each protein. GAPDH is an internal control for protein loading. The result from PANC1 pancreatic cancer cell line is shown in a , and the result from MIA PaCa2 (Mia2) pancreatic cancer cell line is shown in ( b )

Techniques Used: Expressing, Western Blot

Induction of apoptosis, pancreatic cancer cell killing and cell viability inhibition by FL118: a and b , FL118 treatment results in activation of caspase-3 and cleavage of PARP. Subconfluent pancreatic cancer cells (A, PANC1; B, MIA PaCa2) were treated with FL118 as shown, and the activation of casepase-3 and cleavage of PARP were detected by western blots. GAPDG is the internal control for total protein loading. c , FL118 induces pancreatic cancer cell death. Subconfluent PANC1 and Mia2 pancreatic cancer cells were treated with vehicle or with FL118 at 10, 10 and 500 nM for 48 h. Then the sub-G1 DNA content (later apoptotic dead cells) was determined by flow cytometry. Relative sub-G1 DNA production levels were analyzed and the data were derived from 3 independent testing and shown as histogram as mean ± SD. d , FL118 inhibits pancreatic cancer cell viability. Subconfluent Mia2, PANC1 and BxPC3 pancreatic cancer cells as well as normal human dermal fibroblast cells were treated with vehicle (no FL118) or with a series of FL118 concentrations as shown for 72 h. Then the cell viability was determined using MTT assay. The data were shown as histogram with mean ± SD derived from 3 independent testing assays
Figure Legend Snippet: Induction of apoptosis, pancreatic cancer cell killing and cell viability inhibition by FL118: a and b , FL118 treatment results in activation of caspase-3 and cleavage of PARP. Subconfluent pancreatic cancer cells (A, PANC1; B, MIA PaCa2) were treated with FL118 as shown, and the activation of casepase-3 and cleavage of PARP were detected by western blots. GAPDG is the internal control for total protein loading. c , FL118 induces pancreatic cancer cell death. Subconfluent PANC1 and Mia2 pancreatic cancer cells were treated with vehicle or with FL118 at 10, 10 and 500 nM for 48 h. Then the sub-G1 DNA content (later apoptotic dead cells) was determined by flow cytometry. Relative sub-G1 DNA production levels were analyzed and the data were derived from 3 independent testing and shown as histogram as mean ± SD. d , FL118 inhibits pancreatic cancer cell viability. Subconfluent Mia2, PANC1 and BxPC3 pancreatic cancer cells as well as normal human dermal fibroblast cells were treated with vehicle (no FL118) or with a series of FL118 concentrations as shown for 72 h. Then the cell viability was determined using MTT assay. The data were shown as histogram with mean ± SD derived from 3 independent testing assays

Techniques Used: Inhibition, Activation Assay, Western Blot, Flow Cytometry, Cytometry, Derivative Assay, MTT Assay

Effects of FL118 on the expression of proteins that are involved the pancreatic cancer cell DNA damage and repair: a , Subconfluent PANC1 pancreatic cancer cells were treated with FL118 as shown, and the expression of ERCC1, ERCC6, γ-H2AX, ChK1, ChK2, ATM, ATR, RAD51 and DNA Pol II was detected by western blots using corresponding antibodies for each protein. GAPDH is an internal control for protein loading. b, Decrease of ERCC6 expression can be rescued with proteasome inhibitor MG132. Subconfluent SKOV3 cells were treated with FL118 and MG132 alone or in combination as shown for 8 h, followed by western blot analyses with ERCC6 antibody. Tubulin is the internal control for protein loading. c , Comparison of FL118 and topotecan (TOP) effects on ERCC6 expression. Subconfluent SKOV3 cells were treated with FL118 or TOP as shown for 8 h, followed by western blot analyses with ERCC6 antibody. Tubulin is the internal control for protein loading
Figure Legend Snippet: Effects of FL118 on the expression of proteins that are involved the pancreatic cancer cell DNA damage and repair: a , Subconfluent PANC1 pancreatic cancer cells were treated with FL118 as shown, and the expression of ERCC1, ERCC6, γ-H2AX, ChK1, ChK2, ATM, ATR, RAD51 and DNA Pol II was detected by western blots using corresponding antibodies for each protein. GAPDH is an internal control for protein loading. b, Decrease of ERCC6 expression can be rescued with proteasome inhibitor MG132. Subconfluent SKOV3 cells were treated with FL118 and MG132 alone or in combination as shown for 8 h, followed by western blot analyses with ERCC6 antibody. Tubulin is the internal control for protein loading. c , Comparison of FL118 and topotecan (TOP) effects on ERCC6 expression. Subconfluent SKOV3 cells were treated with FL118 or TOP as shown for 8 h, followed by western blot analyses with ERCC6 antibody. Tubulin is the internal control for protein loading

Techniques Used: Expressing, Western Blot

2) Product Images from "Anticancer effects and underlying mechanism of Colchicine on human gastric cancer cell lines in vitro and in vivo"

Article Title: Anticancer effects and underlying mechanism of Colchicine on human gastric cancer cell lines in vitro and in vivo

Journal: Bioscience Reports

doi: 10.1042/BSR20181802

The effect of Colchicine on tumor growth and apoptosis in vivo ( A ) The antitumor effects of Colchicine on TV of xenograft model of NCI-N87 cells. ( B ) The antitumor effects of Colchicine on tumor weight of xenograft model of NCI-N87 cells. ( C ) Apoptotic cells were detected in xenograft tumor tissue using the TUNEL assay (200×). ( D ) Quantitated results of the TUNEL assay. Data are means ± S.D. ( n =10). * P
Figure Legend Snippet: The effect of Colchicine on tumor growth and apoptosis in vivo ( A ) The antitumor effects of Colchicine on TV of xenograft model of NCI-N87 cells. ( B ) The antitumor effects of Colchicine on tumor weight of xenograft model of NCI-N87 cells. ( C ) Apoptotic cells were detected in xenograft tumor tissue using the TUNEL assay (200×). ( D ) Quantitated results of the TUNEL assay. Data are means ± S.D. ( n =10). * P

Techniques Used: In Vivo, TUNEL Assay

The effect of Colchicine on cell apoptosis ( A ) Morphological analysis of the nuclei of NCI-N87 cells stained by Hoechst 33342 after various concentrations of Colchicine treatment for 48 h (200×). ( B ) Quantitative analysis of the percentage of apoptotic NCI-N87 cells stained by Hoechst 33342 after various concentrations of Colchicine treatment for 48 h. ( C ) Flow cytometry analysis of NCI-N87 cells after various concentrations of Colchicine treatment for 48 h. ( D ) The percentage of apoptotic cells in each group after various concentrations of Colchicine treatment for 48 h. Data are means ± S.D. ( n =5). ** P
Figure Legend Snippet: The effect of Colchicine on cell apoptosis ( A ) Morphological analysis of the nuclei of NCI-N87 cells stained by Hoechst 33342 after various concentrations of Colchicine treatment for 48 h (200×). ( B ) Quantitative analysis of the percentage of apoptotic NCI-N87 cells stained by Hoechst 33342 after various concentrations of Colchicine treatment for 48 h. ( C ) Flow cytometry analysis of NCI-N87 cells after various concentrations of Colchicine treatment for 48 h. ( D ) The percentage of apoptotic cells in each group after various concentrations of Colchicine treatment for 48 h. Data are means ± S.D. ( n =5). ** P

Techniques Used: Staining, Flow Cytometry, Cytometry

3) Product Images from "The Presence of CD14 Overcomes Evasion of Innate Immune Responses by Virulent Francisella tularensis in Human Dendritic Cells In Vitro and Pulmonary Cells In Vivo ▿ in Human Dendritic Cells In Vitro and Pulmonary Cells In Vivo ▿ †"

Article Title: The Presence of CD14 Overcomes Evasion of Innate Immune Responses by Virulent Francisella tularensis in Human Dendritic Cells In Vitro and Pulmonary Cells In Vivo ▿ in Human Dendritic Cells In Vitro and Pulmonary Cells In Vivo ▿ †

Journal: Infection and Immunity

doi: 10.1128/IAI.00750-09

CD14 aids in elicitation of proinflammatory cytokines in vivo. (A) Alveolar macrophages, lung DC, and peritoneal cells from naïve mice were assessed for cell surface expression of CD14 by flow cytometry. Peritoneal cells expressed uniform levels of CD14 on their cell surface. In contrast, alveolar macrophages and lung DC were weakly positive for CD14 on their cell surface. (B) CD14 facilitates production of cytokines following in vivo infection with F . tularensis SchuS4. Mice were infected with F . tularensis SchuS4 in the presence of sCD14. Uninfected, treated animals served as negative controls. Additional controls consisted of SchuS4-infected and uninfected mice that received PBS. Two days after infection, cytokines present in lung and spleen homogenate were quantified. Administration of sCD14 did not significantly change the amount of cytokine detected in lungs or spleens from uninfected mice. SchuS4-infected mice treated with PBS also failed to produce any significantly different concentrations of cytokines compared to uninfected controls. In contrast, SchuS4-infected animals treated with sCD14 had significantly more TNF-α ( P = 0.0034) and IL-6 ( P = 0.0346) in their lungs compared to the PBS-treated, SchuS4-infected controls. No significant difference in cytokine production among any groups was noted in samples obtained from the spleen. Data are representative of two experiments of similar design. The symbols show the values for individual mice, and the mean value for a group of mice is indicated by a short horizontal line.
Figure Legend Snippet: CD14 aids in elicitation of proinflammatory cytokines in vivo. (A) Alveolar macrophages, lung DC, and peritoneal cells from naïve mice were assessed for cell surface expression of CD14 by flow cytometry. Peritoneal cells expressed uniform levels of CD14 on their cell surface. In contrast, alveolar macrophages and lung DC were weakly positive for CD14 on their cell surface. (B) CD14 facilitates production of cytokines following in vivo infection with F . tularensis SchuS4. Mice were infected with F . tularensis SchuS4 in the presence of sCD14. Uninfected, treated animals served as negative controls. Additional controls consisted of SchuS4-infected and uninfected mice that received PBS. Two days after infection, cytokines present in lung and spleen homogenate were quantified. Administration of sCD14 did not significantly change the amount of cytokine detected in lungs or spleens from uninfected mice. SchuS4-infected mice treated with PBS also failed to produce any significantly different concentrations of cytokines compared to uninfected controls. In contrast, SchuS4-infected animals treated with sCD14 had significantly more TNF-α ( P = 0.0034) and IL-6 ( P = 0.0346) in their lungs compared to the PBS-treated, SchuS4-infected controls. No significant difference in cytokine production among any groups was noted in samples obtained from the spleen. Data are representative of two experiments of similar design. The symbols show the values for individual mice, and the mean value for a group of mice is indicated by a short horizontal line.

Techniques Used: In Vivo, Mouse Assay, Expressing, Flow Cytometry, Cytometry, Infection

Monocytes, but not human dendritic cells, secrete cytokines in response to F . tularensis SchuS4 infection. Primary human monocytes and donor-matched hDC were infected with F . tularensis SchuS4 at a MOI of 50. Twenty-four hours after infection, culture supernatants were analyzed for cytokines. Monocytes infected with F . tularensis SchuS4 secreted significantly more TNF-α, IL-6, IL-1β, MIP1β, and MIP-1α compared to monocytes from uninfected controls ( P
Figure Legend Snippet: Monocytes, but not human dendritic cells, secrete cytokines in response to F . tularensis SchuS4 infection. Primary human monocytes and donor-matched hDC were infected with F . tularensis SchuS4 at a MOI of 50. Twenty-four hours after infection, culture supernatants were analyzed for cytokines. Monocytes infected with F . tularensis SchuS4 secreted significantly more TNF-α, IL-6, IL-1β, MIP1β, and MIP-1α compared to monocytes from uninfected controls ( P

Techniques Used: Infection

CD14 is critical for induction of cytokines during F . tularensis SchuS4 infection of human cells. Human dendritic cells (A) or primary human monocytes (B) were infected with F . tularensis SchuS4 at a MOI of 50 in the presence of sCD14 (DC), anti-CD14 (monocytes), or isotype control (monocytes). (A) sCD14 elicited secretion of significantly greater concentrations of TNF-α and MIP-1α from SchuS4-infected cells compared to the cells from all other groups ( P
Figure Legend Snippet: CD14 is critical for induction of cytokines during F . tularensis SchuS4 infection of human cells. Human dendritic cells (A) or primary human monocytes (B) were infected with F . tularensis SchuS4 at a MOI of 50 in the presence of sCD14 (DC), anti-CD14 (monocytes), or isotype control (monocytes). (A) sCD14 elicited secretion of significantly greater concentrations of TNF-α and MIP-1α from SchuS4-infected cells compared to the cells from all other groups ( P

Techniques Used: Infection

4) Product Images from "A Cyclooxygenase-2 Inhibitor (SC-58125) Blocks Growth of Established Human Colon Cancer Xenografts 1"

Article Title: A Cyclooxygenase-2 Inhibitor (SC-58125) Blocks Growth of Established Human Colon Cancer Xenografts 1

Journal: Neoplasia (New York, N.Y.)

doi:

SC-58125 does not induce apoptosis. LLC (A) or HCA-7 (B) cells untreated (negative control), treated with SC-58635 (positive control), DMSO (vehicle), or with 100 µM SC-58125 for 24 hours (LLC), or 72 hours (HCA-7). Cells were harvested then fixed, and fragmented DNA was labeled using the TUNEL assay with dUTP- FITC as substrate. Analysis was performed using a Becton Dickinson FACScan flow cytometer. Ten thousand gated events were analyzed.
Figure Legend Snippet: SC-58125 does not induce apoptosis. LLC (A) or HCA-7 (B) cells untreated (negative control), treated with SC-58635 (positive control), DMSO (vehicle), or with 100 µM SC-58125 for 24 hours (LLC), or 72 hours (HCA-7). Cells were harvested then fixed, and fragmented DNA was labeled using the TUNEL assay with dUTP- FITC as substrate. Analysis was performed using a Becton Dickinson FACScan flow cytometer. Ten thousand gated events were analyzed.

Techniques Used: High Content Screening, Negative Control, Positive Control, Labeling, TUNEL Assay, Flow Cytometry, Cytometry

5) Product Images from "Macrophages and Dendritic Cells Emerge in the Liver during Intestinal Inflammation and Predispose the Liver to Inflammation"

Article Title: Macrophages and Dendritic Cells Emerge in the Liver during Intestinal Inflammation and Predispose the Liver to Inflammation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0084619

Immune dysregulation in the liver independent of T cell accumulation in the liver. ( A ) Numbers of hepatic mononuclear cells. Data are presented as the mean ± SEM for each group. RAG-2 −/− mice ( n = 4) and RAG-2 −/− LP CD4 + mice ( n = 4). ( B ) Representative data from flow cytometry analysis of Th cells in each organ. Dead cells were excluded by 7AAD staining. ( C ) Numbers of hepatic CD3 + CD4 + Th cells and non-T cells. ( D ) Representative data from flow cytometry analysis of pDCs and Mφs in the liver of each experimental group. Dead cells were excluded using 7AAD staining. Scatter plots for CD11b − CD11c int and CD11b + CD11c − cells are shown in the middle and bottom rows, respectively. ( E ) Proportion of PDCA-1 + CD11b − CD11c int pDCs and F4/80 + CD11b + CD11c − Mφs among whole mononuclear cells. Data are representative of three independent experiments. Values are presented as the mean ± SEM from seven mice in each group. * P
Figure Legend Snippet: Immune dysregulation in the liver independent of T cell accumulation in the liver. ( A ) Numbers of hepatic mononuclear cells. Data are presented as the mean ± SEM for each group. RAG-2 −/− mice ( n = 4) and RAG-2 −/− LP CD4 + mice ( n = 4). ( B ) Representative data from flow cytometry analysis of Th cells in each organ. Dead cells were excluded by 7AAD staining. ( C ) Numbers of hepatic CD3 + CD4 + Th cells and non-T cells. ( D ) Representative data from flow cytometry analysis of pDCs and Mφs in the liver of each experimental group. Dead cells were excluded using 7AAD staining. Scatter plots for CD11b − CD11c int and CD11b + CD11c − cells are shown in the middle and bottom rows, respectively. ( E ) Proportion of PDCA-1 + CD11b − CD11c int pDCs and F4/80 + CD11b + CD11c − Mφs among whole mononuclear cells. Data are representative of three independent experiments. Values are presented as the mean ± SEM from seven mice in each group. * P

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

GF condition abrogates the compositional changes of hepatic APCs in acute colitis models. ( A ) H E staining of colon sections taken from mice treated with water (left) or DSS (right). Magnification, ×100. ( B ) H E staining of liver sections from water- (left) and DSS-treated (right) mice. Magnification, ×100. ( C ) Number of liver mononuclear cells. ( D ) Proportion and ( E ) absolute number of PDCA-1 + CD11b − CD11c int pDCs and CD11b + CD11c − Mφs among whole mononuclear cells. ( F, G ) Comparisons between SPF and GF in the histology (F) and the numbers of Mφs (G) in DSS-treated mice. Data are representative of two independent experiments. Values are presented as the mean ± SEM for each group ( n = 5, water-treated GF group; n = 4, DSS-treated GF group; n = 5, DSS-treated SPF group). N.S., no significant difference.
Figure Legend Snippet: GF condition abrogates the compositional changes of hepatic APCs in acute colitis models. ( A ) H E staining of colon sections taken from mice treated with water (left) or DSS (right). Magnification, ×100. ( B ) H E staining of liver sections from water- (left) and DSS-treated (right) mice. Magnification, ×100. ( C ) Number of liver mononuclear cells. ( D ) Proportion and ( E ) absolute number of PDCA-1 + CD11b − CD11c int pDCs and CD11b + CD11c − Mφs among whole mononuclear cells. ( F, G ) Comparisons between SPF and GF in the histology (F) and the numbers of Mφs (G) in DSS-treated mice. Data are representative of two independent experiments. Values are presented as the mean ± SEM for each group ( n = 5, water-treated GF group; n = 4, DSS-treated GF group; n = 5, DSS-treated SPF group). N.S., no significant difference.

Techniques Used: Staining, Mouse Assay

Accumulation of liver macrophages in acute colitis models. ( A ) H E specimens of the colon taken from mice treated with water (left) or 2% DSS (right). Magnification, ×40 ( B ) H E specimens of livers from mice treated with water (left) and DSS (right). Magnification, ×100 ( C ) The number of liver mononuclear cells from water- and DSS-treated mice. ( D ) The absolute number of PDCA-1 + CD11b − CD11c int pDCs and CD11b + CD11c − Mφs among whole mononuclear cells. Data are representative of three independent experiments. ( E ) Levels of mRNA transcripts for IFN-γ, TNF, and IL-6 in the liver. Values are presented as the mean ± SEM for each group ( n = 4, water-treated group; n = 5, DSS-treated group). * P
Figure Legend Snippet: Accumulation of liver macrophages in acute colitis models. ( A ) H E specimens of the colon taken from mice treated with water (left) or 2% DSS (right). Magnification, ×40 ( B ) H E specimens of livers from mice treated with water (left) and DSS (right). Magnification, ×100 ( C ) The number of liver mononuclear cells from water- and DSS-treated mice. ( D ) The absolute number of PDCA-1 + CD11b − CD11c int pDCs and CD11b + CD11c − Mφs among whole mononuclear cells. Data are representative of three independent experiments. ( E ) Levels of mRNA transcripts for IFN-γ, TNF, and IL-6 in the liver. Values are presented as the mean ± SEM for each group ( n = 4, water-treated group; n = 5, DSS-treated group). * P

Techniques Used: Mouse Assay

Hepatic Mφ/cDCs cells under colitic conditions induce a Th1 inflammatory response. ( A ) FACS analysis of PDCA-1 + CD11b − CD11c int pDCs from the livers of WT (left column) mice. We also analyzed CD11b + CD11c − Mφs from the livers of ConA-treated (middle) and IL-10 −/− (right) mice, respectively. Dead cells were excluded with 7AAD staining. ( B ) Proliferation of naïve CFSE-labeled splenic CD4 + T cells from OT-II mice, and co-cultured WT pDCs, ConA Mφs, or IL-10 −/− Mφs in the presence of OVA. Dead cells were excluded with 7AAD staining and CD4 + T cells gated on CD3 + CD4 + cells are shown (B and C). Data are representative of three independent experiments. ( C ) Intracellular IFN-γ and IL-17A expression in CD4 + T cells co-cultured with WT pDCs, ConA Mφs, or IL-10 −/− Mφs in the presence of OVA. Data are representative of three independent experiments. ( D ) Proportion of IFN-γ + IL-17A − , IFN-γ − IL-17A + , and IFN-γ + IL-17A + cells among the Th cell population. ( E ) Cytokine concentrations in the culture supernatant of OT-II CD4 + T cells that were co-cultured with WT pDCs or ConA Mφs. Data are representative of three independent experiments. Each experiment was performed using duplicate samples. * P
Figure Legend Snippet: Hepatic Mφ/cDCs cells under colitic conditions induce a Th1 inflammatory response. ( A ) FACS analysis of PDCA-1 + CD11b − CD11c int pDCs from the livers of WT (left column) mice. We also analyzed CD11b + CD11c − Mφs from the livers of ConA-treated (middle) and IL-10 −/− (right) mice, respectively. Dead cells were excluded with 7AAD staining. ( B ) Proliferation of naïve CFSE-labeled splenic CD4 + T cells from OT-II mice, and co-cultured WT pDCs, ConA Mφs, or IL-10 −/− Mφs in the presence of OVA. Dead cells were excluded with 7AAD staining and CD4 + T cells gated on CD3 + CD4 + cells are shown (B and C). Data are representative of three independent experiments. ( C ) Intracellular IFN-γ and IL-17A expression in CD4 + T cells co-cultured with WT pDCs, ConA Mφs, or IL-10 −/− Mφs in the presence of OVA. Data are representative of three independent experiments. ( D ) Proportion of IFN-γ + IL-17A − , IFN-γ − IL-17A + , and IFN-γ + IL-17A + cells among the Th cell population. ( E ) Cytokine concentrations in the culture supernatant of OT-II CD4 + T cells that were co-cultured with WT pDCs or ConA Mφs. Data are representative of three independent experiments. Each experiment was performed using duplicate samples. * P

Techniques Used: FACS, Mouse Assay, Staining, Labeling, Cell Culture, Expressing

6) Product Images from "T cell immunodominance is dictated by the positively selecting self-peptide"

Article Title: T cell immunodominance is dictated by the positively selecting self-peptide

