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CD3 + <t>CD4</t> + cells were isolated from L-Tg-FIR and FIR mice and stained for Foxp3 expression. (A) Representative <t>FACS</t> plot depicts percent Foxp3 expression from WT and L-Tg splenocytes. (B) MFI of Foxp3 expression is plotted from different tissues taken from
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1) Product Images from "Differential levels of Tl1a affect the expansion and function of regulatory T cells in modulating murine colitis"

Article Title: Differential levels of Tl1a affect the expansion and function of regulatory T cells in modulating murine colitis

Journal: Inflammatory bowel diseases

doi: 10.1097/MIB.0000000000000653

CD3 + CD4 + cells were isolated from L-Tg-FIR and FIR mice and stained for Foxp3 expression. (A) Representative FACS plot depicts percent Foxp3 expression from WT and L-Tg splenocytes. (B) MFI of Foxp3 expression is plotted from different tissues taken from
Figure Legend Snippet: CD3 + CD4 + cells were isolated from L-Tg-FIR and FIR mice and stained for Foxp3 expression. (A) Representative FACS plot depicts percent Foxp3 expression from WT and L-Tg splenocytes. (B) MFI of Foxp3 expression is plotted from different tissues taken from

Techniques Used: Isolation, Mouse Assay, Staining, Expressing, FACS

2) Product Images from "FGF1 Mediates Overnutrition-Induced Compensatory β-Cell Differentiation"

Article Title: FGF1 Mediates Overnutrition-Induced Compensatory β-Cell Differentiation

Journal: Diabetes

doi: 10.2337/db15-0085

FGF1 is necessary for overnutrition-induced β-cell differentiation. A : RT-PCR analysis of fgf1 , insa , gcga , and amy2a expression in FACS-sorted β-cells. nc, no template control. B : Overnutrition-induced mild hyperglycemia in fgf1 mu1/mu1 fish. Wild-type and fgf1 mu1/mu1 larvae were cultured for 8 h in nutrient-free or 5% egg yolk solution at 6 dpf, and their total free glucose levels were determined immediately after. n = 10. Representative confocal projections of β-cells of 6-dpf ( C ) and 4-week-old ( E ) Tg(−1.2ins:H2B-mCherry) or fgf1 mu1/mu1 ;Tg(−1.2ins:H2B-mCherry) larvae cultured for 8 h in nutrient-free medium or 5% egg yolk. Scale bar indicates 10 µm in C and 20 μm in E . Quantification of β-cell number from 6-dpf larvae ( D ) or 4-week-old fish ( F ) suggested a loss of overnutrition-induced β-cell differentiation in fgf1 -deficient fish. n = 7–24 in D and n = 10 in F . * P
Figure Legend Snippet: FGF1 is necessary for overnutrition-induced β-cell differentiation. A : RT-PCR analysis of fgf1 , insa , gcga , and amy2a expression in FACS-sorted β-cells. nc, no template control. B : Overnutrition-induced mild hyperglycemia in fgf1 mu1/mu1 fish. Wild-type and fgf1 mu1/mu1 larvae were cultured for 8 h in nutrient-free or 5% egg yolk solution at 6 dpf, and their total free glucose levels were determined immediately after. n = 10. Representative confocal projections of β-cells of 6-dpf ( C ) and 4-week-old ( E ) Tg(−1.2ins:H2B-mCherry) or fgf1 mu1/mu1 ;Tg(−1.2ins:H2B-mCherry) larvae cultured for 8 h in nutrient-free medium or 5% egg yolk. Scale bar indicates 10 µm in C and 20 μm in E . Quantification of β-cell number from 6-dpf larvae ( D ) or 4-week-old fish ( F ) suggested a loss of overnutrition-induced β-cell differentiation in fgf1 -deficient fish. n = 7–24 in D and n = 10 in F . * P

Techniques Used: Cell Differentiation, Reverse Transcription Polymerase Chain Reaction, Expressing, FACS, Fluorescence In Situ Hybridization, Cell Culture

3) Product Images from "MASTL inhibition promotes mitotic catastrophe through PP2A activation to inhibit cancer growth and radioresistance in breast cancer cells"