Journal: eLife

doi: 10.7554/eLife.01457

The evolving concepts of the relationship between positively selecting self-peptide and post-selection T cell repertoire. The figures show the progress of our understanding about the relationship between thymic self-peptides and post-selection CD4 + T cell repertoire, and are arranged in chorological order. The first finding from late 90′ revealed that one positively selecting self-peptide was capable of selecting many different TCRs ( Ignatowicz et al., 1996 ; Ignatowicz et al., 1997 ; Liu et al., 1997 ; Huseby et al., 2005 ). A few years later, Barton and Rudensky proposed that even though one self-peptide may select many TCRs, a diverse low abundant self-peptide repertoire is required to generate a full TCR repertoire ( Barton and Rudensky, 1999 ). Subsequently, with the publication of a series of single chain mice, the model was further refined because even though the TCR repertoire selected by one single ligand was not reduced significantly enough to allow an visualization of a repertoire ‘hole’, the postselection T cells in each single chain mouse showed different degrees of self-peptide reactivity in mixed lymphocyte culture ( Barton et al., 2002 ). These studies showed that a diverse collection of positively selecting self-peptides were necessary to generate a full T cell repertoire ( Barton and Rudensky, 1999 ; Barton et al., 2002 ). This present study involving the gp250 self-peptide showed increased frequency of Vα11 + Vβ3 + CD4 + T cells with MCC-preferred CDR3 features in gp250 SC mice. The Vα11 + Vβ3 + CD4 + T cells with specific CDR3 features were the dominant responders to promote MCC primary and memory responses. Therefore, gp250’s selecting capability provided a possible explanation to elucidate Vα11 + TCR-driven MCC immunodominance: a positively selecting self-peptide may favor the selection of TCR pairs and CDR3 features that were specific for MCC responses. DOI: http://dx.doi.org/10.7554/eLife.01457.013
Figure Legend Snippet: The evolving concepts of the relationship between positively selecting self-peptide and post-selection T cell repertoire. The figures show the progress of our understanding about the relationship between thymic self-peptides and post-selection CD4 + T cell repertoire, and are arranged in chorological order. The first finding from late 90′ revealed that one positively selecting self-peptide was capable of selecting many different TCRs ( Ignatowicz et al., 1996 ; Ignatowicz et al., 1997 ; Liu et al., 1997 ; Huseby et al., 2005 ). A few years later, Barton and Rudensky proposed that even though one self-peptide may select many TCRs, a diverse low abundant self-peptide repertoire is required to generate a full TCR repertoire ( Barton and Rudensky, 1999 ). Subsequently, with the publication of a series of single chain mice, the model was further refined because even though the TCR repertoire selected by one single ligand was not reduced significantly enough to allow an visualization of a repertoire ‘hole’, the postselection T cells in each single chain mouse showed different degrees of self-peptide reactivity in mixed lymphocyte culture ( Barton et al., 2002 ). These studies showed that a diverse collection of positively selecting self-peptides were necessary to generate a full T cell repertoire ( Barton and Rudensky, 1999 ; Barton et al., 2002 ). This present study involving the gp250 self-peptide showed increased frequency of Vα11 + Vβ3 + CD4 + T cells with MCC-preferred CDR3 features in gp250 SC mice. The Vα11 + Vβ3 + CD4 + T cells with specific CDR3 features were the dominant responders to promote MCC primary and memory responses. Therefore, gp250’s selecting capability provided a possible explanation to elucidate Vα11 + TCR-driven MCC immunodominance: a positively selecting self-peptide may favor the selection of TCR pairs and CDR3 features that were specific for MCC responses. DOI: http://dx.doi.org/10.7554/eLife.01457.013

Techniques Used: Selection, Mouse Assay

The gp250 self-peptide skews the selection of MCC-reactive preferred TCR pairs. ( A ) The Vα11 and Vβ3 expression of thymic CD4SP or peripheral CD4 + T cells in B6.K or gp250 SC mice. The data are representative of three experiments. ( B ) The quantification by percentage of Vα11 + CD4SP thymocytes or CD4 + peripheral T cells in B6.K or gp250 SC mice. n = 7; mean ± SD; two-tailed Mann–Whitney test. ( C ) The distribution of Vβ usages among Vα11 + CD4SP thymocytes or CD4 + peripheral T cells in B6.K or gp250 SC mice. The pie chart was plotted with the mean of three experiments (n = 7). ( D and E ) The quantification of increased dominant and subdominant MCC-reactive TCR pairs of CD4SP thymocytes or CD4 + peripheral T cells in B6.K or gp250 SC mice. n = 7; mean ± SD; two-tailed Mann–Whitney test. ( F ) The Vα11 and Vβ3 expression of thymic CD4SP or peripheral CD4 + T cells in B6.K or gp250 SC bone marrow chimeras reconstituted with B6.K HSC cells. The data are representative of three experiments. n = 4; mean ± SD; two-tailed Mann–Whitney test. DOI: http://dx.doi.org/10.7554/eLife.01457.008
Figure Legend Snippet: The gp250 self-peptide skews the selection of MCC-reactive preferred TCR pairs. ( A ) The Vα11 and Vβ3 expression of thymic CD4SP or peripheral CD4 + T cells in B6.K or gp250 SC mice. The data are representative of three experiments. ( B ) The quantification by percentage of Vα11 + CD4SP thymocytes or CD4 + peripheral T cells in B6.K or gp250 SC mice. n = 7; mean ± SD; two-tailed Mann–Whitney test. ( C ) The distribution of Vβ usages among Vα11 + CD4SP thymocytes or CD4 + peripheral T cells in B6.K or gp250 SC mice. The pie chart was plotted with the mean of three experiments (n = 7). ( D and E ) The quantification of increased dominant and subdominant MCC-reactive TCR pairs of CD4SP thymocytes or CD4 + peripheral T cells in B6.K or gp250 SC mice. n = 7; mean ± SD; two-tailed Mann–Whitney test. ( F ) The Vα11 and Vβ3 expression of thymic CD4SP or peripheral CD4 + T cells in B6.K or gp250 SC bone marrow chimeras reconstituted with B6.K HSC cells. The data are representative of three experiments. n = 4; mean ± SD; two-tailed Mann–Whitney test. DOI: http://dx.doi.org/10.7554/eLife.01457.008

Techniques Used: Selection, Expressing, Mouse Assay, Two Tailed Test, MANN-WHITNEY

gp250-mediated positive selection greatly expands MCC-tetramer + CD4 + T cells. ( A and B ) The frequency of MCC-tetramer + CD4SP thymocytes or peripheral CD4 + T cells in B6.K or gp250 SC mice. The plot was gated on the live CD4 + CD3 + CD8 − population. The data are representative of at least five experiments. Bar graph shows the summary ( B ; thymus, n = 12; spleen, n = 17; mean ± SD; two-tailed Mann–Whitney test). ( C and D ) The frequency of Hb-tetramer + CD4SP thymocytes or peripheral CD4 + T cells in B6.K or gp250 SC mice. The plot was gated on live CD4 + CD3 + CD8 − population. The data are representative of at least three experiments. ( D ): n = 3; mean ± SD; two-tailed Mann–Whitney test. ( E and F ) The frequency of MCC-tetramer + peripheral CD4 + T cells in B6.K or gp250 SC bone marrow chimeras reconstituted with B6.K bone marrows. ( F ): n = 4; mean ± SD; two-tailed Mann–Whitney test. DOI: http://dx.doi.org/10.7554/eLife.01457.006
Figure Legend Snippet: gp250-mediated positive selection greatly expands MCC-tetramer + CD4 + T cells. ( A and B ) The frequency of MCC-tetramer + CD4SP thymocytes or peripheral CD4 + T cells in B6.K or gp250 SC mice. The plot was gated on the live CD4 + CD3 + CD8 − population. The data are representative of at least five experiments. Bar graph shows the summary ( B ; thymus, n = 12; spleen, n = 17; mean ± SD; two-tailed Mann–Whitney test). ( C and D ) The frequency of Hb-tetramer + CD4SP thymocytes or peripheral CD4 + T cells in B6.K or gp250 SC mice. The plot was gated on live CD4 + CD3 + CD8 − population. The data are representative of at least three experiments. ( D ): n = 3; mean ± SD; two-tailed Mann–Whitney test. ( E and F ) The frequency of MCC-tetramer + peripheral CD4 + T cells in B6.K or gp250 SC bone marrow chimeras reconstituted with B6.K bone marrows. ( F ): n = 4; mean ± SD; two-tailed Mann–Whitney test. DOI: http://dx.doi.org/10.7554/eLife.01457.006

Techniques Used: Selection, Mouse Assay, Two Tailed Test, MANN-WHITNEY

The majority of gp250-selected MCC-specific CD4 + T cells express Vα11 + TCR. ( A and B ) The Vα11 expression of MCC-tetramer + peripheral CD4 + T cells in B6.K or gp250 SC mice. ( B ) n = 6; mean ± SD; two-tailed Mann–Whitney test. ( C and D ) The CD69 upregulation of sorted MCC-tetramer − or MCC-tetramer + CD4 + T cells from B6.K or gp250 SC splenocytes. Data are representative of three experiments. ( D ): n = 3, paired t tests. DOI: http://dx.doi.org/10.7554/eLife.01457.007
Figure Legend Snippet: The majority of gp250-selected MCC-specific CD4 + T cells express Vα11 + TCR. ( A and B ) The Vα11 expression of MCC-tetramer + peripheral CD4 + T cells in B6.K or gp250 SC mice. ( B ) n = 6; mean ± SD; two-tailed Mann–Whitney test. ( C and D ) The CD69 upregulation of sorted MCC-tetramer − or MCC-tetramer + CD4 + T cells from B6.K or gp250 SC splenocytes. Data are representative of three experiments. ( D ): n = 3, paired t tests. DOI: http://dx.doi.org/10.7554/eLife.01457.007

Techniques Used: Expressing, Mouse Assay, Two Tailed Test, MANN-WHITNEY

The I-E k α and covalently-linked gp250/I-E k β transgenes restore surface expression of I-E k in gp250 SC mice class II-deficient mice. The surface expression of I-E k on B cells ( A and B ) and dendritic cells ( C and D ) from the spleens of gp250 SC mice. The B cells and dendritic cells from MHCII −/− CD74 −/− mice were used as negative controls. The plots are representative of three independent experiments ( A and C ) and summarized in the bar graph ( B and D ; mean ± SD; n = 3; two-tailed Mann–Whitney test). DOI: http://dx.doi.org/10.7554/eLife.01457.003
Figure Legend Snippet: The I-E k α and covalently-linked gp250/I-E k β transgenes restore surface expression of I-E k in gp250 SC mice class II-deficient mice. The surface expression of I-E k on B cells ( A and B ) and dendritic cells ( C and D ) from the spleens of gp250 SC mice. The B cells and dendritic cells from MHCII −/− CD74 −/− mice were used as negative controls. The plots are representative of three independent experiments ( A and C ) and summarized in the bar graph ( B and D ; mean ± SD; n = 3; two-tailed Mann–Whitney test). DOI: http://dx.doi.org/10.7554/eLife.01457.003

Techniques Used: Expressing, Mouse Assay, Two Tailed Test, MANN-WHITNEY

TCR Vβ usages of peripheral T cells in gp250 SC mice. ( A and B ) Vβs expression of peripheral CD4 + T cells in B6.K or gp250 SC mice. The frequencies of three Vβs were decreased ( A ) and three were increased ( B ). n = 9; data are representative of three experiments. n = 4; mean ± SD; two-tailed Mann–Whitney test. ( C and D ) B6.K HSC cells were used to reconstitute lethally irradiated gp250 SC or B6.K mice. After a 12-week reconstitution, Vβx expression repertoire in CD4 + or CD8 + T cells were examined ( D ), and the frequencies of Vβs that were increased in gp250 SC mice ( B ) were shown in ( C ). n = 5; the data are representative of three experiments. n = 4; mean ± SD; two-tailed Mann–Whitney test. DOI: http://dx.doi.org/10.7554/eLife.01457.005
Figure Legend Snippet: TCR Vβ usages of peripheral T cells in gp250 SC mice. ( A and B ) Vβs expression of peripheral CD4 + T cells in B6.K or gp250 SC mice. The frequencies of three Vβs were decreased ( A ) and three were increased ( B ). n = 9; data are representative of three experiments. n = 4; mean ± SD; two-tailed Mann–Whitney test. ( C and D ) B6.K HSC cells were used to reconstitute lethally irradiated gp250 SC or B6.K mice. After a 12-week reconstitution, Vβx expression repertoire in CD4 + or CD8 + T cells were examined ( D ), and the frequencies of Vβs that were increased in gp250 SC mice ( B ) were shown in ( C ). n = 5; the data are representative of three experiments. n = 4; mean ± SD; two-tailed Mann–Whitney test. DOI: http://dx.doi.org/10.7554/eLife.01457.005

Techniques Used: Mouse Assay, Expressing, Two Tailed Test, MANN-WHITNEY, Irradiation

The gp250 self-peptide skews the selection of TCRs exhibiting conserved MCC-reactive features. ( A ) The pie chart of amino acid usages at CDR3 α89 and α91 of bulk-sorted peripheral Vα11 + Vβ3 + CD4 + T cells in B6.K or gp250 SC mice. The gp250 SC data were obtained by single cell repertoire analysis. ( B ) The frequency of MCC-reactive preferred CDR3α amino acids, α89N and α91E from gp250-selected peripheral Vα11 + Vβ3 + CD4 + T cells in single cell analysis (left) or bulk population analysis (right). ( C ) The pie chart of amino acid usages at CDR3 β97 and β99 from bulk-sorted peripheral Vα11 + Vβ3 + CD4 + T cells in B6.K or gp250 SC mice. The data were representative of two independent experiments. ( D ) The frequencies of CDR3 β97N, β99A, and β99E of Vα11 + Vβ3 + CD4 + T cells in B6.K or gp250 SC mice. ( B and D ) The single cell B10.A data were plotted based on McHeyzer-Williams et al. (1999) published data. DOI: http://dx.doi.org/10.7554/eLife.01457.009
Figure Legend Snippet: The gp250 self-peptide skews the selection of TCRs exhibiting conserved MCC-reactive features. ( A ) The pie chart of amino acid usages at CDR3 α89 and α91 of bulk-sorted peripheral Vα11 + Vβ3 + CD4 + T cells in B6.K or gp250 SC mice. The gp250 SC data were obtained by single cell repertoire analysis. ( B ) The frequency of MCC-reactive preferred CDR3α amino acids, α89N and α91E from gp250-selected peripheral Vα11 + Vβ3 + CD4 + T cells in single cell analysis (left) or bulk population analysis (right). ( C ) The pie chart of amino acid usages at CDR3 β97 and β99 from bulk-sorted peripheral Vα11 + Vβ3 + CD4 + T cells in B6.K or gp250 SC mice. The data were representative of two independent experiments. ( D ) The frequencies of CDR3 β97N, β99A, and β99E of Vα11 + Vβ3 + CD4 + T cells in B6.K or gp250 SC mice. ( B and D ) The single cell B10.A data were plotted based on McHeyzer-Williams et al. (1999) published data. DOI: http://dx.doi.org/10.7554/eLife.01457.009

Techniques Used: Selection, Mouse Assay, Single-cell Analysis

7) Product Images from "Two Distinct Functional Patterns of Hepatitis C Virus (HCV)-Specific T Cell Responses in Seronegative, Aviremic Patients"

Article Title: Two Distinct Functional Patterns of Hepatitis C Virus (HCV)-Specific T Cell Responses in Seronegative, Aviremic Patients

Journal: PLoS ONE

doi: 10.1371/journal.pone.0062319

Polyfunctionality of HCV-specific T cells in seronegative, aviremic hemodialysis patients. PBMCs were stimulated with an epitope peptide or a peptide mix, and production of IFN-γ, TNF-α, IL-2, and MIP-1β was simultaneously assessed in multi-cytokine ICS in order to evaluate polyfunctionality of HCV-specific CD4 + T cells. In each patient, the fraction of T cells positive for a given number of functions is presented as bar graphs. In the patient with CD8 + T cell responses (CMI-4), cytotoxic degranulation function was also evaluated by CD107a staining in addition to multi-cytokine ICS for IFN-γ, TNF-α, IL-2, and MIP-1β.
Figure Legend Snippet: Polyfunctionality of HCV-specific T cells in seronegative, aviremic hemodialysis patients. PBMCs were stimulated with an epitope peptide or a peptide mix, and production of IFN-γ, TNF-α, IL-2, and MIP-1β was simultaneously assessed in multi-cytokine ICS in order to evaluate polyfunctionality of HCV-specific CD4 + T cells. In each patient, the fraction of T cells positive for a given number of functions is presented as bar graphs. In the patient with CD8 + T cell responses (CMI-4), cytotoxic degranulation function was also evaluated by CD107a staining in addition to multi-cytokine ICS for IFN-γ, TNF-α, IL-2, and MIP-1β.

Techniques Used: Staining

Polyfunctionality assay of HCV-specific T cells in the patients of group I. Representative examples of polyfunctionality assays of CD4 + T cells in CMI-1 (A and B) and of CD8 + T cells in CMI-4 (C and D) are presented. In CMI-1, PBMCs were stimulated with NS5B 2966–2980 epitope peptide, and IFN-γ, TNF-α, IL-2, and MIP-1β production was assessed (A and B). In CMI-4, PBMCs were stimulated with NS3 1541–1555 epitope peptide, and CD107a, IFN-γ, TNF-α, IL-2, and MIP-1β production was assessed (C and D). In the culture supernatant of NS3 1541–1555 peptide-stimulated PBMCs, concentration of granzyme B was determined by CBA (E). The data are presented by FACS dot plots and the pie graphs show the fraction of T cells positive for a given number of functions (A and C). Detailed analysis of polyfunctionality is presented with every possible combination of functions (B and D).
Figure Legend Snippet: Polyfunctionality assay of HCV-specific T cells in the patients of group I. Representative examples of polyfunctionality assays of CD4 + T cells in CMI-1 (A and B) and of CD8 + T cells in CMI-4 (C and D) are presented. In CMI-1, PBMCs were stimulated with NS5B 2966–2980 epitope peptide, and IFN-γ, TNF-α, IL-2, and MIP-1β production was assessed (A and B). In CMI-4, PBMCs were stimulated with NS3 1541–1555 epitope peptide, and CD107a, IFN-γ, TNF-α, IL-2, and MIP-1β production was assessed (C and D). In the culture supernatant of NS3 1541–1555 peptide-stimulated PBMCs, concentration of granzyme B was determined by CBA (E). The data are presented by FACS dot plots and the pie graphs show the fraction of T cells positive for a given number of functions (A and C). Detailed analysis of polyfunctionality is presented with every possible combination of functions (B and D).

Techniques Used: Concentration Assay, Crocin Bleaching Assay, FACS

Polyfunctionality assay of HCV-specific T cells in the patients of group II. Representative example of polyfunctionality assays of CD4 + T cells in CMI-8 is presented. PBMCs were stimulated with NS3 peptide mix, and IFN-γ, TNF-α, IL-2, and MIP-1β production were assessed. The data are presented by FACS dot plots and the pie graph shows the fraction of T cells positive for a given number of functions (A). Detailed analysis of polyfunctionality is presented with every possible combination of functions (B).
Figure Legend Snippet: Polyfunctionality assay of HCV-specific T cells in the patients of group II. Representative example of polyfunctionality assays of CD4 + T cells in CMI-8 is presented. PBMCs were stimulated with NS3 peptide mix, and IFN-γ, TNF-α, IL-2, and MIP-1β production were assessed. The data are presented by FACS dot plots and the pie graph shows the fraction of T cells positive for a given number of functions (A). Detailed analysis of polyfunctionality is presented with every possible combination of functions (B).

Techniques Used: FACS

Cytokine profile of HCV-specific T cells in seronegative, aviremic hemodialysis patients. (A) After stimulating PBMCs with an epitope peptide or a peptide mix for 3 days, culture supernatant was harvested, and the concentrations of IFN-γ and TNF-α determined in the culture supernatant by CBA. There are two groups of patients in terms of cytokine profile: group I, patients with IFN-γ and TNF-α responses; group II, patients with TNF-α-predominant responses. (B) TNF-α ICS was performed in the patients of group II. FACS dot plots are representative data from two patients, CMI-7 and CMI-8.
Figure Legend Snippet: Cytokine profile of HCV-specific T cells in seronegative, aviremic hemodialysis patients. (A) After stimulating PBMCs with an epitope peptide or a peptide mix for 3 days, culture supernatant was harvested, and the concentrations of IFN-γ and TNF-α determined in the culture supernatant by CBA. There are two groups of patients in terms of cytokine profile: group I, patients with IFN-γ and TNF-α responses; group II, patients with TNF-α-predominant responses. (B) TNF-α ICS was performed in the patients of group II. FACS dot plots are representative data from two patients, CMI-7 and CMI-8.

Techniques Used: Crocin Bleaching Assay, FACS

8) Product Images from "In vivo imaging of mice infected with bioluminescent Trypanosoma cruzi unveils novel sites of infection"

Article Title: In vivo imaging of mice infected with bioluminescent Trypanosoma cruzi unveils novel sites of infection

Journal: Parasites & Vectors

doi: 10.1186/1756-3305-7-89

Analyses of cytokines in the acute phase of infection. Comparison of cytokine expression in the blood of mice infected with Dm28c-WT (A) and Dm28c-luc (B) 20 dpi. Statistically significant differences were observed for IL-5, IL-6, and IFN-γ when the p value was
Figure Legend Snippet: Analyses of cytokines in the acute phase of infection. Comparison of cytokine expression in the blood of mice infected with Dm28c-WT (A) and Dm28c-luc (B) 20 dpi. Statistically significant differences were observed for IL-5, IL-6, and IFN-γ when the p value was

Techniques Used: Infection, Expressing, Mouse Assay

9) Product Images from "Serum Amyloid P Aids Complement-Mediated Immunity to Streptococcus pneumoniae"

Article Title: Serum Amyloid P Aids Complement-Mediated Immunity to Streptococcus pneumoniae

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.0030120

Cytokine Levels in BALF Recovered from Wild-Type or Apcs −/− Mice 4 h or 24 h after i.n. Inoculation with 1.0 × 10 6 cfu of ST2 S. pneumoniae Error bars represent SDs, and the p -values (2-tailed t tests) for comparison between wild-type and Apcs −/− mice are given below the title for each panel. (A) Cytokine levels 4 h after inoculation. (B) Cytokine levels 24 h after inoculation.
Figure Legend Snippet: Cytokine Levels in BALF Recovered from Wild-Type or Apcs −/− Mice 4 h or 24 h after i.n. Inoculation with 1.0 × 10 6 cfu of ST2 S. pneumoniae Error bars represent SDs, and the p -values (2-tailed t tests) for comparison between wild-type and Apcs −/− mice are given below the title for each panel. (A) Cytokine levels 4 h after inoculation. (B) Cytokine levels 24 h after inoculation.

Techniques Used: Mouse Assay

Effects of SAP on C3b Deposition on S. pneumoniae Measured Using Flow Cytometry (A) Time course of the proportion of ST2 S. pneumoniae bacteria positive for C3b after incubation in serum from wild-type and Apcs −/− mice. For the comparison of results for wild-type versus Apcs −/− mice, p
Figure Legend Snippet: Effects of SAP on C3b Deposition on S. pneumoniae Measured Using Flow Cytometry (A) Time course of the proportion of ST2 S. pneumoniae bacteria positive for C3b after incubation in serum from wild-type and Apcs −/− mice. For the comparison of results for wild-type versus Apcs −/− mice, p

Techniques Used: Flow Cytometry, Cytometry, Incubation, Mouse Assay

Effect of SAP on Phagocytosis of S. pneumoniae (A–C) Phagocytosis (presented as proportion of HL60 cells associated with fluorescent bacteria) of (A) ST2, (B) ST4, and (C) ST23F after incubation in different dilutions of serum from wild-type (circles) or Apcs −/− (squares) mice. Results for incubation in HBSS are shown by the triangle symbol, and for the ST2 strain the results for a 50:50 mix of serum from wild-type and Apcs −/− mice (diamonds) are also included. (D) Example of a flow cytometry histogram of phagocytosis of ST2 S. pneumoniae by HL60 cells after incubation in HBSS or serum from wild-type or Apcs −/− mice. (E) Effect of addition of 5 or 50 μg/ml exogenous hSAP on phagocytosis of the ST2 S. pneumoniae strain in serum from Apcs −/− mice. (F) Effect of addition of hSAP (50 μg/ml) on phagocytosis of the ST2 S. pneumoniae strain in serum from Apcs −/− .C1qa −/− mice. Grey column, results for Apcs −/− .C1qa −/− serum; white column, results for Apcs −/− .C1qa −/− serum in the presence of hSAP. For panels (A–C), (E), and (F), asterisks mark significant p -values for comparisons of results for wild-type or mixed serum to Apcs −/− serum (2-tailed t tests, * p
Figure Legend Snippet: Effect of SAP on Phagocytosis of S. pneumoniae (A–C) Phagocytosis (presented as proportion of HL60 cells associated with fluorescent bacteria) of (A) ST2, (B) ST4, and (C) ST23F after incubation in different dilutions of serum from wild-type (circles) or Apcs −/− (squares) mice. Results for incubation in HBSS are shown by the triangle symbol, and for the ST2 strain the results for a 50:50 mix of serum from wild-type and Apcs −/− mice (diamonds) are also included. (D) Example of a flow cytometry histogram of phagocytosis of ST2 S. pneumoniae by HL60 cells after incubation in HBSS or serum from wild-type or Apcs −/− mice. (E) Effect of addition of 5 or 50 μg/ml exogenous hSAP on phagocytosis of the ST2 S. pneumoniae strain in serum from Apcs −/− mice. (F) Effect of addition of hSAP (50 μg/ml) on phagocytosis of the ST2 S. pneumoniae strain in serum from Apcs −/− .C1qa −/− mice. Grey column, results for Apcs −/− .C1qa −/− serum; white column, results for Apcs −/− .C1qa −/− serum in the presence of hSAP. For panels (A–C), (E), and (F), asterisks mark significant p -values for comparisons of results for wild-type or mixed serum to Apcs −/− serum (2-tailed t tests, * p

Techniques Used: Incubation, Mouse Assay, Flow Cytometry, Cytometry

10) Product Images from "Synergistic antitumor activity of oncolytic reovirus and chemotherapeutic agents in non-small cell lung cancer cells"

Article Title: Synergistic antitumor activity of oncolytic reovirus and chemotherapeutic agents in non-small cell lung cancer cells

Journal: Molecular Cancer

doi: 10.1186/1476-4598-8-47

Flow cytometric analysis of DNA content and caspase-3 activation in NSCLC cells treated with either ReoT3D (MOI = 20) or paclitaxel alone (0.1 or 1 μM), or both in combination . (a) Shown are scattergrams (top) and histograms (bottom), ungated, of NCI-H23 cells treated with the indicated agent(s) for 24 hours. Harvested cells were fixed, permeabilized and stained with FITC-conjugated anti-active caspase-3 antibody followed by PI staining. Each histogram depicts the DNA content of cells in G1 (2N) and G2/M (4N), while the scattergrams demonstrate the proportion of cells with activated caspase-3 in different cell cycle phases. Values shown in each quadrant of scattergrams represent the percentages of cells. Of note, the histograms also show the apoptotic subdiploid peak (sub-2N), especially enhanced in paclitaxel-treated NCI-H23 cells. (b) The effects of ReoT3D-paclitaxel combination on caspase-3 activation and DNA content were compared among NCI-H23, NCI-H460, EKVX and NCI-322M. These NSCLC cells were analyzed as described in (a). Shown are the percentages of cells positive or negative for activated caspase-3 in sub- G1/G1 or post-G1 phases. The data shown in (a) and (b) are representative of 2 experiments.
Figure Legend Snippet: Flow cytometric analysis of DNA content and caspase-3 activation in NSCLC cells treated with either ReoT3D (MOI = 20) or paclitaxel alone (0.1 or 1 μM), or both in combination . (a) Shown are scattergrams (top) and histograms (bottom), ungated, of NCI-H23 cells treated with the indicated agent(s) for 24 hours. Harvested cells were fixed, permeabilized and stained with FITC-conjugated anti-active caspase-3 antibody followed by PI staining. Each histogram depicts the DNA content of cells in G1 (2N) and G2/M (4N), while the scattergrams demonstrate the proportion of cells with activated caspase-3 in different cell cycle phases. Values shown in each quadrant of scattergrams represent the percentages of cells. Of note, the histograms also show the apoptotic subdiploid peak (sub-2N), especially enhanced in paclitaxel-treated NCI-H23 cells. (b) The effects of ReoT3D-paclitaxel combination on caspase-3 activation and DNA content were compared among NCI-H23, NCI-H460, EKVX and NCI-322M. These NSCLC cells were analyzed as described in (a). Shown are the percentages of cells positive or negative for activated caspase-3 in sub- G1/G1 or post-G1 phases. The data shown in (a) and (b) are representative of 2 experiments.

Techniques Used: Flow Cytometry, Activation Assay, Staining

11) Product Images from "The heat shock protein 90 inhibitor BIIB021 suppresses the growth of T and natural killer cell lymphomas"

Article Title: The heat shock protein 90 inhibitor BIIB021 suppresses the growth of T and natural killer cell lymphomas

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2015.00280

BIIB021 induced apoptosis in some EBV-positive and -negative T and NK cell lines . EBV-positive T cell lines (SNT13 and SNT16), an EBV-negative T cell line (Jurkat), EBV-positive NK cell lines (KAI3 and SNK6), and an EBV-negative NK cell line (KHYG1) were cultured with DMSO or 5 μM BIIB021 for 48 h. Apoptosis was measured by flow cytometry after annexin V-phycoerythrin (annexin V) and 7-aminoactinomycin D (7-AAD) staining.
Figure Legend Snippet: BIIB021 induced apoptosis in some EBV-positive and -negative T and NK cell lines . EBV-positive T cell lines (SNT13 and SNT16), an EBV-negative T cell line (Jurkat), EBV-positive NK cell lines (KAI3 and SNK6), and an EBV-negative NK cell line (KHYG1) were cultured with DMSO or 5 μM BIIB021 for 48 h. Apoptosis was measured by flow cytometry after annexin V-phycoerythrin (annexin V) and 7-aminoactinomycin D (7-AAD) staining.

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

12) Product Images from "Silenced suppressor of cytokine signaling 1 (SOCS1) enhances the maturation and antifungal immunity of dendritic cells in response to Candida albicans in vitro"

Article Title: Silenced suppressor of cytokine signaling 1 (SOCS1) enhances the maturation and antifungal immunity of dendritic cells in response to Candida albicans in vitro

Journal: Immunologic Research

doi: 10.1007/s12026-014-8562-8

SOCS1 siRNA treatment DCs co-cultured with C. albicans induced strong Th1 responses in MLR. Proliferative capacity of allogeneic T cells from C57 mice co-cultured with DCs in MLR. a – c IFN-γ, IL-4, and IL-17 concentrations in the culture medium were measured by flow cytometry. Cells were stained with anti-IL-4-PE, anti-IFN-γ-FITC, and IL-17-FITC to detect intracellular cytokine production by CD4 + T cells. Mean ± SD of data from seven independent experiments is shown. d – f Supernatants were collected on day 5 of the culture, and IFN-γ, IL-4, and IL-17 concentrations analyzed using cytometric bead arrays. Mean ± SD of data from seven independent experiments is shown. ** p
Figure Legend Snippet: SOCS1 siRNA treatment DCs co-cultured with C. albicans induced strong Th1 responses in MLR. Proliferative capacity of allogeneic T cells from C57 mice co-cultured with DCs in MLR. a – c IFN-γ, IL-4, and IL-17 concentrations in the culture medium were measured by flow cytometry. Cells were stained with anti-IL-4-PE, anti-IFN-γ-FITC, and IL-17-FITC to detect intracellular cytokine production by CD4 + T cells. Mean ± SD of data from seven independent experiments is shown. d – f Supernatants were collected on day 5 of the culture, and IFN-γ, IL-4, and IL-17 concentrations analyzed using cytometric bead arrays. Mean ± SD of data from seven independent experiments is shown. ** p

Techniques Used: Cell Culture, Mouse Assay, Flow Cytometry, Cytometry, Staining

Exogenous anti-IL-12 or anti-IFN-γ mAbs reversed the enhancement of IFN-γ production by CD4 + T cells. a , b DCs were exposed to SOCS1 siRNA and were treated with C. albicans for 24 h. At day 10, T cells were stimulated with PMA and ionomycin for 5 h in the presence of brefeldin A. Cells were stained with anti-IFN-γ-FITC and IL-17-FITC to detect intracellular cytokine production by flow cytometry. Data were Mean ± SD of seven independent experiments. * p
Figure Legend Snippet: Exogenous anti-IL-12 or anti-IFN-γ mAbs reversed the enhancement of IFN-γ production by CD4 + T cells. a , b DCs were exposed to SOCS1 siRNA and were treated with C. albicans for 24 h. At day 10, T cells were stimulated with PMA and ionomycin for 5 h in the presence of brefeldin A. Cells were stained with anti-IFN-γ-FITC and IL-17-FITC to detect intracellular cytokine production by flow cytometry. Data were Mean ± SD of seven independent experiments. * p

Techniques Used: Staining, Flow Cytometry, Cytometry

13) Product Images from "In Vitro Evaluation of Endothelial Progenitor Cells from Adipose Tissue as Potential Angiogenic Cell Sources for Bladder Angiogenesis"

Article Title: In Vitro Evaluation of Endothelial Progenitor Cells from Adipose Tissue as Potential Angiogenic Cell Sources for Bladder Angiogenesis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0117644

Flow cytometric analysis of ADEPCs and ADSCs. (A) The single cell population with high forward scatter of ADEPCs and ADSCs could be obtained through our isolation technique. (B) ADEPCs were positive for CD34 (87.3±3.9%), Stro-1 (89.1±4.6%), VEGFR-2 (16.3±5.7%), eNOS (91.5±3.7%) and CD31 (90.6±4.1%) but negative for α-SMA, CD14 and CD45. ADSCs were positive for CD34 (15.2±2.3%), Stro-1 (88.7±5.2%) and α-SMA (80.1±2.9%), but negative for VEGFR-2, eNOS, CD31, CD14 and CD45. (C) Quantification of surface markers (means expressed as percentages; n = 6).
Figure Legend Snippet: Flow cytometric analysis of ADEPCs and ADSCs. (A) The single cell population with high forward scatter of ADEPCs and ADSCs could be obtained through our isolation technique. (B) ADEPCs were positive for CD34 (87.3±3.9%), Stro-1 (89.1±4.6%), VEGFR-2 (16.3±5.7%), eNOS (91.5±3.7%) and CD31 (90.6±4.1%) but negative for α-SMA, CD14 and CD45. ADSCs were positive for CD34 (15.2±2.3%), Stro-1 (88.7±5.2%) and α-SMA (80.1±2.9%), but negative for VEGFR-2, eNOS, CD31, CD14 and CD45. (C) Quantification of surface markers (means expressed as percentages; n = 6).

Techniques Used: Flow Cytometry, Isolation

14) Product Images from "Antineoplastic activity of the DNA methyltransferase inhibitor 5-aza-2?-deoxycytidine in anaplastic large cell lymphoma"

Article Title: Antineoplastic activity of the DNA methyltransferase inhibitor 5-aza-2?-deoxycytidine in anaplastic large cell lymphoma

Journal: Biochimie

doi: 10.1016/j.biochi.2012.05.029

5-Aza-CdR treatment in vivo leads to inhibition of DNMT1 and apoptosis and results in demethylation of tumor suppressor p16INK4A. A. Cell cycle analysis shows that 5-aza-CdR treatment leads to increased apoptosis. Tumor samples were prepared for FACS analysis as described in the methods section and analyzed with a BD FACSCanto II flow cytometer using the BD FACS Diva Software. Values are means ± SEM. Each value is the mean of three replicates for control and 5-aza-CdR day 5 tumors and of two replicates for 5-aza-CdR day 3 tumors. B. DNMT1 is not detected in tumor protein extracts derived from 5-aza-CdR treated mice. Proteins were extracted from tumor tissue from two tumors treated with 5-aza-CdR starting on day 3 and three tumors treated with 5-aza-CdR on day 5 as described in materials and methods. DNMT1 protein levels were analysed by Western Blot. C. Analysis of the p16 INK4A promotor region by COBRA shows demethylation after 5-aza-CdR treatment. DNA was extracted from tumor tissue, bisulfite converted and analysed by COBRA. Restriction fragments were analysed using the Agilent 2100 Bioanalyzer platform. Normal PBMCs and in vitro by M.SssI methylated PBMCs were used as unmethylated and methylated controls, respectively. Note the re-appearance of the unmethylated fragment in treated samples indicated by the arrow. D. The percentage of methylated fragments is significantly reduced in 5-aza-CdR treated tumors. Percentages of methylated and unmethylated fragments in relation to total peak areas in electropherograms were calculated with Agilent software as described in the methods section.
Figure Legend Snippet: 5-Aza-CdR treatment in vivo leads to inhibition of DNMT1 and apoptosis and results in demethylation of tumor suppressor p16INK4A. A. Cell cycle analysis shows that 5-aza-CdR treatment leads to increased apoptosis. Tumor samples were prepared for FACS analysis as described in the methods section and analyzed with a BD FACSCanto II flow cytometer using the BD FACS Diva Software. Values are means ± SEM. Each value is the mean of three replicates for control and 5-aza-CdR day 5 tumors and of two replicates for 5-aza-CdR day 3 tumors. B. DNMT1 is not detected in tumor protein extracts derived from 5-aza-CdR treated mice. Proteins were extracted from tumor tissue from two tumors treated with 5-aza-CdR starting on day 3 and three tumors treated with 5-aza-CdR on day 5 as described in materials and methods. DNMT1 protein levels were analysed by Western Blot. C. Analysis of the p16 INK4A promotor region by COBRA shows demethylation after 5-aza-CdR treatment. DNA was extracted from tumor tissue, bisulfite converted and analysed by COBRA. Restriction fragments were analysed using the Agilent 2100 Bioanalyzer platform. Normal PBMCs and in vitro by M.SssI methylated PBMCs were used as unmethylated and methylated controls, respectively. Note the re-appearance of the unmethylated fragment in treated samples indicated by the arrow. D. The percentage of methylated fragments is significantly reduced in 5-aza-CdR treated tumors. Percentages of methylated and unmethylated fragments in relation to total peak areas in electropherograms were calculated with Agilent software as described in the methods section.

Techniques Used: In Vivo, Inhibition, Cell Cycle Assay, FACS, Flow Cytometry, Cytometry, Software, Derivative Assay, Mouse Assay, Western Blot, Combined Bisulfite Restriction Analysis Assay, In Vitro, Methylation

15) Product Images from "The dose dependent in vitro responses of MCF-7 and MDA-MB-231 cell lines to extracts of Vatica diospyroides symington type SS fruit include effects on mode of cell death"

Article Title: The dose dependent in vitro responses of MCF-7 and MDA-MB-231 cell lines to extracts of Vatica diospyroides symington type SS fruit include effects on mode of cell death

Journal: Pharmacognosy Magazine

doi: 10.4103/0973-1296.157718

Scatter plots indicative of apoptotic patterns for (a) MCF-7 cells untreated and treated with cotyledon extract, and (b) MDA-MB-231 cells untreated and treated with pericarp extract of Vatica diospyroides type SS fruit. Treatment dose levels were half 50% growth inhibition concentrations (IC 50 ), IC 50 and 2-fold IC 50 , for 24 h. The cells were stained with AnnexinV-FITC/PI and analyzed by flow cytometry. Viable, early apoptotic, late apoptotic, and nonviable cells are positioned in the lower left, lower right, upper right and upper left segments of the diagram. Yellow arrows indicate apoptotic progression of cells, whereas red arrow shows necrotic transition. Five thousand events were counted in each assay
Figure Legend Snippet: Scatter plots indicative of apoptotic patterns for (a) MCF-7 cells untreated and treated with cotyledon extract, and (b) MDA-MB-231 cells untreated and treated with pericarp extract of Vatica diospyroides type SS fruit. Treatment dose levels were half 50% growth inhibition concentrations (IC 50 ), IC 50 and 2-fold IC 50 , for 24 h. The cells were stained with AnnexinV-FITC/PI and analyzed by flow cytometry. Viable, early apoptotic, late apoptotic, and nonviable cells are positioned in the lower left, lower right, upper right and upper left segments of the diagram. Yellow arrows indicate apoptotic progression of cells, whereas red arrow shows necrotic transition. Five thousand events were counted in each assay

Techniques Used: Multiple Displacement Amplification, Inhibition, Staining, Flow Cytometry, Cytometry

16) Product Images from "Combining a CD20 Chimeric Antigen Receptor and an Inducible Caspase 9 Suicide Switch to Improve the Efficacy and Safety of T Cell Adoptive Immunotherapy for Lymphoma"

Article Title: Combining a CD20 Chimeric Antigen Receptor and an Inducible Caspase 9 Suicide Switch to Improve the Efficacy and Safety of T Cell Adoptive Immunotherapy for Lymphoma

Journal: PLoS ONE

doi: 10.1371/journal.pone.0082742

CD20-specific effector activity of transduced T cells in vitro. ( A ) Transduced T cells exhibit CD20-specific cytolytic activity in vitro. iC9-CD20CAR-Δ19 T cells transduced with CD20-CAR (CD20-CAR T), or non-transduced (mock) T cells were co-cultured with EL4, Daudi, and Granta in various effector to target (E:T) ratios. Mean percent killing (± SD of triplicate culture) was determined by 4-hour standard 51 C release assay. ( B ) Anti-tumor effect of the transduced T cells. Mock or iC9-CD20CAR-Δ19 transduced T cells were co-cultured with CD20 + Ramos cells in various Effector to Target ratios (E:T) for 48 hours. Cells were then stained with antibodies recognizing CD22 and CD3. Flow cytometric analysis was used to determine the presence of Ramos cells (CD22 + CD3 − ) and T cells (CD22 − CD3 + ). Similar results were obtained with three independent experiments. ( C ) Cytokine production. Expanded T cells secreted IL2, IFN-γ and TNF-α after co-culture with CD20-expressing Ramos target cells for 48 hours (mean ± SD of triplicate samples). There were negligible levels of cytokine production when the expanded T cells were cultured alone with no target cells. Results are representative of three independent experiments.
Figure Legend Snippet: CD20-specific effector activity of transduced T cells in vitro. ( A ) Transduced T cells exhibit CD20-specific cytolytic activity in vitro. iC9-CD20CAR-Δ19 T cells transduced with CD20-CAR (CD20-CAR T), or non-transduced (mock) T cells were co-cultured with EL4, Daudi, and Granta in various effector to target (E:T) ratios. Mean percent killing (± SD of triplicate culture) was determined by 4-hour standard 51 C release assay. ( B ) Anti-tumor effect of the transduced T cells. Mock or iC9-CD20CAR-Δ19 transduced T cells were co-cultured with CD20 + Ramos cells in various Effector to Target ratios (E:T) for 48 hours. Cells were then stained with antibodies recognizing CD22 and CD3. Flow cytometric analysis was used to determine the presence of Ramos cells (CD22 + CD3 − ) and T cells (CD22 − CD3 + ). Similar results were obtained with three independent experiments. ( C ) Cytokine production. Expanded T cells secreted IL2, IFN-γ and TNF-α after co-culture with CD20-expressing Ramos target cells for 48 hours (mean ± SD of triplicate samples). There were negligible levels of cytokine production when the expanded T cells were cultured alone with no target cells. Results are representative of three independent experiments.

Techniques Used: Activity Assay, In Vitro, Transduction, Cell Culture, Release Assay, Staining, Flow Cytometry, Co-Culture Assay, Expressing

Expansion of transduced T cells using the NIH3T3-based AAPCs. ( A ) Rapid expansion of transduced T cells. Eight days after activation with CD3/28 beads, transduced T cells were cultured in plates coated with irradiated NIH3T3-20/80 (broken line) cells or NIH3T3-IV cells (solid line) in the presence of IL-2 (20 U/ml) and IL-15 (10 ng/ml on day 1 and 1 ng/ml subsequently). Cell counts were enumerated every 7 days by trypan blue exclusion. Absolute numbers of cells at various time points are depicted as mean ± SD of triplicate wells. Results are representative of three independent experiments. ( B ) Preferential enrichment of transduced T cells when co-cultured with 3T3-derived AAPCs. Eight days after activation with CD3/28 beads, transduced T cells were cultured in plates coated with irradiated NIH3T3-20/80 cells or NIH3T3-IV cells. The percentage of CD19 + cells was determined every 7 days by flow cytometirc analysis using a PE-conjugated mouse anti-human CD19 antibody. Results are shown as the mean percentage of CD19 + cells ± SD of triplicate wells at various time points and are representative of three independent experiments. ( C ) Central Memory Phenotype of expanded T cells (CD45RO + CD28 + CD62L + ). Flow cytometric analysis of T cells expanded after 2 re-stimulation cycles using NIH3T3 AAPCs was performed to determine the surface immunophenotype. The percent positivity (mean ± SD of triplicate cultures) for CD28, CD62L, CD45RO, and CD45RA was determined by corresponding antibody staining. Cells were gated on CD3 + CD19 + T cells. Similar results were obtained with 4 independent experiments using cells cultured after either 2 or 3 re-stimulation cycles. Results were concordant in 5 experiments.
Figure Legend Snippet: Expansion of transduced T cells using the NIH3T3-based AAPCs. ( A ) Rapid expansion of transduced T cells. Eight days after activation with CD3/28 beads, transduced T cells were cultured in plates coated with irradiated NIH3T3-20/80 (broken line) cells or NIH3T3-IV cells (solid line) in the presence of IL-2 (20 U/ml) and IL-15 (10 ng/ml on day 1 and 1 ng/ml subsequently). Cell counts were enumerated every 7 days by trypan blue exclusion. Absolute numbers of cells at various time points are depicted as mean ± SD of triplicate wells. Results are representative of three independent experiments. ( B ) Preferential enrichment of transduced T cells when co-cultured with 3T3-derived AAPCs. Eight days after activation with CD3/28 beads, transduced T cells were cultured in plates coated with irradiated NIH3T3-20/80 cells or NIH3T3-IV cells. The percentage of CD19 + cells was determined every 7 days by flow cytometirc analysis using a PE-conjugated mouse anti-human CD19 antibody. Results are shown as the mean percentage of CD19 + cells ± SD of triplicate wells at various time points and are representative of three independent experiments. ( C ) Central Memory Phenotype of expanded T cells (CD45RO + CD28 + CD62L + ). Flow cytometric analysis of T cells expanded after 2 re-stimulation cycles using NIH3T3 AAPCs was performed to determine the surface immunophenotype. The percent positivity (mean ± SD of triplicate cultures) for CD28, CD62L, CD45RO, and CD45RA was determined by corresponding antibody staining. Cells were gated on CD3 + CD19 + T cells. Similar results were obtained with 4 independent experiments using cells cultured after either 2 or 3 re-stimulation cycles. Results were concordant in 5 experiments.