Article Title: MASTL inhibition promotes mitotic catastrophe through PP2A activation to inhibit cancer growth and radioresistance in breast cancer cells

Journal: BMC Cancer

doi: 10.1186/s12885-018-4600-6

MASTL depletion increases the radiosensitivity of breast cancer cells. MCF7 cells were transfected with either 5 nmol/l control siRNA or MASTL.5 siRNA. Cells were treated 0, 3 or 4 Gy irradiation for 42 h. a , b Cell proliferation was determined by FACS analysis and then analyzed by immunoblotted with the indicated antibodies. c The results of the clonogenic assay. Representative images of the cells treated the indicated conditions (left panel). The number of colonies was measured (right panel). d The sphere forming assay was performed ( > 110 sphere colonies per data point). Scale bar = 100 μm. Representative images of sphere forming assay. e PP2A-Aα/β proteins were immunoprecipitated using an anti-PP2A-Aα/β antibody and analyzed for PP2A activity. The data represent typical results and are presented as the mean ± standard deviation of three independent experiments; ** P
Figure Legend Snippet: MASTL depletion increases the radiosensitivity of breast cancer cells. MCF7 cells were transfected with either 5 nmol/l control siRNA or MASTL.5 siRNA. Cells were treated 0, 3 or 4 Gy irradiation for 42 h. a , b Cell proliferation was determined by FACS analysis and then analyzed by immunoblotted with the indicated antibodies. c The results of the clonogenic assay. Representative images of the cells treated the indicated conditions (left panel). The number of colonies was measured (right panel). d The sphere forming assay was performed ( > 110 sphere colonies per data point). Scale bar = 100 μm. Representative images of sphere forming assay. e PP2A-Aα/β proteins were immunoprecipitated using an anti-PP2A-Aα/β antibody and analyzed for PP2A activity. The data represent typical results and are presented as the mean ± standard deviation of three independent experiments; ** P

Techniques Used: Transfection, Irradiation, FACS, Clonogenic Assay, Immunoprecipitation, Activity Assay, Standard Deviation

MASTL depletion does not affect the viability of normal cells. HUVEC, HDFn, MCF7, and MCF10A cells were transfected with either control siRNA or MASTL.5 siRNA for 48 h. a , f The cells were analyzed by immunoblotting with the indicated antibodies. b , c , g Cell viability was determined by WST-8 assay. d , e , h , i The cell cycle was analyzed by FACS. The data represent typical results and are presented as the mean ± standard deviation of three independent experiments; ** P
Figure Legend Snippet: MASTL depletion does not affect the viability of normal cells. HUVEC, HDFn, MCF7, and MCF10A cells were transfected with either control siRNA or MASTL.5 siRNA for 48 h. a , f The cells were analyzed by immunoblotting with the indicated antibodies. b , c , g Cell viability was determined by WST-8 assay. d , e , h , i The cell cycle was analyzed by FACS. The data represent typical results and are presented as the mean ± standard deviation of three independent experiments; ** P

Techniques Used: Transfection, FACS, Standard Deviation

4) Product Images from "Engineering genetically-encoded synthetic biomarkers for breath-based cancer detection"

Article Title: Engineering genetically-encoded synthetic biomarkers for breath-based cancer detection