Techniques Used: Activation Assay, Cell Culture, Irradiation, Derivative Assay, Flow Cytometry, Staining

17) Product Images from "Polycaprolactone scaffold engineered for sustained release of resveratrol: therapeutic enhancement in bone tissue engineering"

Article Title: Polycaprolactone scaffold engineered for sustained release of resveratrol: therapeutic enhancement in bone tissue engineering

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S49460

Immunophenotyping of cell surface markers of hBMSCs using flow cytometric analysis. ( I ) Positive markers for mesenchymal stem cells: ( A ) CD90-PERCP, ( B ) CD105-APC, ( C ) CD73-PE, ( D ) CD44-FITC, ( E ) CD29-PE, showing higher percentage expression (control and sample as labeled). ( II ) Negative markers for mesenchymal stem cells: ( A ) CD34-PE, ( B ) CD45-APC-Cy7, ( C ) CD133-APC-A, ( D ) CD31-FITC, ( E ) HLA-DR-PERCP show a low percentage expression (control and sample as labeled). Note: The surface marker profile shows that the cells obtained at third passage were mesenchymal stem cells. BM1-P3-DMEM-LG represents bone marrow cells obtained at third passage in DMEM-LG medium. Abbreviations: DMEM-LG, Dulbecco’s Minimum Essential Medium-Low Glucose; hBMSCs, human bone marrow-derived mesenchymal stem cells; SSC, side scatter; BM, bone marrow.
Figure Legend Snippet: Immunophenotyping of cell surface markers of hBMSCs using flow cytometric analysis. ( I ) Positive markers for mesenchymal stem cells: ( A ) CD90-PERCP, ( B ) CD105-APC, ( C ) CD73-PE, ( D ) CD44-FITC, ( E ) CD29-PE, showing higher percentage expression (control and sample as labeled). ( II ) Negative markers for mesenchymal stem cells: ( A ) CD34-PE, ( B ) CD45-APC-Cy7, ( C ) CD133-APC-A, ( D ) CD31-FITC, ( E ) HLA-DR-PERCP show a low percentage expression (control and sample as labeled). Note: The surface marker profile shows that the cells obtained at third passage were mesenchymal stem cells. BM1-P3-DMEM-LG represents bone marrow cells obtained at third passage in DMEM-LG medium. Abbreviations: DMEM-LG, Dulbecco’s Minimum Essential Medium-Low Glucose; hBMSCs, human bone marrow-derived mesenchymal stem cells; SSC, side scatter; BM, bone marrow.

Techniques Used: Flow Cytometry, Expressing, Labeling, Marker, Derivative Assay

18) Product Images from "Dysregulation of Th17 Cells during the Early Post-Transplant Period in Patients under Calcineurin Inhibitor Based Immunosuppression"

Article Title: Dysregulation of Th17 Cells during the Early Post-Transplant Period in Patients under Calcineurin Inhibitor Based Immunosuppression

Journal: PLoS ONE

doi: 10.1371/journal.pone.0042011

Distribution of T naïve , T CM , T EM subpopulations and IL-17 + /T naïve , IL-17 + /T EM and IL-17 + /T CM , subpopulations of CD4 + T lymphocytes at 1 and 3 month after transplantation compared to before transplantation. PBMC from patients before KT, patients at 1month after KT and patients at 3 month after KT were stimulated for 4 h ex vivo with PMA and ionomycin in the presence of GolgiStop.CD4 + lymphocytes were stained with mAbs to CD45RA and CCR7, which identified three subsets. In addition, analysis of IL-17 in CD4 + T cell subsets by intracellular flow cytometry was done. After surface staining with CD45 and CCR7 mAbs, cells were fixated and permeabilized and intracellular accumulated cytokines were detected with IL-17 mAbs. (A) T naïve /CD4 + T (CD45RA + CCR7 + /CD4 + Tcells), (B) IL-17 + /T naïve , (C) T CM /CD4 + T (CD45RA − CCR7 + /CD4 + Tcells), (D) IL-17 + /T CM + , (E) T EM /CD4 + T (CD45RA − CCR7 − /CD4 + Tcells), (F) IL-17 + /T EM + . Bars show the means. * P
Figure Legend Snippet: Distribution of T naïve , T CM , T EM subpopulations and IL-17 + /T naïve , IL-17 + /T EM and IL-17 + /T CM , subpopulations of CD4 + T lymphocytes at 1 and 3 month after transplantation compared to before transplantation. PBMC from patients before KT, patients at 1month after KT and patients at 3 month after KT were stimulated for 4 h ex vivo with PMA and ionomycin in the presence of GolgiStop.CD4 + lymphocytes were stained with mAbs to CD45RA and CCR7, which identified three subsets. In addition, analysis of IL-17 in CD4 + T cell subsets by intracellular flow cytometry was done. After surface staining with CD45 and CCR7 mAbs, cells were fixated and permeabilized and intracellular accumulated cytokines were detected with IL-17 mAbs. (A) T naïve /CD4 + T (CD45RA + CCR7 + /CD4 + Tcells), (B) IL-17 + /T naïve , (C) T CM /CD4 + T (CD45RA − CCR7 + /CD4 + Tcells), (D) IL-17 + /T CM + , (E) T EM /CD4 + T (CD45RA − CCR7 − /CD4 + Tcells), (F) IL-17 + /T EM + . Bars show the means. * P

Techniques Used: Transplantation Assay, Ex Vivo, Staining, Flow Cytometry, Cytometry

Expression of IL-1beta, and HMGB1 associated with Th17 cell at 1 and 3 month after transplantation compared to before transplantation. PBMC from patients before KT, patients at 1month after KT and patients at 3 month after KT were stimulated for 4 h ex vivo with PMA and ionomycin in the presence of GolgiStop. PBMC from all groups were treated as described in Figure 1 and Materials and Methods . The expression of IL-1beta (A), HMGB1(B) mRNA was measured using real-time PCR. Bars show the means.
Figure Legend Snippet: Expression of IL-1beta, and HMGB1 associated with Th17 cell at 1 and 3 month after transplantation compared to before transplantation. PBMC from patients before KT, patients at 1month after KT and patients at 3 month after KT were stimulated for 4 h ex vivo with PMA and ionomycin in the presence of GolgiStop. PBMC from all groups were treated as described in Figure 1 and Materials and Methods . The expression of IL-1beta (A), HMGB1(B) mRNA was measured using real-time PCR. Bars show the means.

Techniques Used: Expressing, Transplantation Assay, Ex Vivo, Real-time Polymerase Chain Reaction

Distribution of lymphocyte and CD4+ T cell subtype at 1 and 3 month after transplantation compared to before transplantation. PBMC from patients before KT, patients at 1month after KT and patients at 3 month after KT were stimulated for 4 h ex vivo with PMA and ionomycin in the presence of Golgi Stop. The percentage of Target cells was measured by flowcytometry. The frequency (%) of Lymphocyte/Leukocyte cells (A), CD4 + T/Lymphocyte cells (B), IFN-γ + /CD4 + T cells (C), IL-4 + /CD4 + T cells (D) CD25 + FOXP3 + /CD4 + T cells (E) and IL-17 + /CD4 + T cells (F) in patients before KT, patients at 1month after KT and patients at 3 month after KT. Bars show the means. * P
Figure Legend Snippet: Distribution of lymphocyte and CD4+ T cell subtype at 1 and 3 month after transplantation compared to before transplantation. PBMC from patients before KT, patients at 1month after KT and patients at 3 month after KT were stimulated for 4 h ex vivo with PMA and ionomycin in the presence of Golgi Stop. The percentage of Target cells was measured by flowcytometry. The frequency (%) of Lymphocyte/Leukocyte cells (A), CD4 + T/Lymphocyte cells (B), IFN-γ + /CD4 + T cells (C), IL-4 + /CD4 + T cells (D) CD25 + FOXP3 + /CD4 + T cells (E) and IL-17 + /CD4 + T cells (F) in patients before KT, patients at 1month after KT and patients at 3 month after KT. Bars show the means. * P

Techniques Used: Transplantation Assay, Ex Vivo

Effects of Tac in Th1, Th2, Th17 and Treg subpopulations of CD4+T lymphocytes from the peripheral blood of healthy donors. PBMC were preincubated for 1 h in the presence of Tac and stimulated with 1 µg/ml anti-CD3 and anti-CD28. Flow cytometry of intracellular IFN-r (A), IL-4 (B), Th17 (C) and Treg (D) in CD4+ T cells stimulated in the presence of plate-bound anti-CD3 plus anti-CD28, assessed after 48 h and then stimulated for 4 h with PMA and ionomycin in the presence of GolgiStop. The data are representative of three independent experiments. The values are expressed as the mean ± SEM. * P
Figure Legend Snippet: Effects of Tac in Th1, Th2, Th17 and Treg subpopulations of CD4+T lymphocytes from the peripheral blood of healthy donors. PBMC were preincubated for 1 h in the presence of Tac and stimulated with 1 µg/ml anti-CD3 and anti-CD28. Flow cytometry of intracellular IFN-r (A), IL-4 (B), Th17 (C) and Treg (D) in CD4+ T cells stimulated in the presence of plate-bound anti-CD3 plus anti-CD28, assessed after 48 h and then stimulated for 4 h with PMA and ionomycin in the presence of GolgiStop. The data are representative of three independent experiments. The values are expressed as the mean ± SEM. * P

Techniques Used: Flow Cytometry, Cytometry

Effect of Tac in Th17 subpopulations of CD4+T lymphocytes from the peripheral blood of early-post transplant recipients. We used flow cytometry to examine how Tacrolimus regulates in vitro Th17 subpopulations of CD4+ T lymphocytes in Th17-polarizing condition. PBMC from renal transplant recipients were preincubated for 1 h in the presence of Tacrolimus and stimulated with Th17-polarizing condition for 48 h. Anti-CD3 (1 µg/ml), anti-CD28 (1 µg/ml), IL-1b (20 ng/ml), IL-6 (20 ng/ml) and IL-23 (20 ng/ml) were added to stimulate the differentiation of Th17 cells. Neutralizing antibodies to IFN-gamma (2 µg/ml) and IL-4 (2 µg/ml) were added in some experiments (R D Systems). Flow cytometry of intracellular Th17 in CD4+ T cells stimulated in the presence of Th17-polarizing condition, assessed after 48 h and then stimulated for 4 h with PMA and ionomycin in the presence of GolgiStop. The data are representative of three independent experiments. The values are expressed as the mean ± SEM.
Figure Legend Snippet: Effect of Tac in Th17 subpopulations of CD4+T lymphocytes from the peripheral blood of early-post transplant recipients. We used flow cytometry to examine how Tacrolimus regulates in vitro Th17 subpopulations of CD4+ T lymphocytes in Th17-polarizing condition. PBMC from renal transplant recipients were preincubated for 1 h in the presence of Tacrolimus and stimulated with Th17-polarizing condition for 48 h. Anti-CD3 (1 µg/ml), anti-CD28 (1 µg/ml), IL-1b (20 ng/ml), IL-6 (20 ng/ml) and IL-23 (20 ng/ml) were added to stimulate the differentiation of Th17 cells. Neutralizing antibodies to IFN-gamma (2 µg/ml) and IL-4 (2 µg/ml) were added in some experiments (R D Systems). Flow cytometry of intracellular Th17 in CD4+ T cells stimulated in the presence of Th17-polarizing condition, assessed after 48 h and then stimulated for 4 h with PMA and ionomycin in the presence of GolgiStop. The data are representative of three independent experiments. The values are expressed as the mean ± SEM.

Techniques Used: Flow Cytometry, Cytometry, In Vitro

Flow cytometric analysis of T cell subsets. PBMCs were stained with anti-CD4 PE-cy7, anti-CD25 APC, anti-IFN-γ FITC, anti-IL-17 PE, anti-IL-4 APC and anti-Foxp3 FITC. CD4+ cells were gated for further analysis. PBMC from patients before KT, patients at 1month after KT and patients at 3 month after KT were stimulated for 4 h ex vivo with PMA and ionomycin in the presence of Golgi Stop. The percentage of Target cells was measured by flowcytometry. The frequency (%) of Lymphocyte/Leukocyte cells, CD4 + T/Lymphocyte cells, IL-17+/CD4 + T cells, IFN-γ + /CD4 + T cells, IL-4 + /CD4 + T cells (A) and CD25 + FOXP3 + /CD4 + T cells (B) in patients before KT, patients at 1month after KT and patients at 3 month after KT. After surface staining with anti-CD4, CD45 and CCR7 mAbs, cells were fixated and permeabilized and intracellular accumulated cytokines were detected with IL-17 mAbs. T naïve /CD4 + T (CD45RA + CCR7 + /CD4 + Tcells), IL-17 + /T naïve , T CM /CD4 + T (CD45RA − CCR7 + /CD4 + Tcells), IL-17 + /T CM + and T EM /CD4 + T (CD45RA − CCR7 − /CD4 + Tcells), IL-17 + /T EM + (C).
Figure Legend Snippet: Flow cytometric analysis of T cell subsets. PBMCs were stained with anti-CD4 PE-cy7, anti-CD25 APC, anti-IFN-γ FITC, anti-IL-17 PE, anti-IL-4 APC and anti-Foxp3 FITC. CD4+ cells were gated for further analysis. PBMC from patients before KT, patients at 1month after KT and patients at 3 month after KT were stimulated for 4 h ex vivo with PMA and ionomycin in the presence of Golgi Stop. The percentage of Target cells was measured by flowcytometry. The frequency (%) of Lymphocyte/Leukocyte cells, CD4 + T/Lymphocyte cells, IL-17+/CD4 + T cells, IFN-γ + /CD4 + T cells, IL-4 + /CD4 + T cells (A) and CD25 + FOXP3 + /CD4 + T cells (B) in patients before KT, patients at 1month after KT and patients at 3 month after KT. After surface staining with anti-CD4, CD45 and CCR7 mAbs, cells were fixated and permeabilized and intracellular accumulated cytokines were detected with IL-17 mAbs. T naïve /CD4 + T (CD45RA + CCR7 + /CD4 + Tcells), IL-17 + /T naïve , T CM /CD4 + T (CD45RA − CCR7 + /CD4 + Tcells), IL-17 + /T CM + and T EM /CD4 + T (CD45RA − CCR7 − /CD4 + Tcells), IL-17 + /T EM + (C).

Techniques Used: Flow Cytometry, Staining, Ex Vivo

19) Product Images from "Minocycline Down-Regulates Topical Mucosal Inflammation during the Application of Microbicide Candidates"

Article Title: Minocycline Down-Regulates Topical Mucosal Inflammation during the Application of Microbicide Candidates

Journal: PLoS ONE

doi: 10.1371/journal.pone.0043211

Dynamic profiles of different inflammatory cytokines after intravaginal application of different gel formulations. The inflammatory cytokines ((a) IL-10, (b) IL-2, (c) TNF-α, (d) IL-4, (e) IFN-γ, (f) IL-6, (g) IL-17A) for each mouse were quantified by CBA assay. The X-axis indicates the days after the administration of gels. Y-axis indicates the production of corresponding inflammatory cytokines. Data were represented as means ± SD from triplicate samples of CVLs pooled from 5 mice. In the graph, CS indicates gel containing 60 mg/ml CS alone; CSm indicates combined gel containing both 60 mg/ml CS and 50 μg/ml minocycline; HEC indicates a HEC placebo gel and HECm indicates HEC gel containing 50 μg/ml minocycline. ANOVA and Tukey's post-hoc tests were performed among different groups. The significant difference ( p
Figure Legend Snippet: Dynamic profiles of different inflammatory cytokines after intravaginal application of different gel formulations. The inflammatory cytokines ((a) IL-10, (b) IL-2, (c) TNF-α, (d) IL-4, (e) IFN-γ, (f) IL-6, (g) IL-17A) for each mouse were quantified by CBA assay. The X-axis indicates the days after the administration of gels. Y-axis indicates the production of corresponding inflammatory cytokines. Data were represented as means ± SD from triplicate samples of CVLs pooled from 5 mice. In the graph, CS indicates gel containing 60 mg/ml CS alone; CSm indicates combined gel containing both 60 mg/ml CS and 50 μg/ml minocycline; HEC indicates a HEC placebo gel and HECm indicates HEC gel containing 50 μg/ml minocycline. ANOVA and Tukey's post-hoc tests were performed among different groups. The significant difference ( p

Techniques Used: Crocin Bleaching Assay, Mouse Assay

20) Product Images from "ABCB1 haplotypes are associated with P-gp activity and affect a major molecular response in chronic myeloid leukemia patients treated with a standard dose of imatinib"

Article Title: ABCB1 haplotypes are associated with P-gp activity and affect a major molecular response in chronic myeloid leukemia patients treated with a standard dose of imatinib

Journal: Oncology Letters

doi: 10.3892/ol.2014.1857

Effect of ATP-binding cassette subfamily B member 1 haplotypes on P-gp activity. P-gp activity was measured as a percentage of the Rh123 efflux and the medians were compared using the Mann-Whitney U test. The haplotype groups were as follows: Wild-type, 1236CC/3435CC/2677GG; and mutated, 1236CT/3435CT/2677GT and 1236TT/3435TT/2677TT. P-gp, P-glycoprotein; Rh123, rhodamine 123.
Figure Legend Snippet: Effect of ATP-binding cassette subfamily B member 1 haplotypes on P-gp activity. P-gp activity was measured as a percentage of the Rh123 efflux and the medians were compared using the Mann-Whitney U test. The haplotype groups were as follows: Wild-type, 1236CC/3435CC/2677GG; and mutated, 1236CT/3435CT/2677GT and 1236TT/3435TT/2677TT. P-gp, P-glycoprotein; Rh123, rhodamine 123.

Techniques Used: Binding Assay, Activity Assay, MANN-WHITNEY

21) Product Images from "Robust Phenotypic Activation of Eosinophils during Experimental Toxocara canis Infection"

Article Title: Robust Phenotypic Activation of Eosinophils during Experimental Toxocara canis Infection

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00064

Impact of in vitro stimulation with Toxocara canis antigens on bone marrow-derived eosinophils. In vitro T. canis antigen stimulation was performed on bone marrow - derived eosinophils from T. canis -infected as well as non-infected control groups. Flow cytometric evaluation of major histocompatibility complex (MHC-II), CD80, CD86, and CD69 within Siglec-F + -eosinophils was performed by ImageStream Mark II and analyzed by the Ideas software. Activation and co-stimulatory-related molecules were assessed in Siglec-F + -eosinophils by mean fluorescence intensity (MFI) assessed or percentage of positive cells as verified by histograms.
Figure Legend Snippet: Impact of in vitro stimulation with Toxocara canis antigens on bone marrow-derived eosinophils. In vitro T. canis antigen stimulation was performed on bone marrow - derived eosinophils from T. canis -infected as well as non-infected control groups. Flow cytometric evaluation of major histocompatibility complex (MHC-II), CD80, CD86, and CD69 within Siglec-F + -eosinophils was performed by ImageStream Mark II and analyzed by the Ideas software. Activation and co-stimulatory-related molecules were assessed in Siglec-F + -eosinophils by mean fluorescence intensity (MFI) assessed or percentage of positive cells as verified by histograms.

Techniques Used: In Vitro, Derivative Assay, Infection, Flow Cytometry, Software, Activation Assay, Fluorescence

Impact of in vitro Toxocara canis antigen stimulation on cell surface molecule expression by bone marrow-derived eosinophils. In vitro T. canis antigen stimulation was performed on bone marrow - derived eosinophils from T. canis -infected as well as non-infected control groups. Control cultures (CC) and antigen-stimulated ones (TcAg) were incubated as described in Section “ Materials and Methods ” and examined by flow cytometric evaluation of CD69, major histocompatibility complex (MHC-II), CD80, and CD86 within Siglec-F + -eosinophils displaying either FSC HIGH (dark shades) or FSC LOW (light shades) phenotype. Activation and antigen presentation-related molecules were also assessed in Siglec-F + -eosinophils by mean fluorescence intensity (MFI) or percentage of positive cells and plotted as bar graphs. In addition, fold-changes of TcAg versus CC were calculated to assess the impact of T. canis Ag stimuli and expressed as index of stimulation (TcAg/CC INDEX), which was plotted as bar graphs.
Figure Legend Snippet: Impact of in vitro Toxocara canis antigen stimulation on cell surface molecule expression by bone marrow-derived eosinophils. In vitro T. canis antigen stimulation was performed on bone marrow - derived eosinophils from T. canis -infected as well as non-infected control groups. Control cultures (CC) and antigen-stimulated ones (TcAg) were incubated as described in Section “ Materials and Methods ” and examined by flow cytometric evaluation of CD69, major histocompatibility complex (MHC-II), CD80, and CD86 within Siglec-F + -eosinophils displaying either FSC HIGH (dark shades) or FSC LOW (light shades) phenotype. Activation and antigen presentation-related molecules were also assessed in Siglec-F + -eosinophils by mean fluorescence intensity (MFI) or percentage of positive cells and plotted as bar graphs. In addition, fold-changes of TcAg versus CC were calculated to assess the impact of T. canis Ag stimuli and expressed as index of stimulation (TcAg/CC INDEX), which was plotted as bar graphs.

Techniques Used: In Vitro, Expressing, Derivative Assay, Infection, Incubation, Flow Cytometry, Activation Assay, Fluorescence

22) Product Images from "Cathelicidins prime platelets to mediate arterial thrombosis and tissue inflammation"

Article Title: Cathelicidins prime platelets to mediate arterial thrombosis and tissue inflammation

Journal: Nature Communications

doi: 10.1038/s41467-018-03925-2

Cathelicidins promote lung injury. a , b Representative histology images of lung tissue obtained from patients with acute pneumonia and two control subjects without tissue inflammation. Patient 1 presented with aspiration pneumonia. Immunohistochemistry for LL-37 ( a , b ) and hematoxylin-eosin (H E) staining ( b ). Bars, 100 µm ( a ) or 50 µm ( b ). c – t Mouse model of acute lung injury (ALI) induced by intratracheal injection of 0.1 M HCl. c Representative images of H E stained mouse lung tissue after ALI in wild type and CRAMP − / − mice. Bar, 50 µm. d Alveolar neutrophil counts, e number of interstitial (including pulmonary intravascular) neutrophils, and f alveolar permeability after ALI in wild type ( n = 6) and CRAMP − / − mice ( n = 7). g Alveolar neutrophils and h interstitial (including pulmonary intravascular) neutrophils after ALI in wild type (wt → wt) and CRAMP − / − (ko → wt) chimeras ( n = 7). i – k Analysis of citrullinated histone H3 (citH3) staining in Ly6G+ neutrophils. i Representative image of lung tissue after ALI. CitH3 (green), Ly6G (red), DAPI (nuclear stain, blue). Bar, 10 µm. j , k Quantification of citH3 staining in Ly6G+ neutrophils ( n = 6). l Alveolar permeability after ALI ( n = 7). m , n Flow cytometry analysis of platelet–neutrophil aggregates after ALI in m systemic circulation ( n = 6) and n the pulmonary vascular compartment ( n = 7). o – r Flow cytometry analysis of o P-selectin surface expression, p GPIIb/IIIa activation, q HMGB1 surface expression, and r phosphatidylserine (PS) exposure on platelets. s Survival curves and t oxygen saturations after 30 min after ALI induction ( n = 6). Graphs show mean and SEM. P- values were determined by Mann–Whitney U -test ( d , f , g , h , l , n ), unpaired t -test ( e , j , k , m , o , p , q , r , t ), or Log-rank (Mantel-Cox) test ( s )
Figure Legend Snippet: Cathelicidins promote lung injury. a , b Representative histology images of lung tissue obtained from patients with acute pneumonia and two control subjects without tissue inflammation. Patient 1 presented with aspiration pneumonia. Immunohistochemistry for LL-37 ( a , b ) and hematoxylin-eosin (H E) staining ( b ). Bars, 100 µm ( a ) or 50 µm ( b ). c – t Mouse model of acute lung injury (ALI) induced by intratracheal injection of 0.1 M HCl. c Representative images of H E stained mouse lung tissue after ALI in wild type and CRAMP − / − mice. Bar, 50 µm. d Alveolar neutrophil counts, e number of interstitial (including pulmonary intravascular) neutrophils, and f alveolar permeability after ALI in wild type ( n = 6) and CRAMP − / − mice ( n = 7). g Alveolar neutrophils and h interstitial (including pulmonary intravascular) neutrophils after ALI in wild type (wt → wt) and CRAMP − / − (ko → wt) chimeras ( n = 7). i – k Analysis of citrullinated histone H3 (citH3) staining in Ly6G+ neutrophils. i Representative image of lung tissue after ALI. CitH3 (green), Ly6G (red), DAPI (nuclear stain, blue). Bar, 10 µm. j , k Quantification of citH3 staining in Ly6G+ neutrophils ( n = 6). l Alveolar permeability after ALI ( n = 7). m , n Flow cytometry analysis of platelet–neutrophil aggregates after ALI in m systemic circulation ( n = 6) and n the pulmonary vascular compartment ( n = 7). o – r Flow cytometry analysis of o P-selectin surface expression, p GPIIb/IIIa activation, q HMGB1 surface expression, and r phosphatidylserine (PS) exposure on platelets. s Survival curves and t oxygen saturations after 30 min after ALI induction ( n = 6). Graphs show mean and SEM. P- values were determined by Mann–Whitney U -test ( d , f , g , h , l , n ), unpaired t -test ( e , j , k , m , o , p , q , r , t ), or Log-rank (Mantel-Cox) test ( s )

Techniques Used: Immunohistochemistry, Staining, Injection, Mouse Assay, Permeability, Flow Cytometry, Cytometry, Expressing, Activation Assay, MANN-WHITNEY

Cathelicidin-dependent signaling in platelets. a – i LL-37 induced signaling in isolated human platelets. a – c Flow cytometry analysis of platelet P-selectin surface expression in the presence of a the calcium chelator BAPTA ( n = 5) or a phospholipase C inhibitor (U-73122, n = 6), b pertussis or cholera toxin to inhibit G-protein signaling ( n = 4), or c inhibitors of tyrosine kinases Src-family kinases (Dasatinib, n = 7) and Syk (R406, n = 5). d Representative western blots of phosphorylated Src-family kinase and phosphorylated Syk upon incubation of platelets with LL-37. Collagen was used as positive control for tyrosine kinase phosphorylation, β-actin served as loading control. Images are representative of three independent blots. e – i Flow cytometry analysis of LL-37 platelet P-selectin surface expression in the presence of e STAT3 small molecule inhibitor (Stattic, n = 6), f GPVI antibody (HGP5C4, n = 9), g GPIIb/IIIa antibody (Abciximab or Tirofiban, n = 4), h formyl-peptid-receptor (FPR1 or FPR2) antibody, and i inhibitors against the purinergic P2X 7 -receptor (Boc-MLF 10, WRW4 or A438079, n = 4). j – l CRAMP-induced signaling in isolated mouse platelets. j P-selectin surface expression in the presence of a GPVI depleting antibody (JAQ1, n = 6). k P-selectin surface expression and l phospholipase C phosphorylation in platelet lysates from platelet-specific Syk-deficient mice and respective littermates after stimulation with CRAMP (flow cytometry: n = 10 Syk −/− and n = 5 Syk + / + animals, western blot analysis n = 5 each). Graphs show mean and SEM. P- values were determined by unpaired ( a , f , j – l ) or paired ( c , e , h ) t -test
Figure Legend Snippet: Cathelicidin-dependent signaling in platelets. a – i LL-37 induced signaling in isolated human platelets. a – c Flow cytometry analysis of platelet P-selectin surface expression in the presence of a the calcium chelator BAPTA ( n = 5) or a phospholipase C inhibitor (U-73122, n = 6), b pertussis or cholera toxin to inhibit G-protein signaling ( n = 4), or c inhibitors of tyrosine kinases Src-family kinases (Dasatinib, n = 7) and Syk (R406, n = 5). d Representative western blots of phosphorylated Src-family kinase and phosphorylated Syk upon incubation of platelets with LL-37. Collagen was used as positive control for tyrosine kinase phosphorylation, β-actin served as loading control. Images are representative of three independent blots. e – i Flow cytometry analysis of LL-37 platelet P-selectin surface expression in the presence of e STAT3 small molecule inhibitor (Stattic, n = 6), f GPVI antibody (HGP5C4, n = 9), g GPIIb/IIIa antibody (Abciximab or Tirofiban, n = 4), h formyl-peptid-receptor (FPR1 or FPR2) antibody, and i inhibitors against the purinergic P2X 7 -receptor (Boc-MLF 10, WRW4 or A438079, n = 4). j – l CRAMP-induced signaling in isolated mouse platelets. j P-selectin surface expression in the presence of a GPVI depleting antibody (JAQ1, n = 6). k P-selectin surface expression and l phospholipase C phosphorylation in platelet lysates from platelet-specific Syk-deficient mice and respective littermates after stimulation with CRAMP (flow cytometry: n = 10 Syk −/− and n = 5 Syk + / + animals, western blot analysis n = 5 each). Graphs show mean and SEM. P- values were determined by unpaired ( a , f , j – l ) or paired ( c , e , h ) t -test

Techniques Used: Isolation, Flow Cytometry, Cytometry, Expressing, Western Blot, Incubation, Positive Control, Mouse Assay

Cathelicidins induce platelet activation and secretion. a – f Incubation of isolated human platelets for 15 min with increasing concentrations of LL-37 or scrambled control peptide (Scra; applied Scra concentration as indicated or equivalent to the maximal LL-37 concentration). ADP (5 µmol/L), collagen (Coll, 5 µg/mL), thrombin (Thr, Thrombin 0.05 U/mL), or GPVI receptor activating antibody HGP4C9 (1 µg/mL) were used for comparison. a Flow cytometric analysis of P-selectin surface expression ( n = 6). b CD40L release into the supernatant as measured by ELISA ( n = 3). Flow cytometry analysis of c CD40 ligand (CD40L) surface expression ( n = 4), d intracellular IL-1β ( n = 5), e HMGB1 surface expression ( n = 4), and f GPIIb/IIIa receptor activation ( n = 6). g – i Flow cytometry analysis of CRAMP-induced activation of isolated mouse platelets. g P-selectin ( n = 5), h CD40L ( n = 4), and i activated GPIIb/IIIa ( n = 7). Thrombin (Thr, 0.5 U/mL) was used for comparison. Graphs show mean and SEM. P- values were determined by one-way repeated measures ANOVA with Bonferroni correction ( a , c – e ), paired t -test ( b , f ), or unpaired t -test ( g – i )
Figure Legend Snippet: Cathelicidins induce platelet activation and secretion. a – f Incubation of isolated human platelets for 15 min with increasing concentrations of LL-37 or scrambled control peptide (Scra; applied Scra concentration as indicated or equivalent to the maximal LL-37 concentration). ADP (5 µmol/L), collagen (Coll, 5 µg/mL), thrombin (Thr, Thrombin 0.05 U/mL), or GPVI receptor activating antibody HGP4C9 (1 µg/mL) were used for comparison. a Flow cytometric analysis of P-selectin surface expression ( n = 6). b CD40L release into the supernatant as measured by ELISA ( n = 3). Flow cytometry analysis of c CD40 ligand (CD40L) surface expression ( n = 4), d intracellular IL-1β ( n = 5), e HMGB1 surface expression ( n = 4), and f GPIIb/IIIa receptor activation ( n = 6). g – i Flow cytometry analysis of CRAMP-induced activation of isolated mouse platelets. g P-selectin ( n = 5), h CD40L ( n = 4), and i activated GPIIb/IIIa ( n = 7). Thrombin (Thr, 0.5 U/mL) was used for comparison. Graphs show mean and SEM. P- values were determined by one-way repeated measures ANOVA with Bonferroni correction ( a , c – e ), paired t -test ( b , f ), or unpaired t -test ( g – i )

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

23) Product Images from "Protease Activity of Campylobacter jejuni HtrA Modulates Distinct Intestinal and Systemic Immune Responses in Infected Secondary Abiotic IL-10 Deficient Mice"

Article Title: Protease Activity of Campylobacter jejuni HtrA Modulates Distinct Intestinal and Systemic Immune Responses in Infected Secondary Abiotic IL-10 Deficient Mice

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2019.00079

Systemic secretion of pro-inflammatory cytokines in C. jejuni infected secondary abiotic IL-10 −/− mice. Secondary abiotic IL-10 −/− mice were perorally infected either with the C. jejuni 11168 HtrA−S197A strain (closed circles) or the isogenic htrA mutant 11168 HtrA−S197A (open circles) by gavage on days 0 and 1. (A) MCP-1, (B) IL-6, (C) TNF, and (D) IFN-γ concentrations were measured in serum samples taken at day 6 postinfection. Naive mice (open diamonds) served as uninfected controls. Medians (black bars), levels of significance ( p -value) determined by the Mann-Whitney U -test and numbers of analyzed animals (in parentheses) are indicated. Data representative for four independent experiments are shown.
Figure Legend Snippet: Systemic secretion of pro-inflammatory cytokines in C. jejuni infected secondary abiotic IL-10 −/− mice. Secondary abiotic IL-10 −/− mice were perorally infected either with the C. jejuni 11168 HtrA−S197A strain (closed circles) or the isogenic htrA mutant 11168 HtrA−S197A (open circles) by gavage on days 0 and 1. (A) MCP-1, (B) IL-6, (C) TNF, and (D) IFN-γ concentrations were measured in serum samples taken at day 6 postinfection. Naive mice (open diamonds) served as uninfected controls. Medians (black bars), levels of significance ( p -value) determined by the Mann-Whitney U -test and numbers of analyzed animals (in parentheses) are indicated. Data representative for four independent experiments are shown.

Techniques Used: Infection, Mouse Assay, Mutagenesis, MANN-WHITNEY

Colonic secretion of pro-inflammatory cytokines in C. jejuni infected secondary abiotic IL-10 −/− mice. Secondary abiotic IL-10 −/− mice were perorally infected either with the C. jejuni 11168 HtrA−S197A strain (closed circles) or the isogenic htrA mutant 11168 HtrA−S197A (open circles) by gavage on days 0 and 1. (A) MCP-1, (B) IL-6, (C) TNF, and (D) IFN-γ concentrations were measured in supernatants derived from colonic ex vivo biopsies at day 6 postinfection. Naive mice (open diamonds) served as uninfected controls. Medians (black bars), levels of significance ( p -value) determined by the Mann-Whitney U -test and numbers of analyzed animals (in parentheses) are indicated. Data representative for four independent experiments are shown.
Figure Legend Snippet: Colonic secretion of pro-inflammatory cytokines in C. jejuni infected secondary abiotic IL-10 −/− mice. Secondary abiotic IL-10 −/− mice were perorally infected either with the C. jejuni 11168 HtrA−S197A strain (closed circles) or the isogenic htrA mutant 11168 HtrA−S197A (open circles) by gavage on days 0 and 1. (A) MCP-1, (B) IL-6, (C) TNF, and (D) IFN-γ concentrations were measured in supernatants derived from colonic ex vivo biopsies at day 6 postinfection. Naive mice (open diamonds) served as uninfected controls. Medians (black bars), levels of significance ( p -value) determined by the Mann-Whitney U -test and numbers of analyzed animals (in parentheses) are indicated. Data representative for four independent experiments are shown.

Techniques Used: Infection, Mouse Assay, Mutagenesis, Derivative Assay, Ex Vivo, MANN-WHITNEY

24) Product Images from "Folate Deficiency Triggered Apoptosis of Synoviocytes: Role of Overproduction of Reactive Oxygen Species Generated via NADPH Oxidase/Mitochondrial Complex II and Calcium Perturbation"

Article Title: Folate Deficiency Triggered Apoptosis of Synoviocytes: Role of Overproduction of Reactive Oxygen Species Generated via NADPH Oxidase/Mitochondrial Complex II and Calcium Perturbation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0146440

Folate deficiency provokes apoptotic lethality in synoviocytes. HIG-82 synoviocytes (1.5×10 5 ) were plated in 60-mm cultured dishes for 24 h. The culture medium was replaced with FC, MFD, and FD media and then continued cultivating for additional 48 h. (A) Cells were then collected, washed with PBS, fixed in PBS-methanol (1:2 v/v) solution and maintained at 4°C for at least 18 h. After one washed with PBS, the cell pellets were then stained with a PI solution containing PBS, PI (40μg/mL), and DNase-free RNase A (40μg/mL) for 30 min at RT in the dark. The cell pellets were then analyzed using a Becton-Dickinson FACSan flowcytometer. The epirubicin (500 nM) treatment (Epi) is a positive control assay of apoptosis. The blank bar, gray bar, right slash bar and left slash bar represent FC, MFD, FD and Epi treatment, respectively. The percentages of subG1 population determined by the PI fluorescent intensity in apoptosis cells which was weaker than that of cells in the G1 phase. The percentages of apoptosis cells were characterized as the percentages of cells in the SubG1 region of the DNA distribution histograms. The FD subG1 bar graph is compared with FC or MFD. A p
Figure Legend Snippet: Folate deficiency provokes apoptotic lethality in synoviocytes. HIG-82 synoviocytes (1.5×10 5 ) were plated in 60-mm cultured dishes for 24 h. The culture medium was replaced with FC, MFD, and FD media and then continued cultivating for additional 48 h. (A) Cells were then collected, washed with PBS, fixed in PBS-methanol (1:2 v/v) solution and maintained at 4°C for at least 18 h. After one washed with PBS, the cell pellets were then stained with a PI solution containing PBS, PI (40μg/mL), and DNase-free RNase A (40μg/mL) for 30 min at RT in the dark. The cell pellets were then analyzed using a Becton-Dickinson FACSan flowcytometer. The epirubicin (500 nM) treatment (Epi) is a positive control assay of apoptosis. The blank bar, gray bar, right slash bar and left slash bar represent FC, MFD, FD and Epi treatment, respectively. The percentages of subG1 population determined by the PI fluorescent intensity in apoptosis cells which was weaker than that of cells in the G1 phase. The percentages of apoptosis cells were characterized as the percentages of cells in the SubG1 region of the DNA distribution histograms. The FD subG1 bar graph is compared with FC or MFD. A p

Techniques Used: Cell Culture, Staining, Positive Control Assay

25) Product Images from "Isolation of syncytiotrophoblast microvesicles and exosomes and their characterisation by multicolour flow cytometry and fluorescence Nanoparticle Tracking Analysis"

Article Title: Isolation of syncytiotrophoblast microvesicles and exosomes and their characterisation by multicolour flow cytometry and fluorescence Nanoparticle Tracking Analysis

Journal: Methods (San Diego, Calif.)

doi: 10.1016/j.ymeth.2015.03.028

Immunobead depletion and Fluorescence-NTA (fl-NTA) measurements ( n = 3 replicates) of 10,000× g (10KP) and 150,000× g (150KP) fraction pools. (Ai) Representative immunoblot image of placental alkaline phosphatase (PLAP) in the 10KP pool showing bound EV (PLAP positive) or those remaining in the supernatant (SN; PLAP negative) with increasing doses of IgG1 Dynabeads or anti-PLAP Dynabeads. (Aii) NTA of 10KP pool alone (black line) or post incubation with a saturating concentration [4 × 10 7 ] of IgG1 Dynabeads (blue line) or anti-PLAP Dynabeads (red line). (Aiii) fl-NTA of the 10KP pool measured in; scatter mode: alone (black line), labelled with IgG1-Qdot605 control (blue solid line) or labelled with anti-PLAP-Qdot605 antibody (red solid line) and fluorescence mode: labelled with IgG1-Qdot605 control (blue dash) or anti-PLAP-Qdot605 antibody (red dash). (Bi) Representative immunoblot image of PLAP in the 150KP pool showing bound EV (PLAP positive) or those remaining in the supernatant (SN; PLAP negative) in response to an increasing dose (0.2–2 × 10 7 ) of IgG1 Dynabeads or anti-PLAP Dynabeads. (Bii) NTA of 150KP pool alone (black line) or post incubation with a saturating concentration [1 × 10 7 ] of IgG1 Dynabeads (blue line) or anti-PLAP Dynabeads (red line). (Biii) Fl-NTA of the 150KP pool measured in; scatter mode: alone (black line), labelled with IgG1-Qdot605 control (blue solid line) or labelled with anti-PLAP-Qdot605 antibody (red solid line) and fluorescence mode: labelled with IgG1-Qdot605 control (blue dash) or anti-PLAP-Qdot605 antibody (red dash).
Figure Legend Snippet: Immunobead depletion and Fluorescence-NTA (fl-NTA) measurements ( n = 3 replicates) of 10,000× g (10KP) and 150,000× g (150KP) fraction pools. (Ai) Representative immunoblot image of placental alkaline phosphatase (PLAP) in the 10KP pool showing bound EV (PLAP positive) or those remaining in the supernatant (SN; PLAP negative) with increasing doses of IgG1 Dynabeads or anti-PLAP Dynabeads. (Aii) NTA of 10KP pool alone (black line) or post incubation with a saturating concentration [4 × 10 7 ] of IgG1 Dynabeads (blue line) or anti-PLAP Dynabeads (red line). (Aiii) fl-NTA of the 10KP pool measured in; scatter mode: alone (black line), labelled with IgG1-Qdot605 control (blue solid line) or labelled with anti-PLAP-Qdot605 antibody (red solid line) and fluorescence mode: labelled with IgG1-Qdot605 control (blue dash) or anti-PLAP-Qdot605 antibody (red dash). (Bi) Representative immunoblot image of PLAP in the 150KP pool showing bound EV (PLAP positive) or those remaining in the supernatant (SN; PLAP negative) in response to an increasing dose (0.2–2 × 10 7 ) of IgG1 Dynabeads or anti-PLAP Dynabeads. (Bii) NTA of 150KP pool alone (black line) or post incubation with a saturating concentration [1 × 10 7 ] of IgG1 Dynabeads (blue line) or anti-PLAP Dynabeads (red line). (Biii) Fl-NTA of the 150KP pool measured in; scatter mode: alone (black line), labelled with IgG1-Qdot605 control (blue solid line) or labelled with anti-PLAP-Qdot605 antibody (red solid line) and fluorescence mode: labelled with IgG1-Qdot605 control (blue dash) or anti-PLAP-Qdot605 antibody (red dash).

Techniques Used: Fluorescence, Incubation, Concentration Assay

Representative flow cytometric analyses of fractionated maternal side placental perfusate. (Ai) Forward scatter (FSC) vs. side scatter (SSC) profile of maternal side placental perfusate, (Aii) histogram showing 100% labelling of red blood cells (RBC), (Aiii) maternal side placental perfusate supernatant (1500SN) following depletion of contaminating RBC by 2 × 1500× g centrifugation for 10 min, (Aiv) 1500SN bio-maleimide positive labelling, (Av) FSC vs. SSC profile of 10,000× g pellet (10KP) and (Avi) 10KP bio-maleimide positive labelling. (Bi) 1500SN bio-maleimide positive EV displayed on two-parameter plots and phenotype identified: syncytiotrophoblast derived EV (STBEV), PLAP + /HLA Class I − , (Bii) RBC EV, CD235a/b + /PLAP − , (Biii) platelet EV (CD41 + /PLAP − ), (Biv) leukocyte/endothelial EV (HLA Class I + /CD41 − ). (Ci) 10KP bio-maleimide positive EV displayed on two-parameter plots and phenotype identified: STBEV, PLAP + /HLA Class I − , (Cii) RBC EV, CD235a/b + /PLAP − , (Ciii) platelet EV (CD41 + /PLAP), (Civ) leukocyte/endothelial EV (HLA Class I + /CD41 − ). Histograms show the mean ± SE for percentage positive EV and two-parameter plots show the mean for percentage positive EV ( n = 8). Percentages have been adjusted to account for background contaminating particulates.
Figure Legend Snippet: Representative flow cytometric analyses of fractionated maternal side placental perfusate. (Ai) Forward scatter (FSC) vs. side scatter (SSC) profile of maternal side placental perfusate, (Aii) histogram showing 100% labelling of red blood cells (RBC), (Aiii) maternal side placental perfusate supernatant (1500SN) following depletion of contaminating RBC by 2 × 1500× g centrifugation for 10 min, (Aiv) 1500SN bio-maleimide positive labelling, (Av) FSC vs. SSC profile of 10,000× g pellet (10KP) and (Avi) 10KP bio-maleimide positive labelling. (Bi) 1500SN bio-maleimide positive EV displayed on two-parameter plots and phenotype identified: syncytiotrophoblast derived EV (STBEV), PLAP + /HLA Class I − , (Bii) RBC EV, CD235a/b + /PLAP − , (Biii) platelet EV (CD41 + /PLAP − ), (Biv) leukocyte/endothelial EV (HLA Class I + /CD41 − ). (Ci) 10KP bio-maleimide positive EV displayed on two-parameter plots and phenotype identified: STBEV, PLAP + /HLA Class I − , (Cii) RBC EV, CD235a/b + /PLAP − , (Ciii) platelet EV (CD41 + /PLAP), (Civ) leukocyte/endothelial EV (HLA Class I + /CD41 − ). Histograms show the mean ± SE for percentage positive EV and two-parameter plots show the mean for percentage positive EV ( n = 8). Percentages have been adjusted to account for background contaminating particulates.

Techniques Used: Flow Cytometry, Centrifugation, Derivative Assay

Nanoparticle Tracking Analysis size distribution profiles (mean data n = 8) for maternal side placental perfusate derived samples collected at the following stages of the fractionation process. (A) 2 × 1500× g supernatant (1500SN), (B) 150,000× g supernatant (150KSN), (Ci) 10,000× g pellet (10KP) and 150,000× g pellet (150KP). (Cii) Transmission electron micrographs of 10KP and 150KP. Scale bars represent 200 nm.
Figure Legend Snippet: Nanoparticle Tracking Analysis size distribution profiles (mean data n = 8) for maternal side placental perfusate derived samples collected at the following stages of the fractionation process. (A) 2 × 1500× g supernatant (1500SN), (B) 150,000× g supernatant (150KSN), (Ci) 10,000× g pellet (10KP) and 150,000× g pellet (150KP). (Cii) Transmission electron micrographs of 10KP and 150KP. Scale bars represent 200 nm.