Journal: bioRxiv

doi: 10.1101/2021.09.01.456741

Vector design, transfection, and limonene production by HeLa cells. ( A) Schematic of experimental methodology. (Top) Cultured HeLa cells were transfected with a vector containing LS and eGFP genes under the control of a CAG promoter. Antibiotic and FACS selection for stably transfected clones (sorting on eGFP-expressing cells) resulted in a HeLa cell line containing both LS and eGFP (HeLa-LS-eGFP cells, subsequently referred to as HeLa-LS cells). ( Bottom ) HeLa-LS cells were subsequently transfected with a vector containing the tHMGR and tRFP genes under the control of an EF1α promoter. Antibiotic and FACS selection (based on dual expression of eGFP and tRFP) resulted in a HeLa cell line containing LS, tHMGR, eGFP, and tRFP (HeLa-LS-tHMGR-eGFP-tRFP, subsequently referred to as HeLa-LS-tHMGR). Solid phase microextraction (SPME) fibers were used to sample the culture headspace of confluent stably transfected HeLa-LS and HeLa-LS-tHMGR cells for 30 minutes, and were then analyzed for limonene by GC-MS. (B) (i) Piggybac transposon DNA vector containing truncated limonene synthase (LS) and enhanced green fluorescent protein (eGFP) driven by a CAG promoter, and puromycin resistance gene driven by a CMV promoter. (ii) Piggybac transposon DNA vector containing truncated HMG CoA reductase (tHMGR) and turbo red fluorescent protein (tRFP) driven by an EF1α promoter, and hygromycin resistance gene driven by a CMV promoter. (C) Bright-field and fluorescence images showing HeLa-LS and HeLa-LS-tHMGR cells after antibiotic selection and FACS sorting, compared with untransfected control HeLa cells. Scale bar = 200 um for HeLa control and 400 μm for HeLa-LS and HeLa-LS-tHMGR. (D) Mass spectrum from an SPME fiber exposed to the headspace of confluent HeLa-LS cells (top, red) compared with the reference spectrum of limonene from a mass spectrum library (Mnova database) (bottom, black). Note the characteristic peaks at m/z = 68, 93, and 136. (E) Selected ion monitoring (SIM) mode chromatogram of an SPME headspace sample from HeLa-LS cells (left) and from a pure limonene standard (right), showing matching ion ratios and retention times. (F) Calibration curve relating headspace limonene concentration as measured by SIFT-MS to the quantity of limonene spiked into culture media in a T75 flask ( y = 0.62 x 0.86 , R 2 =0.99). Over the range of limonene production by cultured cells (1 to 1000 ng, red bracket), the relationship is well-modeled by y = 0.28 x (R 2 =0.99). (G) Headspace concentration of limonene as a function of cell number for HeLa-LS ( y = [1.56×10 −6 ] x + 1.06, R 2 = 0.99) and HeLa-LS-tHMGR cells ( y = [3.21×10 −6 ] x + 2.70, R 2 = 0.98) after incubation at 37°C for 24 hours. Limonene measured from HeLa-LS-tHMGR cells was approximately double that from HeLa-LS cells over the cell density range examined.
Figure Legend Snippet: Vector design, transfection, and limonene production by HeLa cells. ( A) Schematic of experimental methodology. (Top) Cultured HeLa cells were transfected with a vector containing LS and eGFP genes under the control of a CAG promoter. Antibiotic and FACS selection for stably transfected clones (sorting on eGFP-expressing cells) resulted in a HeLa cell line containing both LS and eGFP (HeLa-LS-eGFP cells, subsequently referred to as HeLa-LS cells). ( Bottom ) HeLa-LS cells were subsequently transfected with a vector containing the tHMGR and tRFP genes under the control of an EF1α promoter. Antibiotic and FACS selection (based on dual expression of eGFP and tRFP) resulted in a HeLa cell line containing LS, tHMGR, eGFP, and tRFP (HeLa-LS-tHMGR-eGFP-tRFP, subsequently referred to as HeLa-LS-tHMGR). Solid phase microextraction (SPME) fibers were used to sample the culture headspace of confluent stably transfected HeLa-LS and HeLa-LS-tHMGR cells for 30 minutes, and were then analyzed for limonene by GC-MS. (B) (i) Piggybac transposon DNA vector containing truncated limonene synthase (LS) and enhanced green fluorescent protein (eGFP) driven by a CAG promoter, and puromycin resistance gene driven by a CMV promoter. (ii) Piggybac transposon DNA vector containing truncated HMG CoA reductase (tHMGR) and turbo red fluorescent protein (tRFP) driven by an EF1α promoter, and hygromycin resistance gene driven by a CMV promoter. (C) Bright-field and fluorescence images showing HeLa-LS and HeLa-LS-tHMGR cells after antibiotic selection and FACS sorting, compared with untransfected control HeLa cells. Scale bar = 200 um for HeLa control and 400 μm for HeLa-LS and HeLa-LS-tHMGR. (D) Mass spectrum from an SPME fiber exposed to the headspace of confluent HeLa-LS cells (top, red) compared with the reference spectrum of limonene from a mass spectrum library (Mnova database) (bottom, black). Note the characteristic peaks at m/z = 68, 93, and 136. (E) Selected ion monitoring (SIM) mode chromatogram of an SPME headspace sample from HeLa-LS cells (left) and from a pure limonene standard (right), showing matching ion ratios and retention times. (F) Calibration curve relating headspace limonene concentration as measured by SIFT-MS to the quantity of limonene spiked into culture media in a T75 flask ( y = 0.62 x 0.86 , R 2 =0.99). Over the range of limonene production by cultured cells (1 to 1000 ng, red bracket), the relationship is well-modeled by y = 0.28 x (R 2 =0.99). (G) Headspace concentration of limonene as a function of cell number for HeLa-LS ( y = [1.56×10 −6 ] x + 1.06, R 2 = 0.99) and HeLa-LS-tHMGR cells ( y = [3.21×10 −6 ] x + 2.70, R 2 = 0.98) after incubation at 37°C for 24 hours. Limonene measured from HeLa-LS-tHMGR cells was approximately double that from HeLa-LS cells over the cell density range examined.