Techniques Used: Derivative Assay, Fractionation, Transmission Assay

26) Product Images from "Survey of small intestinal and systemic immune responses following murine Arcobacter butzleri infection"

Article Title: Survey of small intestinal and systemic immune responses following murine Arcobacter butzleri infection

Journal: Gut Pathogens

doi: 10.1186/s13099-015-0075-z

Kinetics of pro-inflammatory cytokine responses in the ileum following murine A. butzleri infection. Gnotobiotic IL-10 −/− mice were generated by antibiotic treatment and orally infected either with A. butzleri strain CCUG 30485 ( circles ) or strain C1 ( squares ). Uninfected gnotobiotic IL-10 −/− mice served as negative control ( black diamonds ). Concentrations (pg per mg ileal tissue) of a TNF, b IL-6, c MCP-1 and d IFN-γ were determined in supernatant of ex vivo ileal biopsies at day 6 p.i. ( filled symbols ) and day 16 p.i. ( open symbols ). Numbers of analyzed animals are given in parentheses . Medians ( black bars ) and significance levels as determined by the Mann–Whitney U test are indicated. Data shown were pooled from three independent experiments
Figure Legend Snippet: Kinetics of pro-inflammatory cytokine responses in the ileum following murine A. butzleri infection. Gnotobiotic IL-10 −/− mice were generated by antibiotic treatment and orally infected either with A. butzleri strain CCUG 30485 ( circles ) or strain C1 ( squares ). Uninfected gnotobiotic IL-10 −/− mice served as negative control ( black diamonds ). Concentrations (pg per mg ileal tissue) of a TNF, b IL-6, c MCP-1 and d IFN-γ were determined in supernatant of ex vivo ileal biopsies at day 6 p.i. ( filled symbols ) and day 16 p.i. ( open symbols ). Numbers of analyzed animals are given in parentheses . Medians ( black bars ) and significance levels as determined by the Mann–Whitney U test are indicated. Data shown were pooled from three independent experiments

Techniques Used: Infection, Mouse Assay, Generated, Negative Control, Ex Vivo, MANN-WHITNEY

Kinetic of IFN-γ responses in ex vivo biopsies of mesenteric lymphnodes and spleens of A. butzleri infected mice. Gnotobiotic IL-10 −/− mice were generated by antibiotic treatment and orally infected either with A. butzleri strain CCUG 30485 ( circles ) or strain C1 ( squares ). Uninfected gnotobiotic IL-10 −/− mice served as negative control ( black diamonds ). Concentrations (pg per mg of total protein) of IFN-γ were determined in supernatant of ex vivo biopsies derived from a mesenteric lymphnodes (MLNs) and b spleens at day 6 p.i. ( filled symbols ) and day 16 p.i. ( open symbols ). Numbers of analyzed animals are given in parentheses . Medians ( black bars ) and significance levels as determined by the Mann–Whitney U test are indicated. Data shown were pooled from three independent experiments
Figure Legend Snippet: Kinetic of IFN-γ responses in ex vivo biopsies of mesenteric lymphnodes and spleens of A. butzleri infected mice. Gnotobiotic IL-10 −/− mice were generated by antibiotic treatment and orally infected either with A. butzleri strain CCUG 30485 ( circles ) or strain C1 ( squares ). Uninfected gnotobiotic IL-10 −/− mice served as negative control ( black diamonds ). Concentrations (pg per mg of total protein) of IFN-γ were determined in supernatant of ex vivo biopsies derived from a mesenteric lymphnodes (MLNs) and b spleens at day 6 p.i. ( filled symbols ) and day 16 p.i. ( open symbols ). Numbers of analyzed animals are given in parentheses . Medians ( black bars ) and significance levels as determined by the Mann–Whitney U test are indicated. Data shown were pooled from three independent experiments

Techniques Used: Ex Vivo, Infection, Mouse Assay, Generated, Negative Control, Derivative Assay, MANN-WHITNEY

27) Product Images from "IGFBP7 induces apoptosis of acute myeloid leukemia cells and synergizes with chemotherapy in suppression of leukemia cell survival"

Article Title: IGFBP7 induces apoptosis of acute myeloid leukemia cells and synergizes with chemotherapy in suppression of leukemia cell survival

Journal: Cell Death & Disease

doi: 10.1038/cddis.2014.268

Expression of membrane IGF-1R in leukemic cell lines. ( a ) Flow cytrometric analysis of leukemic cell lines labeled with IGF-1R-phycoerythrin (PE; gray) compared with cells labeled with IgG-PE control antibody (white). ( b ) Detection of phosphorylated IGF-1R using immunoblotting after IGF-1 ligand stimulation (50 ng/ml). ( c ) Cell lines were treated with the indicated concentrations of NVP-AEW541 and cell viability was measured using a MTT assay
Figure Legend Snippet: Expression of membrane IGF-1R in leukemic cell lines. ( a ) Flow cytrometric analysis of leukemic cell lines labeled with IGF-1R-phycoerythrin (PE; gray) compared with cells labeled with IgG-PE control antibody (white). ( b ) Detection of phosphorylated IGF-1R using immunoblotting after IGF-1 ligand stimulation (50 ng/ml). ( c ) Cell lines were treated with the indicated concentrations of NVP-AEW541 and cell viability was measured using a MTT assay

Techniques Used: Expressing, Flow Cytometry, Labeling, MTT Assay

28) Product Images from "Cytotoxicity of 11-epi-Sinulariolide Acetate Isolated from Cultured Soft Corals on HA22T Cells through the Endoplasmic Reticulum Stress Pathway and Mitochondrial Dysfunction"

Article Title: Cytotoxicity of 11-epi-Sinulariolide Acetate Isolated from Cultured Soft Corals on HA22T Cells through the Endoplasmic Reticulum Stress Pathway and Mitochondrial Dysfunction

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms17111787

Treatment with 11- epi -SA stimulates the endoplasmic reticulum (ER) stress-mediated apoptotic pathway. HA22T cells were treated without or with 11- epi -SA at different concentrations ( left ) and different time points ( middle ) as indicated. The cells were then harvested to assess the presence ER stress-related apoptotic pathway proteins as indicated. ( A ) ER-related proteins, 94 kDa glucose-regulated protein (GRP94), GRP78/BiP, CALR, calnexin, and PDI were examined by specific antibodies; ( B ) ER stress-mediated apoptotic proteins, protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), phospho-PERK (p-PERK), eukaryotic initiation factor 2 alpha (eIF2α), p-eIF2α, activating transcription factor (ATF) 6 fragment (ATF6-f), ATF4, and CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) were detected by specific antibodies; ( C ) Some other ER stress-related proteins, inositol-requiring enzyme 1 alpha (IRE1-α), phospho-apoptosis signal-regulating kinase 1 (p-ASK1), c-Jun N-terminal kinase (JNK), phospho-JNK (p-JNK), c-Jun and phosphor-c-Jun (p-c-Jun) were detected by specific antibodies. Please note that cells treated with tunicamycin (Tm) or thapsigargin (Tg) were considered to indicate ER stress response as shown in the right side of each panel.
Figure Legend Snippet: Treatment with 11- epi -SA stimulates the endoplasmic reticulum (ER) stress-mediated apoptotic pathway. HA22T cells were treated without or with 11- epi -SA at different concentrations ( left ) and different time points ( middle ) as indicated. The cells were then harvested to assess the presence ER stress-related apoptotic pathway proteins as indicated. ( A ) ER-related proteins, 94 kDa glucose-regulated protein (GRP94), GRP78/BiP, CALR, calnexin, and PDI were examined by specific antibodies; ( B ) ER stress-mediated apoptotic proteins, protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), phospho-PERK (p-PERK), eukaryotic initiation factor 2 alpha (eIF2α), p-eIF2α, activating transcription factor (ATF) 6 fragment (ATF6-f), ATF4, and CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) were detected by specific antibodies; ( C ) Some other ER stress-related proteins, inositol-requiring enzyme 1 alpha (IRE1-α), phospho-apoptosis signal-regulating kinase 1 (p-ASK1), c-Jun N-terminal kinase (JNK), phospho-JNK (p-JNK), c-Jun and phosphor-c-Jun (p-c-Jun) were detected by specific antibodies. Please note that cells treated with tunicamycin (Tm) or thapsigargin (Tg) were considered to indicate ER stress response as shown in the right side of each panel.

Techniques Used: Binding Assay

Treatment with 11- epi -SA activates mitochondria-related-apoptotic pathway proteins. ( A ) HA22T cells were treated without or with 11- epi -SA at different concentrations ( left ) and different time points ( right ) as indicated. The cells were then harvested and protein expression level was assessed by Western blotting using specific antibodies as indicated, and β-actin as loading control; ( B ) Caspase-dependent pathway proteins were activated in the 11- epi -SA-treated cells. The cell lysates were harvested after 11- epi -SA treatment as indicated above, followed by the examination of poly(ADP-ribose) polymerase 1 (PARP-1), caspase-3, -9, and cytochrome c proteins by using the specific antibodies as indicated. β-actin was used as the loading control.
Figure Legend Snippet: Treatment with 11- epi -SA activates mitochondria-related-apoptotic pathway proteins. ( A ) HA22T cells were treated without or with 11- epi -SA at different concentrations ( left ) and different time points ( right ) as indicated. The cells were then harvested and protein expression level was assessed by Western blotting using specific antibodies as indicated, and β-actin as loading control; ( B ) Caspase-dependent pathway proteins were activated in the 11- epi -SA-treated cells. The cell lysates were harvested after 11- epi -SA treatment as indicated above, followed by the examination of poly(ADP-ribose) polymerase 1 (PARP-1), caspase-3, -9, and cytochrome c proteins by using the specific antibodies as indicated. β-actin was used as the loading control.

Techniques Used: Expressing, Western Blot

11- epi -SA-induced apoptotic pathway in HA22T cancer cells. The anticancer effect of 11- epi -SA is mediated by the induction of mitochondrial dysfunction and the ER stress signaling pathway. TRAF2, tumor necrosis factor receptor-associated factor 2.
Figure Legend Snippet: 11- epi -SA-induced apoptotic pathway in HA22T cancer cells. The anticancer effect of 11- epi -SA is mediated by the induction of mitochondrial dysfunction and the ER stress signaling pathway. TRAF2, tumor necrosis factor receptor-associated factor 2.

Techniques Used:

Appearance of apoptotic characteristics in 11- epi -SA-treated HA22T cells. ( A ) Detection of DNA fragments after 11- epi -SA treatment for 24 h at the indicated concentrations. M, marker; C, control; ( B ) Detection of apoptotic HA22T cells after 11- epi -SA treatment by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) analysis.
Figure Legend Snippet: Appearance of apoptotic characteristics in 11- epi -SA-treated HA22T cells. ( A ) Detection of DNA fragments after 11- epi -SA treatment for 24 h at the indicated concentrations. M, marker; C, control; ( B ) Detection of apoptotic HA22T cells after 11- epi -SA treatment by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) analysis.

Techniques Used: Marker

Treatment with 11- epi -SA inhibits cell migration and induces the appearance of apoptotic characteristics in HA22T cells. ( A ) Mock- or 11- epi -SA-treated HA22T cells in the areas between two dotted lines are cells that have migrated during the indicated time periods. Images represent the apparent reduction of HA22T cell migration after treatment with 3, 6, and 9 μg/mL 11- epi -SA for 24 and 48 h, respectively; ( B ) Quantitative measurement of the migration of the HA22T cells is shown and compared with that of the DMSO-treated cells. The result shows dose-dependent suppression of HA22T cell migration (# p
Figure Legend Snippet: Treatment with 11- epi -SA inhibits cell migration and induces the appearance of apoptotic characteristics in HA22T cells. ( A ) Mock- or 11- epi -SA-treated HA22T cells in the areas between two dotted lines are cells that have migrated during the indicated time periods. Images represent the apparent reduction of HA22T cell migration after treatment with 3, 6, and 9 μg/mL 11- epi -SA for 24 and 48 h, respectively; ( B ) Quantitative measurement of the migration of the HA22T cells is shown and compared with that of the DMSO-treated cells. The result shows dose-dependent suppression of HA22T cell migration (# p

Techniques Used: Migration

29) Product Images from "Mitogen-activated protein kinases (MAPKs) are modulated during Francisella tularensis infection, but inhibition of extracellular-signal-regulated kinases (ERKs) is of limited therapeutic benefit"

Article Title: Mitogen-activated protein kinases (MAPKs) are modulated during Francisella tularensis infection, but inhibition of extracellular-signal-regulated kinases (ERKs) is of limited therapeutic benefit

Journal: European Journal of Clinical Microbiology & Infectious Diseases

doi: 10.1007/s10096-016-2754-1

The effect of PD0325901 treatment (0.05 μM) on bacterial burdens and cytokine secretion in the organs of F. tularensis LVS-infected Balb/c mice. Groups of four mice infected via the intranasal route with 2.5 × 10 4 CFU F. tularensis LVS were treated daily with PBS from 24 h pre-challenge (PBS), PD0325901 from 24 h pre-challenge [PD(−1)]or from 48 h post-challenge [PD(+2)]. Naive mice were used as a control. On day 4 post-challenge the organs were removed, homogenised and bacterial burdens were determined ( a ). The homogenates were also screened for the levels of IL-6 ( b ), MCP-1 ( c ), IFN-γ ( d ), and TNF-α ( e ). Significant difference from PBS-treated group was determined using a one-tailed, unpaired t -test (* = p
Figure Legend Snippet: The effect of PD0325901 treatment (0.05 μM) on bacterial burdens and cytokine secretion in the organs of F. tularensis LVS-infected Balb/c mice. Groups of four mice infected via the intranasal route with 2.5 × 10 4 CFU F. tularensis LVS were treated daily with PBS from 24 h pre-challenge (PBS), PD0325901 from 24 h pre-challenge [PD(−1)]or from 48 h post-challenge [PD(+2)]. Naive mice were used as a control. On day 4 post-challenge the organs were removed, homogenised and bacterial burdens were determined ( a ). The homogenates were also screened for the levels of IL-6 ( b ), MCP-1 ( c ), IFN-γ ( d ), and TNF-α ( e ). Significant difference from PBS-treated group was determined using a one-tailed, unpaired t -test (* = p

Techniques Used: Infection, Mouse Assay, One-tailed Test

30) Product Images from "Successful Treatment of Corticosteroid with Antiviral Therapy for a Neonatal Liver Failure with Disseminated Herpes Simplex Virus Infection"

Article Title: Successful Treatment of Corticosteroid with Antiviral Therapy for a Neonatal Liver Failure with Disseminated Herpes Simplex Virus Infection

Journal: AJP Reports

doi: 10.1055/s-0034-1398392

Sequential changes of the liver function and serum cytokine levels during the initial treatment course (A), and those of HSV DNA copy number in serum (•) and cerebrospinal fluid (▪) during the overall treatment course (B). ALT, alanine aminotransferase; AST, aspartate aminotransferase; IVIG, intravenous high-dose immunoglobulin therapy; HSV: herpes simplex virus; IFN, interferon; IL, interleukin; PT–INR, prothrombin time–international normalized ratio.
Figure Legend Snippet: Sequential changes of the liver function and serum cytokine levels during the initial treatment course (A), and those of HSV DNA copy number in serum (•) and cerebrospinal fluid (▪) during the overall treatment course (B). ALT, alanine aminotransferase; AST, aspartate aminotransferase; IVIG, intravenous high-dose immunoglobulin therapy; HSV: herpes simplex virus; IFN, interferon; IL, interleukin; PT–INR, prothrombin time–international normalized ratio.

Techniques Used: AST Assay

31) Product Images from "Ptpn11 Deletion in A Novel Cartilage Cell Causes Metachondromatosis by Activating Hedgehog Signaling"

Article Title: Ptpn11 Deletion in A Novel Cartilage Cell Causes Metachondromatosis by Activating Hedgehog Signaling

Journal: Nature

doi: 10.1038/nature12396

Ptpn11 deletion in Ctsk-expressing cells causes expansion of novel chondroprogenitor cell population within the Perichondrial Groove of Ranvier. a, Flow cytometric analysis showing YFP + cells from pooled epiphyseal cartilage from 5–7 Ctsk-Control/YFP mice; note increased percentage of such cells in 2-week-old Ctsk-KO/YFP mice. CC: Chondroid cells. b, Flow cytometric analysis of YFP + perichondrial cells showing staining for CD31, CD44, CD90, and CD166. Data in panels a and b are from a single experiment; similar results were obtained in 2 additional experiments. c, Immunofluorescence micrograph showing Stro1 and Jagged1 expression in YFP + perichondrial cells. Nuclei are stained with DAPI. Note enhanced intensity of Stro1 and Jagged staining in Ctsk-KO cells. Data shown are from single mice of each genotype; two additional mice were analyzed for each genotype with similar results. d, CCPs give rise to cartilage, fat and bone. FACS-purified YFP + cells from 5–7 mice were subjected to differentiation assays in triplicate. After 2–3 weeks of culture (see Methods), cells were fixed and stained with Alcian blue, Oil red, and Alizarin red to visualize the formation of cartilage, fat, and bone tissue, respectively.
Figure Legend Snippet: Ptpn11 deletion in Ctsk-expressing cells causes expansion of novel chondroprogenitor cell population within the Perichondrial Groove of Ranvier. a, Flow cytometric analysis showing YFP + cells from pooled epiphyseal cartilage from 5–7 Ctsk-Control/YFP mice; note increased percentage of such cells in 2-week-old Ctsk-KO/YFP mice. CC: Chondroid cells. b, Flow cytometric analysis of YFP + perichondrial cells showing staining for CD31, CD44, CD90, and CD166. Data in panels a and b are from a single experiment; similar results were obtained in 2 additional experiments. c, Immunofluorescence micrograph showing Stro1 and Jagged1 expression in YFP + perichondrial cells. Nuclei are stained with DAPI. Note enhanced intensity of Stro1 and Jagged staining in Ctsk-KO cells. Data shown are from single mice of each genotype; two additional mice were analyzed for each genotype with similar results. d, CCPs give rise to cartilage, fat and bone. FACS-purified YFP + cells from 5–7 mice were subjected to differentiation assays in triplicate. After 2–3 weeks of culture (see Methods), cells were fixed and stained with Alcian blue, Oil red, and Alizarin red to visualize the formation of cartilage, fat, and bone tissue, respectively.

Techniques Used: Expressing, Flow Cytometry, Mouse Assay, Staining, Immunofluorescence, FACS, Purification

32) Product Images from "Modulation of Human Immune Responses by Bovine Interleukin-10"

Article Title: Modulation of Human Immune Responses by Bovine Interleukin-10

Journal: PLoS ONE

doi: 10.1371/journal.pone.0018188

Bovine IL-10 dose-dependently inhibits DC surface marker expression and cytokine production. A: Typical example of raw data of flow cytometric analysis. Data shown is CD80 expression during LPS induced DC maturation of one donor. The solid line indicates CD80 staining and the dashed line the isotype control. On top of the graphs is indicated whether medium, LPS or LPS plus different doses of IL-10 (ng/ml) were used. B: Bovine IL-10 dose-dependently modulates DC surface marker expression (CD83, p = 0.002; CD40, p = 0.030; CD80, p = 0.018) during LPS induced maturation. Relative values are shown from three different donors. Mean fluorescent intensities were divided by the isotype control and expressed relative to the positive control (LPS, without IL-10), which was set at 100%. C: Recombinant bovine IL-10 dose dependently modulates the production of cytokines by human DC's during LPS induced maturation (IL-12, p =
Figure Legend Snippet: Bovine IL-10 dose-dependently inhibits DC surface marker expression and cytokine production. A: Typical example of raw data of flow cytometric analysis. Data shown is CD80 expression during LPS induced DC maturation of one donor. The solid line indicates CD80 staining and the dashed line the isotype control. On top of the graphs is indicated whether medium, LPS or LPS plus different doses of IL-10 (ng/ml) were used. B: Bovine IL-10 dose-dependently modulates DC surface marker expression (CD83, p = 0.002; CD40, p = 0.030; CD80, p = 0.018) during LPS induced maturation. Relative values are shown from three different donors. Mean fluorescent intensities were divided by the isotype control and expressed relative to the positive control (LPS, without IL-10), which was set at 100%. C: Recombinant bovine IL-10 dose dependently modulates the production of cytokines by human DC's during LPS induced maturation (IL-12, p =

Techniques Used: Marker, Expressing, Flow Cytometry, Staining, Positive Control, Recombinant

33) Product Images from "The Role of Inflammatory, Anti-Inflammatory, and Regulatory Cytokines in Patients Infected with Cutaneous Leishmaniasis in Amazonas State, Brazil"

Article Title: The Role of Inflammatory, Anti-Inflammatory, and Regulatory Cytokines in Patients Infected with Cutaneous Leishmaniasis in Amazonas State, Brazil

Journal: Journal of Immunology Research

doi: 10.1155/2014/481750

IL-2, TNF- α , and IFN- γ levels in serum samples from patients infected with different Leishmania species and in noninfected control. Cytokine levels (pg/mL) were calculated for each serum sample. Means (horizontal lines) are shown for each group. Ctrl: noninfected controls; L. g : Leishmania guyanensis ; L. n : L . naiffi ; L. a : L. amazonensis .
Figure Legend Snippet: IL-2, TNF- α , and IFN- γ levels in serum samples from patients infected with different Leishmania species and in noninfected control. Cytokine levels (pg/mL) were calculated for each serum sample. Means (horizontal lines) are shown for each group. Ctrl: noninfected controls; L. g : Leishmania guyanensis ; L. n : L . naiffi ; L. a : L. amazonensis .

Techniques Used: Infection

Levels of IL-4, IL-6, IL-10, and IL-17 in serum samples from patients infected with different Leishmania species and in noninfected control. Cytokine levels (pg/mL) were calculated for each serum sample. Means (horizontal lines) are shown for each group. Ctrl: noninfected control; L. g : L . guyanensis ; L. n : L . naiffi ; L. a : L.amazonensis .
Figure Legend Snippet: Levels of IL-4, IL-6, IL-10, and IL-17 in serum samples from patients infected with different Leishmania species and in noninfected control. Cytokine levels (pg/mL) were calculated for each serum sample. Means (horizontal lines) are shown for each group. Ctrl: noninfected control; L. g : L . guyanensis ; L. n : L . naiffi ; L. a : L.amazonensis .

Techniques Used: Infection

34) Product Images from "Pregnant Women Infected with Pandemic H1N1pdm2009 Influenza Virus Displayed Overproduction of Peripheral Blood CD69+ Lymphocytes and Increased Levels of Serum Cytokines"

Article Title: Pregnant Women Infected with Pandemic H1N1pdm2009 Influenza Virus Displayed Overproduction of Peripheral Blood CD69+ Lymphocytes and Increased Levels of Serum Cytokines

Journal: PLoS ONE

doi: 10.1371/journal.pone.0107900

Serum chemokine concentrations in HW, HP, ILI and PH1N1 women. The chemokine CXCL9/MIG (a), CCL2/MCP-1 (b), CXCL8/IL-8 (c) and CXCL10/IP-10 (d) concentrations were quantified using a CBA system with flow cytometry. The Kruskal-Wallis test with Dunn’s multiple comparison post-test was performed using the GraphPad Software. The significance values were *p
Figure Legend Snippet: Serum chemokine concentrations in HW, HP, ILI and PH1N1 women. The chemokine CXCL9/MIG (a), CCL2/MCP-1 (b), CXCL8/IL-8 (c) and CXCL10/IP-10 (d) concentrations were quantified using a CBA system with flow cytometry. The Kruskal-Wallis test with Dunn’s multiple comparison post-test was performed using the GraphPad Software. The significance values were *p

Techniques Used: Crocin Bleaching Assay, Flow Cytometry, Cytometry, Software

35) Product Images from "Effects of inflammatory cytokine IL-27 on the activation of fibroblast-like synoviocytes in rheumatoid arthritis"

Article Title: Effects of inflammatory cytokine IL-27 on the activation of fibroblast-like synoviocytes in rheumatoid arthritis

Journal: Arthritis Research & Therapy

doi: 10.1186/ar3067

Dose- and time-dependent effect of IL-27 on the induction of chemokine release from control and RA-FLS . Control and RA-FLS were cultured with IL-27 (50 ng/ml) for 48 h and induction of (A) CCL2, (C) CXCL9 and (E) CXCL10 was analysed by CBA using flow cytometry. (B, D, F) Control and RA-FLS were stimulated with IL-27 (0 to 100 ng/ml) for 12 to 48 h and inductions of (B) CCL2, (D) CXCL9 and (F) CXCL10 were analysed by CBA using flow cytometry. * P
Figure Legend Snippet: Dose- and time-dependent effect of IL-27 on the induction of chemokine release from control and RA-FLS . Control and RA-FLS were cultured with IL-27 (50 ng/ml) for 48 h and induction of (A) CCL2, (C) CXCL9 and (E) CXCL10 was analysed by CBA using flow cytometry. (B, D, F) Control and RA-FLS were stimulated with IL-27 (0 to 100 ng/ml) for 12 to 48 h and inductions of (B) CCL2, (D) CXCL9 and (F) CXCL10 were analysed by CBA using flow cytometry. * P

Techniques Used: Cell Culture, Crocin Bleaching Assay, Flow Cytometry, Cytometry

36) Product Images from "ChemR23 Dampens Lung Inflammation and Enhances Anti-viral Immunity in a Mouse Model of Acute Viral Pneumonia"

Article Title: ChemR23 Dampens Lung Inflammation and Enhances Anti-viral Immunity in a Mouse Model of Acute Viral Pneumonia

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002358

Reduced levels of type I IFNs and IL-12p40 in ChemR23 −/− infected mice. At selected time points after infection, lungs and/or broncho-alveolar lavage fluids obtained from wild-type (WT) (white bars) and ChemR23 −/− mice (black bars) were assessed for cytokine transcripts or proteins. ( A ) Lung IFN-α and IFN-β transcripts were assayed by qRT-PCR (upper panels). IFN-α levels were also determined by ELISA in lung homogenates (lower left panel) and broncho-alveolar lavage fluids (lower right panel). ( B ) Chemokine (KC/CXCL1) and cytokines (IFN-γ, TNF-α, IL-6, IL-12p40 and IL-17) levels were determined by ELISA in lung homogenates. Data are the mean ± SEM for groups of seven animals and are representative of three independent experiments. *, p
Figure Legend Snippet: Reduced levels of type I IFNs and IL-12p40 in ChemR23 −/− infected mice. At selected time points after infection, lungs and/or broncho-alveolar lavage fluids obtained from wild-type (WT) (white bars) and ChemR23 −/− mice (black bars) were assessed for cytokine transcripts or proteins. ( A ) Lung IFN-α and IFN-β transcripts were assayed by qRT-PCR (upper panels). IFN-α levels were also determined by ELISA in lung homogenates (lower left panel) and broncho-alveolar lavage fluids (lower right panel). ( B ) Chemokine (KC/CXCL1) and cytokines (IFN-γ, TNF-α, IL-6, IL-12p40 and IL-17) levels were determined by ELISA in lung homogenates. Data are the mean ± SEM for groups of seven animals and are representative of three independent experiments. *, p

Techniques Used: Infection, Mouse Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Transfer of bone marrow from wild-type mice does not protect ChemR23-deficient mice from PVM infection. ( A ) Irradiated (*) wild-type mice reconstituted with ChemR23 −/− bone marrow (WT*+KO), as well as irradiated KO mice reconstituted with bone marrow from WT mice (KO*+WT), were infected by PVM (1000 PFUs) and monitored for weight loss (left panel). 23 days post-infection, mice were sacrificed and inflammatory cells were counted in BAL fluids (right panel). ( B ) BAL fluids were also harvested at day 14 post-infection in a similar experiment including two additional groups, namely WT and KO mice reconstituted respectively with WT and KO bone marrow (WT*+WT, KO*+KO, respectively). Total cell numbers (left panel) as well as cytokine levels (IL-6 and IFN-γ, middle and right panels) were determined in these samples. Data are the mean ± SEM for groups of at least five animals. *, p
Figure Legend Snippet: Transfer of bone marrow from wild-type mice does not protect ChemR23-deficient mice from PVM infection. ( A ) Irradiated (*) wild-type mice reconstituted with ChemR23 −/− bone marrow (WT*+KO), as well as irradiated KO mice reconstituted with bone marrow from WT mice (KO*+WT), were infected by PVM (1000 PFUs) and monitored for weight loss (left panel). 23 days post-infection, mice were sacrificed and inflammatory cells were counted in BAL fluids (right panel). ( B ) BAL fluids were also harvested at day 14 post-infection in a similar experiment including two additional groups, namely WT and KO mice reconstituted respectively with WT and KO bone marrow (WT*+WT, KO*+KO, respectively). Total cell numbers (left panel) as well as cytokine levels (IL-6 and IFN-γ, middle and right panels) were determined in these samples. Data are the mean ± SEM for groups of at least five animals. *, p

Techniques Used: Mouse Assay, Infection, Irradiation

37) Product Images from "Evaluation of the Innate Immune Modulator Acitretin as a Strategy To Clear the HIV Reservoir"

Article Title: Evaluation of the Innate Immune Modulator Acitretin as a Strategy To Clear the HIV Reservoir

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.01368-17

Acitretin does not selectively kill HIV-reactivated cells. (A) Apoptosis percentages in HIV-reactivated (GFP-positive) and not reactivated (GFP-negative) latently infected Jurkat cells (J-Hig). Cells were incubated for 24 h with acitretin (ACI) (1 to 25 μM), panobinostat (PNB) (0.16 μM), or vorinostat (VOR) (0.16 to 4 μM). Apoptosis percentages shown represent fractions of the total GFP-positive/negative subpopulation. Cells were evaluated by flow cytometry with double staining for HIV reactivation (GFP positive) and cell apoptosis (annexin V positive). (B) Cell distribution of apoptotic and/or HIV-reactivated J-Hig cells from the results shown in panel A, without taking into account the fraction of GFP-positive or GFP-negative cells. Assays were evaluated by flow cytometry. (C) Representative cytometry plots from the data in panel A, showing the four subpopulations, i.e., GFP-positive/annexin V-positive, GFP-positive/annexin V-negative, GFP-negative/annexin V-positive, and GFP-negative/annexin V-negative. (D) Ratio of apoptosis values for the HIV-reactivated (GFP-positive) population and the nonreactivated (GFP-negative) population in J-Hig cells. A drug that is selective against HIV-reactivated cells is expected to have a value of  > 1. (E) Apoptosis percentages in HIV-reactivated (GFP-positive) and nonreactivated (GFP-negative) latently infected primary CD4 +  T cells. Cells were incubated for 12 h with acitretin, panobinostat, or vorinostat. The anti-CD3/anti-CD28 condition was used as a reactivation control. Apoptosis percentages shown represent fractions of the total GFP-positive/negative subpopulation. Values represent means ± SDs of at least three independent experiments performed in triplicate. UN, untreated.
Figure Legend Snippet: Acitretin does not selectively kill HIV-reactivated cells. (A) Apoptosis percentages in HIV-reactivated (GFP-positive) and not reactivated (GFP-negative) latently infected Jurkat cells (J-Hig). Cells were incubated for 24 h with acitretin (ACI) (1 to 25 μM), panobinostat (PNB) (0.16 μM), or vorinostat (VOR) (0.16 to 4 μM). Apoptosis percentages shown represent fractions of the total GFP-positive/negative subpopulation. Cells were evaluated by flow cytometry with double staining for HIV reactivation (GFP positive) and cell apoptosis (annexin V positive). (B) Cell distribution of apoptotic and/or HIV-reactivated J-Hig cells from the results shown in panel A, without taking into account the fraction of GFP-positive or GFP-negative cells. Assays were evaluated by flow cytometry. (C) Representative cytometry plots from the data in panel A, showing the four subpopulations, i.e., GFP-positive/annexin V-positive, GFP-positive/annexin V-negative, GFP-negative/annexin V-positive, and GFP-negative/annexin V-negative. (D) Ratio of apoptosis values for the HIV-reactivated (GFP-positive) population and the nonreactivated (GFP-negative) population in J-Hig cells. A drug that is selective against HIV-reactivated cells is expected to have a value of > 1. (E) Apoptosis percentages in HIV-reactivated (GFP-positive) and nonreactivated (GFP-negative) latently infected primary CD4 + T cells. Cells were incubated for 12 h with acitretin, panobinostat, or vorinostat. The anti-CD3/anti-CD28 condition was used as a reactivation control. Apoptosis percentages shown represent fractions of the total GFP-positive/negative subpopulation. Values represent means ± SDs of at least three independent experiments performed in triplicate. UN, untreated.

Techniques Used: Infection, Incubation, Flow Cytometry, Cytometry, Double Staining

38) Product Images from "Human melanoma immunotherapy using tumor antigen-specific T cells generated in humanized mice"

Article Title: Human melanoma immunotherapy using tumor antigen-specific T cells generated in humanized mice

Journal: Oncotarget

doi: 10.18632/oncotarget.7044

MART-1 TCR + CD8, but not CD4, T cells show antigen-specific responses in humanized mice following MART-1 peptide immunization Hu-mice were immunized with MART-1 peptide or PBS as control 15 weeks after humanization, and CD8 + A. - C. and CD4 + D. - F. T cell responses were assessed by flow cytometry 1 week prior to (before) and 3 weeks after immunization. A. and D. Percentages of tetramer + cells in peripheral blood CD8 + A. and CD4 + D. T cells (mean±SEM; n = 3). B. and E. Expression of CD45RA and CCR7 on tetramer + vs . tetramer − CD8 + B. and CD4 + E. T cells (mean ± SEMs; n = 7). Left , representative staining profiles of the cells prepared before (top) and after (bottom) immunization; Right , percentages of T cell subsets in tetramer + (top) and tetramer − (bottom) T cells prepared before and after immunization. Naïve, CD45RA + CCR7 + naïve T cells; EM, CD45RA − CCR7 − effector memory T cells; CM, CD45RA − CCR7 + central memory T cells; EM/RA, CD45RA + CCR7 − effector memory T cells. C. and F. Percentages of IFN-γ producing tetramer + CD8 + C. and CD4 + F. T cells prepared before and after immunization (mean ± SEMs; n = 5). *, p
Figure Legend Snippet: MART-1 TCR + CD8, but not CD4, T cells show antigen-specific responses in humanized mice following MART-1 peptide immunization Hu-mice were immunized with MART-1 peptide or PBS as control 15 weeks after humanization, and CD8 + A. - C. and CD4 + D. - F. T cell responses were assessed by flow cytometry 1 week prior to (before) and 3 weeks after immunization. A. and D. Percentages of tetramer + cells in peripheral blood CD8 + A. and CD4 + D. T cells (mean±SEM; n = 3). B. and E. Expression of CD45RA and CCR7 on tetramer + vs . tetramer − CD8 + B. and CD4 + E. T cells (mean ± SEMs; n = 7). Left , representative staining profiles of the cells prepared before (top) and after (bottom) immunization; Right , percentages of T cell subsets in tetramer + (top) and tetramer − (bottom) T cells prepared before and after immunization. Naïve, CD45RA + CCR7 + naïve T cells; EM, CD45RA − CCR7 − effector memory T cells; CM, CD45RA − CCR7 + central memory T cells; EM/RA, CD45RA + CCR7 − effector memory T cells. C. and F. Percentages of IFN-γ producing tetramer + CD8 + C. and CD4 + F. T cells prepared before and after immunization (mean ± SEMs; n = 5). *, p

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

Antitumor effect by in vitro expanded MART-1-specific human CD8 T cells from humanized mice Tetramer + CD8 + T cells purified from hu-mice ( n = 3) were expanded in cultures as shown in Fig 3b A. - C. , and the anti-melanoma activity of in vitro expanded CD8 + T cells were assessed by adoptive transfer into melanoma-bearing recipients D. - E. A. Human T cell expansion in cultures at the indicated time points. B. Expression of MART-1-specific TCR, CD8, CD45RA and CCR7 on expanded human T cells. C. Cytotoxicity of the expanded human CD8 + T cells against melanoma cells. D. Left panel , tumor burden in mice receiving 1×10 6 of Mel 624 (HLA-A2 + MART-1 + ; s.c.) cells with or without (control) adoptive transfer of in vitro -expanded tetramer + CD8 T cells (1×10 7 per mouse ; n = 5 per group); Right panel , phenotypic analysis of CD3 + TILs at week 5 post-transfer of in vitro -expanded tetramer + CD8 T cells. E. Survival of mice that received 2×10 5 Mel 624 cells (i.v.) with or without adoptive transfer of 1×10 7 in vitro -expanded tetramer + CD8 T cells ( n = 9 per group). *, p
Figure Legend Snippet: Antitumor effect by in vitro expanded MART-1-specific human CD8 T cells from humanized mice Tetramer + CD8 + T cells purified from hu-mice ( n = 3) were expanded in cultures as shown in Fig 3b A. - C. , and the anti-melanoma activity of in vitro expanded CD8 + T cells were assessed by adoptive transfer into melanoma-bearing recipients D. - E. A. Human T cell expansion in cultures at the indicated time points. B. Expression of MART-1-specific TCR, CD8, CD45RA and CCR7 on expanded human T cells. C. Cytotoxicity of the expanded human CD8 + T cells against melanoma cells. D. Left panel , tumor burden in mice receiving 1×10 6 of Mel 624 (HLA-A2 + MART-1 + ; s.c.) cells with or without (control) adoptive transfer of in vitro -expanded tetramer + CD8 T cells (1×10 7 per mouse ; n = 5 per group); Right panel , phenotypic analysis of CD3 + TILs at week 5 post-transfer of in vitro -expanded tetramer + CD8 T cells. E. Survival of mice that received 2×10 5 Mel 624 cells (i.v.) with or without adoptive transfer of 1×10 7 in vitro -expanded tetramer + CD8 T cells ( n = 9 per group). *, p

Techniques Used: In Vitro, Mouse Assay, Purification, Activity Assay, Adoptive Transfer Assay, Expressing

Generation of MART-1-specific T cells in humanized mice made by transplantation of human FTHY and TCR-engineered CD34 + FLCs A. Schematic preparation of the humanized mouse model with human T cells expressing transgenic TCR specific for MART-1. B. Representative flow cytometric profiles showing reconstitution of human T and B cells (left) and MART-1 TCR + T cells (right) in PBMCs of humanized mice. C. FACS analysis of MART-1 TCR + and MART-1 TCR − T cells for CD45RA and CCR7 expression. D. Shown are percentages of MART-1 TCR + T cells in CD8 + and CD4 + T cell compartments (left) and percentages (mean ± SEMs; n = 13) of CD8 + T cells in MART-1 TCR + and MART-1 TCR − T cell population (right). E. FACS assessment of MART-1 TCR + T cells in CD8 + and CD4 + T cells from the indicated tissues at week 22. F. FACS assessment of MART-1 TCR + T cells in CD4 + CD8 + (DP), CD8SP and CD4SP human thymocytes. ***, p
Figure Legend Snippet: Generation of MART-1-specific T cells in humanized mice made by transplantation of human FTHY and TCR-engineered CD34 + FLCs A. Schematic preparation of the humanized mouse model with human T cells expressing transgenic TCR specific for MART-1. B. Representative flow cytometric profiles showing reconstitution of human T and B cells (left) and MART-1 TCR + T cells (right) in PBMCs of humanized mice. C. FACS analysis of MART-1 TCR + and MART-1 TCR − T cells for CD45RA and CCR7 expression. D. Shown are percentages of MART-1 TCR + T cells in CD8 + and CD4 + T cell compartments (left) and percentages (mean ± SEMs; n = 13) of CD8 + T cells in MART-1 TCR + and MART-1 TCR − T cell population (right). E. FACS assessment of MART-1 TCR + T cells in CD8 + and CD4 + T cells from the indicated tissues at week 22. F. FACS assessment of MART-1 TCR + T cells in CD4 + CD8 + (DP), CD8SP and CD4SP human thymocytes. ***, p

Techniques Used: Mouse Assay, Transplantation Assay, Expressing, Transgenic Assay, Flow Cytometry, FACS

39) Product Images from "Semi Mature Blood Dendritic Cells Exist in Patients with Ductal Pancreatic Adenocarcinoma Owing to Inflammatory Factors Released from the Tumor"

Article Title: Semi Mature Blood Dendritic Cells Exist in Patients with Ductal Pancreatic Adenocarcinoma Owing to Inflammatory Factors Released from the Tumor

Journal: PLoS ONE

doi: 10.1371/journal.pone.0013441

Maturation profile of MDCs and PDCs after exposure to TLR ligand. MDC and PDC populations were sorted on a FACS ARIA cell sorter from T cells, B cells, NK cells and monocytes depleted PBMCs using lineage cocktail (Alexa Fluor 700), HLA DR (APC-Alexa Fluor 750), CD11c (PE-Cy7) and CD123 (PerCP-Cy5.5) mabs. The DC subsets were cultured in separate wells for 24 hours in 1% plasma medium with or without the presence of TLR3 ligand (Poly I:C) for MDCs or TLR9 ligand (C CpG) for PDCs. The cells were harvested and stained using direct conjugated antibodies against CD40, CD83, CD86, CCR5, CCR7, PDL-1, ICOSL, DCIR, and B7H3 and analyzed using multi-color flow cytometry. The data were normalized to the control medium and paired t test was performed for calculation of p values. Statistically significant differences between individuals with PDAC and healthy controls are indicated as; * = p
Figure Legend Snippet: Maturation profile of MDCs and PDCs after exposure to TLR ligand. MDC and PDC populations were sorted on a FACS ARIA cell sorter from T cells, B cells, NK cells and monocytes depleted PBMCs using lineage cocktail (Alexa Fluor 700), HLA DR (APC-Alexa Fluor 750), CD11c (PE-Cy7) and CD123 (PerCP-Cy5.5) mabs. The DC subsets were cultured in separate wells for 24 hours in 1% plasma medium with or without the presence of TLR3 ligand (Poly I:C) for MDCs or TLR9 ligand (C CpG) for PDCs. The cells were harvested and stained using direct conjugated antibodies against CD40, CD83, CD86, CCR5, CCR7, PDL-1, ICOSL, DCIR, and B7H3 and analyzed using multi-color flow cytometry. The data were normalized to the control medium and paired t test was performed for calculation of p values. Statistically significant differences between individuals with PDAC and healthy controls are indicated as; * = p

Techniques Used: FACS, Cell Culture, Staining, Flow Cytometry, Cytometry

40) Product Images from "Antitumor activity of arsenite in combination with tetrandrine against human breast cancer cell line MDA-MB-231 in vitro and in vivo"

Article Title: Antitumor activity of arsenite in combination with tetrandrine against human breast cancer cell line MDA-MB-231 in vitro and in vivo

Journal: Cancer Cell International

doi: 10.1186/s12935-018-0613-0

Effects of As III and Tetra, alone or in combination, on the cell cycle profiling and the expression level of cell cycle related-proteins in MDA-MB-231 cells. a – d After treatment with various concentrations of As III (5, 10 and 15 µM), and Tetra (3.5, 4 and 4.5 µg/ml), alone or in combination (5 µM As III + 3.5 µg/ml Tetra, 10 µM As III + 4 µg/ml Tetra), for 48 h, cell cycle profiling was performed by FACSCanto flow cytometer as described under “ Materials and methods ”. Analyzed data and profiles for each G 0 /G 1 and G 2 /M phase using Diva software and ModFit LT™ ver. 3.0. are shown in the gray area. Cells at S phase are shown as shaded area. A representative FACS histogram from three separate experiments is shown. Significant difference between control and treatment with As III and Tetra, alone or in combination, are shown (* p
Figure Legend Snippet: Effects of As III and Tetra, alone or in combination, on the cell cycle profiling and the expression level of cell cycle related-proteins in MDA-MB-231 cells. a – d After treatment with various concentrations of As III (5, 10 and 15 µM), and Tetra (3.5, 4 and 4.5 µg/ml), alone or in combination (5 µM As III + 3.5 µg/ml Tetra, 10 µM As III + 4 µg/ml Tetra), for 48 h, cell cycle profiling was performed by FACSCanto flow cytometer as described under “ Materials and methods ”. Analyzed data and profiles for each G 0 /G 1 and G 2 /M phase using Diva software and ModFit LT™ ver. 3.0. are shown in the gray area. Cells at S phase are shown as shaded area. A representative FACS histogram from three separate experiments is shown. Significant difference between control and treatment with As III and Tetra, alone or in combination, are shown (* p

Techniques Used: Expressing, Multiple Displacement Amplification, Flow Cytometry, Cytometry, Software, FACS

Related Articles

Flow Cytometry:

Article Title: An ABCG2 non-substrate anticancer agent FL118 targets drug-resistant cancer stem-like cells and overcomes treatment resistance of human pancreatic cancer
Article Snippet: .. Following the antibody labeling, PANC1 cells were washed in PBS with 2% BSA before flow cytometric analysis was carried out on a BD FACSAria™ III cell sorter (BD Science, USA). ..

Article Title: Anticancer effects and underlying mechanism of Colchicine on human gastric cancer cell lines in vitro and in vivo
Article Snippet: .. Flow cytometric analysis of apoptosis The percentage of cells undergoing apoptosis was quantitated with an Annexin V-FITC apoptosis detection kit (BD Bioscience, CA, U.S.A.) according to the manufacturer’s instructions. .. Briefly, following Colchicine incubation for 24 h, the cells were washed twice with cold PBS, mixed with binding buffer (200 μl) at a concentration of 1 × 106 cells/ml, and added with 5 μl of annexin V-FITC and 5 μl of PI in the dark at room temperature for 5 min. At the end of the incubation period, the cells were then transferred into a flow cytometry tube and analyzed on an FACScan flow cytometer (Becton & Dickinson Co., U.S.A.).

Article Title: Peroral Low-Dose Toxoplasma gondii Infection of Human Microbiota-Associated Mice — A Subacute Ileitis Model to Unravel Pathogen–Host Interactions
Article Snippet: .. After 18 h at 37 °C, culture supernatants and serum samples were tested for IFN-γ , TNF, IL-6, monocyte chemoattractant protein-1 (MCP-1), IL-6, and IL-10 by the Mouse Inflammation Cytometric Bead Assay (CBA; BD Biosciences, Heidelberg, Germany) on a BD FACSCanto II flow cytometer (BD Biosciences) as described earlier [ , , ]. ..

Antibody Labeling:

Article Title: An ABCG2 non-substrate anticancer agent FL118 targets drug-resistant cancer stem-like cells and overcomes treatment resistance of human pancreatic cancer
Article Snippet: .. Following the antibody labeling, PANC1 cells were washed in PBS with 2% BSA before flow cytometric analysis was carried out on a BD FACSAria™ III cell sorter (BD Science, USA). ..

Cytometry:

Article Title: Peroral Low-Dose Toxoplasma gondii Infection of Human Microbiota-Associated Mice — A Subacute Ileitis Model to Unravel Pathogen–Host Interactions
Article Snippet: .. After 18 h at 37 °C, culture supernatants and serum samples were tested for IFN-γ , TNF, IL-6, monocyte chemoattractant protein-1 (MCP-1), IL-6, and IL-10 by the Mouse Inflammation Cytometric Bead Assay (CBA; BD Biosciences, Heidelberg, Germany) on a BD FACSCanto II flow cytometer (BD Biosciences) as described earlier [ , , ]. ..

Enzyme-linked Immunosorbent Assay:

Article Title: Amblyomma sculptum Salivary PGE2 Modulates the Dendritic Cell-Rickettsia rickettsii Interactions in vitro and in vivo
Article Snippet: .. Cytokines were evaluated in cell-free supernatants collected after 6 h (for TNF-α) or 18 h (for IL-6, IL-12p40 and IL-12p70) by enzyme-linked immunosorbent assay (ELISA) using BD OptEIA™ ELISA Sets (BD Biosciences, San Diego, CA, United States) according to manufacturer's instructions. .. For flow cytometry analysis, DCs were preincubated for 1 h with medium only, A. sculptum saliva (1:50 dilution) or PGE2 (100 nM-final concentration), followed by incubation with R. rickettsii for 18 h, as described above.