Techniques Used: Plasmid Preparation, Transfection, Cell Culture, FACS, Selection, Stable Transfection, Clone Assay, Expressing, Solid-phase Microextraction, Gas Chromatography-Mass Spectrometry, Fluorescence, Concentration Assay, Incubation

5) Product Images from "αEβ7 Integrin Identifies Subsets of Pro-Inflammatory Colonic CD4+ T Lymphocytes in Ulcerative Colitis"

Article Title: αEβ7 Integrin Identifies Subsets of Pro-Inflammatory Colonic CD4+ T Lymphocytes in Ulcerative Colitis

Journal: Journal of Crohn's & Colitis

doi: 10.1093/ecco-jcc/jjw189

Lymphocytes with a regulatory phenotype are expanded in the colonic lamina propria [LP] during ulcerative colitis and lack αE integrin. Dual stain IHC was performed on formalin-fixed paraffin-embedded colonic biopsies to evaluate co-expression of the αE integrin [red] with FOXP3 [green]. Single and dual expressing cells [yellow, indicated by arrowheads] were enumerated using the Nuance Multispectral Tissue Imaging System combined with automated morphometric analysis. [A] A representative Nuance spectrally unmixed image is shown for a patient with active UC. Scale bar represents 50 μm. Automatic cell counting was used to enumerate [B] FOXP3+αE+, [C] FOXP3+αE− cells and [D] FOXP3−αE+ cells per mm 2 of cell area within the epithelium or LP in control subjects [ n = 16] and patients with active UC [ n = 14]. In a separate study cohort, FACS-sorted unstimulated CD4+αE+ and CD4+αE− colonic lymphocytes were analysed for TReg gene expression in [E] control subjects [ n = 8] and [F] patients with active UC [ n = 10].
Figure Legend Snippet: Lymphocytes with a regulatory phenotype are expanded in the colonic lamina propria [LP] during ulcerative colitis and lack αE integrin. Dual stain IHC was performed on formalin-fixed paraffin-embedded colonic biopsies to evaluate co-expression of the αE integrin [red] with FOXP3 [green]. Single and dual expressing cells [yellow, indicated by arrowheads] were enumerated using the Nuance Multispectral Tissue Imaging System combined with automated morphometric analysis. [A] A representative Nuance spectrally unmixed image is shown for a patient with active UC. Scale bar represents 50 μm. Automatic cell counting was used to enumerate [B] FOXP3+αE+, [C] FOXP3+αE− cells and [D] FOXP3−αE+ cells per mm 2 of cell area within the epithelium or LP in control subjects [ n = 16] and patients with active UC [ n = 14]. In a separate study cohort, FACS-sorted unstimulated CD4+αE+ and CD4+αE− colonic lymphocytes were analysed for TReg gene expression in [E] control subjects [ n = 8] and [F] patients with active UC [ n = 10].