Article Title: IL-10-Engineered Human CD4+ Tr1 Cells Eliminate Myeloid Leukemia in an HLA Class I-Dependent Mechanism
Article Snippet: .. Supernatants were harvested after 48 hr of culture, and levels of IL-4, IL-10, IFN-γ, and IL-17 were determined by ELISA according to the manufacturer’s instructions (BD Biosciences). ..

Article Title: Electrophilic nitroalkene-tocopherol derivatives: synthesis, physicochemical characterization and evaluation of anti-inflammatory signaling responses
Article Snippet: .. MCP-1, IL-6 and TNF-α were measured in the supernatant with a commercially available ELISA kit (BD OptEIA). .. Quantitative real-time PCR RAW264.7 cells were grown 10% FBS/DMEM (Gibco) and then treated for 5 h with NATxME (1, 3 and 10 µM).

Staining:

Article Title: Impact of Zostavax Vaccination on T-Cell Accumulation and Cutaneous Gene Expression in the Skin of Older Humans After Varicella Zoster Virus Antigen–Specific Challenge
Article Snippet: .. Following stimulation, cells were stained using combinations of antibodies, including CD3 (clone UCHT1), CD4 (RPA-T4), and CD8 (SK1), for 30 minutes at 4°C and washed, fixed, and permeabilized (Fix & Perm Cell Permeabilisation Kit, Invitrogen, Paisley, UK) before staining for interleukin 2 (clone 5344.111), interferon γ (IFN-γ; B27), and tumor necrosis factor α (TNF-α; Mab11; all from BD Biosciences, Oxford, UK). .. Multiparameter investigation of skin and blood T-cell phenotypes was performed on an LSR II or BD Fortessa instrument, using FACS Diva software (BD Biosciences, Oxford, UK), and data were subsequently analyzed using FlowJo, version X (TreeStar, Ashland, OR), as previously described [ , ].

Recombinase Polymerase Amplification:

Article Title: Impact of Zostavax Vaccination on T-Cell Accumulation and Cutaneous Gene Expression in the Skin of Older Humans After Varicella Zoster Virus Antigen–Specific Challenge
Article Snippet: .. Following stimulation, cells were stained using combinations of antibodies, including CD3 (clone UCHT1), CD4 (RPA-T4), and CD8 (SK1), for 30 minutes at 4°C and washed, fixed, and permeabilized (Fix & Perm Cell Permeabilisation Kit, Invitrogen, Paisley, UK) before staining for interleukin 2 (clone 5344.111), interferon γ (IFN-γ; B27), and tumor necrosis factor α (TNF-α; Mab11; all from BD Biosciences, Oxford, UK). .. Multiparameter investigation of skin and blood T-cell phenotypes was performed on an LSR II or BD Fortessa instrument, using FACS Diva software (BD Biosciences, Oxford, UK), and data were subsequently analyzed using FlowJo, version X (TreeStar, Ashland, OR), as previously described [ , ].

Crocin Bleaching Assay:

Article Title: Peroral Low-Dose Toxoplasma gondii Infection of Human Microbiota-Associated Mice — A Subacute Ileitis Model to Unravel Pathogen–Host Interactions
Article Snippet: .. After 18 h at 37 °C, culture supernatants and serum samples were tested for IFN-γ , TNF, IL-6, monocyte chemoattractant protein-1 (MCP-1), IL-6, and IL-10 by the Mouse Inflammation Cytometric Bead Assay (CBA; BD Biosciences, Heidelberg, Germany) on a BD FACSCanto II flow cytometer (BD Biosciences) as described earlier [ , , ]. ..

Article Title: Lepidium meyenii Walp Exhibits Anti-Inflammatory Activity against ConA-Induced Acute Hepatitis
Article Snippet: .. Serum and liver tissue concentrations of TNF-α , IFN-γ , IL-1β , IL-2, IL-6, IL-12, and IL-17a were determined using CBA Flex Sets according to the manufacturer's instructions (BD Biosciences Company, USA). .. Western Blot Analysis Liver tissue supernatants were boiled with 2 × SDS loading buffer for 10 min.

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    Becton Dickinson rat monoclonal anti mouse syndecan 4
    ATXβ controls breast cancer cell metastasis through an SDC4-dependent mechanism ( A ) 4T1 cell adhesion to increasing amounts of ATXβ, BSA (400 ng) was used as control (left panels). Data represent the mean of adherent cells/mm 2 ±SD of adherent cells of 3 experiments performed in 8 replicates (right panel). ( B ) Flow cytometry detection of cell surface expression of <t>syndecan-4</t> (SDC4) in 4T1 cells. Cells were immunostained with KY/8.2 monoclonal antibody (anti-SDC4) (black bar), or isotype control antibody MOPC21 (grey bar). NT: not treated cells (open bar). ( C ) Inhibition of 4T1 cell adhesion on ATXβ with KY/8.2 antibody (anti-SDC4). Indicated cell lines were preincubated for 1 h in the presence of KY/8.2 or MOPC21 antibodies (10 µg/mL). Data represent the mean of adherent cells/mm 2 ±SD of adherent cells of 3 experiments performed in 8 replicates ( *** : P
    Rat Monoclonal Anti Mouse Syndecan 4, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson panc1 cells
    FL118 targets both proliferative cancer cells and latent stem cell-like cancer cells: a , FL118-cisplatin combination enhances killing of <t>PANC1</t> cells. Dead cells were determined by trypan blue exclusion. b , GFPcODC-positive cells survive 50-□M cisplatin for 4 days, evaluated with trypan blue exclusion assay. c , FL118-cisplatin kills GFPcODC positive (drug-resistant) cells (arrows). Phase-contrast and GFP imaging of cells were digitally taken. d , FL118-cisplatin combination reduces PANC1 spheroid cell number. *, P
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    Becton Dickinson gpa expression
    Amplification and maturation of CD34 + fetal liver (FL) and adult peripheral blood (AB) derived cells during ex vivo erythroid differentiation. (A) CD34 + immuno-enriched stem cells (FL, solid line; AB, dashed line) were grown in liquid culture according to the two-step protocol. Note that the purity of immuno-enriched cells was not assessed before expansion. Total cells were counted daily from day 4 to day 8 of culture. Mean values of fold-change relative to day 4 of culture with the standard deviation for five experiments are shown. (B) Flow cytometry analysis for transferrin receptor <t>(CD71)</t> and glycophorin A <t>(GPA)</t> surface expression on culture day 8. Flow cytometric gates are denoted as CD71 − /GPA − (lower left quadrant), CD71 + /GPA − (upper left quadrant), CD71 + /GPA + (upper right quadrant) and CD71 − /GPA + (lower right quadrant). (C) Morphology of cells harvested at culture day 8.
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    Becton Dickinson 2 nbdg fluorescence
    NRF2 upregulates Warburg effect in CICs. a Glycolytic enzyme protein levels in Control GFP or NRF2 Over cells cultivated in selection medium for 2 weeks. b Total transcripts were prepared from sh- NRF2 and Control sh-Luc sphere cells (SAS and OECM1 cells) and further analyzed by Real-time RT-PCR. c Total proteins, prepared from sh -NRF2 and control sphere cells (SAS-S, OECM1-S), were analyzed by immunoblotting with the indicated antibodies. d Glucose uptake levels in sh -NRF2 and Control sphere cells were measured by detecting the uptake of fluorescent <t>2-NBDG</t> dye. e Intracellular lactate levels measured in sh -NRF2 and control sphere cells. f Intracellular lactate levels measured in Control GFP or NRF2 Over cells cultivated within selection medium. g LC-MS metabolic profiling showing the metabolic intermediates of glycolysis, PPP, TCA, and glutaminolysis. ( n = 3 independent experiments; t -test, * P
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    ATXβ controls breast cancer cell metastasis through an SDC4-dependent mechanism ( A ) 4T1 cell adhesion to increasing amounts of ATXβ, BSA (400 ng) was used as control (left panels). Data represent the mean of adherent cells/mm 2 ±SD of adherent cells of 3 experiments performed in 8 replicates (right panel). ( B ) Flow cytometry detection of cell surface expression of syndecan-4 (SDC4) in 4T1 cells. Cells were immunostained with KY/8.2 monoclonal antibody (anti-SDC4) (black bar), or isotype control antibody MOPC21 (grey bar). NT: not treated cells (open bar). ( C ) Inhibition of 4T1 cell adhesion on ATXβ with KY/8.2 antibody (anti-SDC4). Indicated cell lines were preincubated for 1 h in the presence of KY/8.2 or MOPC21 antibodies (10 µg/mL). Data represent the mean of adherent cells/mm 2 ±SD of adherent cells of 3 experiments performed in 8 replicates ( *** : P

    Journal: Oncotarget

    Article Title: Autotaxin-β interaction with the cell surface via syndecan-4 impacts on cancer cell proliferation and metastasis

    doi: 10.18632/oncotarget.26039

    Figure Lengend Snippet: ATXβ controls breast cancer cell metastasis through an SDC4-dependent mechanism ( A ) 4T1 cell adhesion to increasing amounts of ATXβ, BSA (400 ng) was used as control (left panels). Data represent the mean of adherent cells/mm 2 ±SD of adherent cells of 3 experiments performed in 8 replicates (right panel). ( B ) Flow cytometry detection of cell surface expression of syndecan-4 (SDC4) in 4T1 cells. Cells were immunostained with KY/8.2 monoclonal antibody (anti-SDC4) (black bar), or isotype control antibody MOPC21 (grey bar). NT: not treated cells (open bar). ( C ) Inhibition of 4T1 cell adhesion on ATXβ with KY/8.2 antibody (anti-SDC4). Indicated cell lines were preincubated for 1 h in the presence of KY/8.2 or MOPC21 antibodies (10 µg/mL). Data represent the mean of adherent cells/mm 2 ±SD of adherent cells of 3 experiments performed in 8 replicates ( *** : P

    Article Snippet: Rat monoclonal anti-mouse syndecan-4 (KY/8.2) was from Becton Dickinson Biosciences (Franklin Lakes, NJ, USA) and MOPC21 antibody was from ICN Pharmaceuticals (Paris, France).

    Techniques: Flow Cytometry, Cytometry, Expressing, Inhibition

    FL118 targets both proliferative cancer cells and latent stem cell-like cancer cells: a , FL118-cisplatin combination enhances killing of PANC1 cells. Dead cells were determined by trypan blue exclusion. b , GFPcODC-positive cells survive 50-□M cisplatin for 4 days, evaluated with trypan blue exclusion assay. c , FL118-cisplatin kills GFPcODC positive (drug-resistant) cells (arrows). Phase-contrast and GFP imaging of cells were digitally taken. d , FL118-cisplatin combination reduces PANC1 spheroid cell number. *, P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: An ABCG2 non-substrate anticancer agent FL118 targets drug-resistant cancer stem-like cells and overcomes treatment resistance of human pancreatic cancer

    doi: 10.1186/s13046-018-0899-8

    Figure Lengend Snippet: FL118 targets both proliferative cancer cells and latent stem cell-like cancer cells: a , FL118-cisplatin combination enhances killing of PANC1 cells. Dead cells were determined by trypan blue exclusion. b , GFPcODC-positive cells survive 50-□M cisplatin for 4 days, evaluated with trypan blue exclusion assay. c , FL118-cisplatin kills GFPcODC positive (drug-resistant) cells (arrows). Phase-contrast and GFP imaging of cells were digitally taken. d , FL118-cisplatin combination reduces PANC1 spheroid cell number. *, P

    Article Snippet: Following the antibody labeling, PANC1 cells were washed in PBS with 2% BSA before flow cytometric analysis was carried out on a BD FACSAria™ III cell sorter (BD Science, USA).

    Techniques: Trypan Blue Exclusion Assay, Imaging

    FL118 downregulates the expression of multiple antiapoptotic proteins and upregulates certain proapoptotic proteins in pancreatic cancer cells: Subconfluent pancreatic cancer cells were treated with FL118 as shown, and the expression of antiapoptotic proteins survivin, Mcl-1, XIAP, and cIAP2 as well as proapoptotic proteins Bad, Bim and Bax was detected by western blots using corresponding antibodies for each protein. GAPDH is an internal control for protein loading. The result from PANC1 pancreatic cancer cell line is shown in a , and the result from MIA PaCa2 (Mia2) pancreatic cancer cell line is shown in ( b )

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: An ABCG2 non-substrate anticancer agent FL118 targets drug-resistant cancer stem-like cells and overcomes treatment resistance of human pancreatic cancer

    doi: 10.1186/s13046-018-0899-8

    Figure Lengend Snippet: FL118 downregulates the expression of multiple antiapoptotic proteins and upregulates certain proapoptotic proteins in pancreatic cancer cells: Subconfluent pancreatic cancer cells were treated with FL118 as shown, and the expression of antiapoptotic proteins survivin, Mcl-1, XIAP, and cIAP2 as well as proapoptotic proteins Bad, Bim and Bax was detected by western blots using corresponding antibodies for each protein. GAPDH is an internal control for protein loading. The result from PANC1 pancreatic cancer cell line is shown in a , and the result from MIA PaCa2 (Mia2) pancreatic cancer cell line is shown in ( b )

    Article Snippet: Following the antibody labeling, PANC1 cells were washed in PBS with 2% BSA before flow cytometric analysis was carried out on a BD FACSAria™ III cell sorter (BD Science, USA).

    Techniques: Expressing, Western Blot

    Induction of apoptosis, pancreatic cancer cell killing and cell viability inhibition by FL118: a and b , FL118 treatment results in activation of caspase-3 and cleavage of PARP. Subconfluent pancreatic cancer cells (A, PANC1; B, MIA PaCa2) were treated with FL118 as shown, and the activation of casepase-3 and cleavage of PARP were detected by western blots. GAPDG is the internal control for total protein loading. c , FL118 induces pancreatic cancer cell death. Subconfluent PANC1 and Mia2 pancreatic cancer cells were treated with vehicle or with FL118 at 10, 10 and 500 nM for 48 h. Then the sub-G1 DNA content (later apoptotic dead cells) was determined by flow cytometry. Relative sub-G1 DNA production levels were analyzed and the data were derived from 3 independent testing and shown as histogram as mean ± SD. d , FL118 inhibits pancreatic cancer cell viability. Subconfluent Mia2, PANC1 and BxPC3 pancreatic cancer cells as well as normal human dermal fibroblast cells were treated with vehicle (no FL118) or with a series of FL118 concentrations as shown for 72 h. Then the cell viability was determined using MTT assay. The data were shown as histogram with mean ± SD derived from 3 independent testing assays

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: An ABCG2 non-substrate anticancer agent FL118 targets drug-resistant cancer stem-like cells and overcomes treatment resistance of human pancreatic cancer

    doi: 10.1186/s13046-018-0899-8

    Figure Lengend Snippet: Induction of apoptosis, pancreatic cancer cell killing and cell viability inhibition by FL118: a and b , FL118 treatment results in activation of caspase-3 and cleavage of PARP. Subconfluent pancreatic cancer cells (A, PANC1; B, MIA PaCa2) were treated with FL118 as shown, and the activation of casepase-3 and cleavage of PARP were detected by western blots. GAPDG is the internal control for total protein loading. c , FL118 induces pancreatic cancer cell death. Subconfluent PANC1 and Mia2 pancreatic cancer cells were treated with vehicle or with FL118 at 10, 10 and 500 nM for 48 h. Then the sub-G1 DNA content (later apoptotic dead cells) was determined by flow cytometry. Relative sub-G1 DNA production levels were analyzed and the data were derived from 3 independent testing and shown as histogram as mean ± SD. d , FL118 inhibits pancreatic cancer cell viability. Subconfluent Mia2, PANC1 and BxPC3 pancreatic cancer cells as well as normal human dermal fibroblast cells were treated with vehicle (no FL118) or with a series of FL118 concentrations as shown for 72 h. Then the cell viability was determined using MTT assay. The data were shown as histogram with mean ± SD derived from 3 independent testing assays

    Article Snippet: Following the antibody labeling, PANC1 cells were washed in PBS with 2% BSA before flow cytometric analysis was carried out on a BD FACSAria™ III cell sorter (BD Science, USA).

    Techniques: Inhibition, Activation Assay, Western Blot, Flow Cytometry, Cytometry, Derivative Assay, MTT Assay

    Effects of FL118 on the expression of proteins that are involved the pancreatic cancer cell DNA damage and repair: a , Subconfluent PANC1 pancreatic cancer cells were treated with FL118 as shown, and the expression of ERCC1, ERCC6, γ-H2AX, ChK1, ChK2, ATM, ATR, RAD51 and DNA Pol II was detected by western blots using corresponding antibodies for each protein. GAPDH is an internal control for protein loading. b, Decrease of ERCC6 expression can be rescued with proteasome inhibitor MG132. Subconfluent SKOV3 cells were treated with FL118 and MG132 alone or in combination as shown for 8 h, followed by western blot analyses with ERCC6 antibody. Tubulin is the internal control for protein loading. c , Comparison of FL118 and topotecan (TOP) effects on ERCC6 expression. Subconfluent SKOV3 cells were treated with FL118 or TOP as shown for 8 h, followed by western blot analyses with ERCC6 antibody. Tubulin is the internal control for protein loading

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: An ABCG2 non-substrate anticancer agent FL118 targets drug-resistant cancer stem-like cells and overcomes treatment resistance of human pancreatic cancer

    doi: 10.1186/s13046-018-0899-8

    Figure Lengend Snippet: Effects of FL118 on the expression of proteins that are involved the pancreatic cancer cell DNA damage and repair: a , Subconfluent PANC1 pancreatic cancer cells were treated with FL118 as shown, and the expression of ERCC1, ERCC6, γ-H2AX, ChK1, ChK2, ATM, ATR, RAD51 and DNA Pol II was detected by western blots using corresponding antibodies for each protein. GAPDH is an internal control for protein loading. b, Decrease of ERCC6 expression can be rescued with proteasome inhibitor MG132. Subconfluent SKOV3 cells were treated with FL118 and MG132 alone or in combination as shown for 8 h, followed by western blot analyses with ERCC6 antibody. Tubulin is the internal control for protein loading. c , Comparison of FL118 and topotecan (TOP) effects on ERCC6 expression. Subconfluent SKOV3 cells were treated with FL118 or TOP as shown for 8 h, followed by western blot analyses with ERCC6 antibody. Tubulin is the internal control for protein loading

    Article Snippet: Following the antibody labeling, PANC1 cells were washed in PBS with 2% BSA before flow cytometric analysis was carried out on a BD FACSAria™ III cell sorter (BD Science, USA).

    Techniques: Expressing, Western Blot

    Amplification and maturation of CD34 + fetal liver (FL) and adult peripheral blood (AB) derived cells during ex vivo erythroid differentiation. (A) CD34 + immuno-enriched stem cells (FL, solid line; AB, dashed line) were grown in liquid culture according to the two-step protocol. Note that the purity of immuno-enriched cells was not assessed before expansion. Total cells were counted daily from day 4 to day 8 of culture. Mean values of fold-change relative to day 4 of culture with the standard deviation for five experiments are shown. (B) Flow cytometry analysis for transferrin receptor (CD71) and glycophorin A (GPA) surface expression on culture day 8. Flow cytometric gates are denoted as CD71 − /GPA − (lower left quadrant), CD71 + /GPA − (upper left quadrant), CD71 + /GPA + (upper right quadrant) and CD71 − /GPA + (lower right quadrant). (C) Morphology of cells harvested at culture day 8.

    Journal: PeerJ

    Article Title: Comparison of gene expression profiles between human erythroid cells derived from fetal liver and adult peripheral blood

    doi: 10.7717/peerj.5527

    Figure Lengend Snippet: Amplification and maturation of CD34 + fetal liver (FL) and adult peripheral blood (AB) derived cells during ex vivo erythroid differentiation. (A) CD34 + immuno-enriched stem cells (FL, solid line; AB, dashed line) were grown in liquid culture according to the two-step protocol. Note that the purity of immuno-enriched cells was not assessed before expansion. Total cells were counted daily from day 4 to day 8 of culture. Mean values of fold-change relative to day 4 of culture with the standard deviation for five experiments are shown. (B) Flow cytometry analysis for transferrin receptor (CD71) and glycophorin A (GPA) surface expression on culture day 8. Flow cytometric gates are denoted as CD71 − /GPA − (lower left quadrant), CD71 + /GPA − (upper left quadrant), CD71 + /GPA + (upper right quadrant) and CD71 − /GPA + (lower right quadrant). (C) Morphology of cells harvested at culture day 8.

    Article Snippet: Erythroid populations comprising cells with high CD71 and GPA expression were FACS sorted using a BD FACSAria™ III cell sorter (BD Biosciences).

    Techniques: Amplification, Derivative Assay, Ex Vivo, Standard Deviation, Flow Cytometry, Cytometry, Expressing

    NRF2 upregulates Warburg effect in CICs. a Glycolytic enzyme protein levels in Control GFP or NRF2 Over cells cultivated in selection medium for 2 weeks. b Total transcripts were prepared from sh- NRF2 and Control sh-Luc sphere cells (SAS and OECM1 cells) and further analyzed by Real-time RT-PCR. c Total proteins, prepared from sh -NRF2 and control sphere cells (SAS-S, OECM1-S), were analyzed by immunoblotting with the indicated antibodies. d Glucose uptake levels in sh -NRF2 and Control sphere cells were measured by detecting the uptake of fluorescent 2-NBDG dye. e Intracellular lactate levels measured in sh -NRF2 and control sphere cells. f Intracellular lactate levels measured in Control GFP or NRF2 Over cells cultivated within selection medium. g LC-MS metabolic profiling showing the metabolic intermediates of glycolysis, PPP, TCA, and glutaminolysis. ( n = 3 independent experiments; t -test, * P

    Journal: Cell Death & Disease

    Article Title: ROS-independent ER stress-mediated NRF2 activation promotes warburg effect to maintain stemness-associated properties of cancer-initiating cells

    doi: 10.1038/s41419-017-0250-x

    Figure Lengend Snippet: NRF2 upregulates Warburg effect in CICs. a Glycolytic enzyme protein levels in Control GFP or NRF2 Over cells cultivated in selection medium for 2 weeks. b Total transcripts were prepared from sh- NRF2 and Control sh-Luc sphere cells (SAS and OECM1 cells) and further analyzed by Real-time RT-PCR. c Total proteins, prepared from sh -NRF2 and control sphere cells (SAS-S, OECM1-S), were analyzed by immunoblotting with the indicated antibodies. d Glucose uptake levels in sh -NRF2 and Control sphere cells were measured by detecting the uptake of fluorescent 2-NBDG dye. e Intracellular lactate levels measured in sh -NRF2 and control sphere cells. f Intracellular lactate levels measured in Control GFP or NRF2 Over cells cultivated within selection medium. g LC-MS metabolic profiling showing the metabolic intermediates of glycolysis, PPP, TCA, and glutaminolysis. ( n = 3 independent experiments; t -test, * P

    Article Snippet: The 2-NBDG fluorescence was measured using a FACScanto flow cytometer (Becton Dickinson).

    Techniques: Selection, Quantitative RT-PCR, Liquid Chromatography with Mass Spectroscopy