Techniques Used: Staining, Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Expressing, Imaging, Cell Counting, FACS

αE integrin expression by CD4+ T lymphocytes is associated with a Th17/Th1 phenotype, which is enhanced in ulcerative colitis. Ex-vivo colonic CD4+ T cells were stimulated and analysed by flow cytometry for dual cytokine production of IL-17A and IFNγ. [A] Representative two-colour FACS plots of IL-17A vs IFNγ for CD4+αE+, CD4+αE− cells. [B] Summary Tukey box plots of double positive IL-17A+IFNγ+ lymphocyte frequency and double negative IL-17A−IFNγ− lymphocyte frequency [ n = 4 control subjects and n = 6 UC patients].
Figure Legend Snippet: αE integrin expression by CD4+ T lymphocytes is associated with a Th17/Th1 phenotype, which is enhanced in ulcerative colitis. Ex-vivo colonic CD4+ T cells were stimulated and analysed by flow cytometry for dual cytokine production of IL-17A and IFNγ. [A] Representative two-colour FACS plots of IL-17A vs IFNγ for CD4+αE+, CD4+αE− cells. [B] Summary Tukey box plots of double positive IL-17A+IFNγ+ lymphocyte frequency and double negative IL-17A−IFNγ− lymphocyte frequency [ n = 4 control subjects and n = 6 UC patients].

Techniques Used: Expressing, Ex Vivo, Flow Cytometry, Cytometry, FACS

Ulcerative colitis is associated with an influx of mucosal leukocytes. Cells isolated from colonic biopsies sampled from control subjects [ n = 14] and UC patients [ n = 10] were evaluated by flow cytometry. Frequency of [A] CD45+ leukocytes, and [B] CD3+ T lymphocytes in the colonic mucosa is shown, along with [C] CD4:CD8 ratio of all CD3+ T cells and [D] representative αE vs CD3 FACS plots of CD45+ leukocytes, with summary graph of the proportion of CD45+αE+ leukocytes that were T cells. [E] In a subset of patients, surface expression of α4β7 dimer on αE+ and αE− T cells from control subjects [ n = 6] and UC patients [ n = 8] was examined.
Figure Legend Snippet: Ulcerative colitis is associated with an influx of mucosal leukocytes. Cells isolated from colonic biopsies sampled from control subjects [ n = 14] and UC patients [ n = 10] were evaluated by flow cytometry. Frequency of [A] CD45+ leukocytes, and [B] CD3+ T lymphocytes in the colonic mucosa is shown, along with [C] CD4:CD8 ratio of all CD3+ T cells and [D] representative αE vs CD3 FACS plots of CD45+ leukocytes, with summary graph of the proportion of CD45+αE+ leukocytes that were T cells. [E] In a subset of patients, surface expression of α4β7 dimer on αE+ and αE− T cells from control subjects [ n = 6] and UC patients [ n = 8] was examined.

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

αE integrin expression by colonic CD4+ T lymphocytes is associated with Th17 differentiation. FACS-sorted, unstimulated CD4+αE+ and CD4+αE− T lymphocytes isolated from active UC colonic biopsies were analysed for gene expression of [A] FOXP3 vs RORC and [B] IL17A vs RORC [ n = 10 patients]. [C] In a separate study cohort, IL-17A protein expression in CD4+αE+ and CD4+αE− T cells was evaluated by FACS in colonic ex-vivo stimulated T lymphocytes from control subjects [ n = 6] and patients with active UC [ n = 8]. Representative two-colour FACS plots from control and UC patients are shown alongside summary Tukey box plots. Representative flow cytometry showing CD4+ T cell CD161 co-expression with [D] intracellular IL-17A, and [E] surface αE staining on a patient with active UC.
Figure Legend Snippet: αE integrin expression by colonic CD4+ T lymphocytes is associated with Th17 differentiation. FACS-sorted, unstimulated CD4+αE+ and CD4+αE− T lymphocytes isolated from active UC colonic biopsies were analysed for gene expression of [A] FOXP3 vs RORC and [B] IL17A vs RORC [ n = 10 patients]. [C] In a separate study cohort, IL-17A protein expression in CD4+αE+ and CD4+αE− T cells was evaluated by FACS in colonic ex-vivo stimulated T lymphocytes from control subjects [ n = 6] and patients with active UC [ n = 8]. Representative two-colour FACS plots from control and UC patients are shown alongside summary Tukey box plots. Representative flow cytometry showing CD4+ T cell CD161 co-expression with [D] intracellular IL-17A, and [E] surface αE staining on a patient with active UC.

Techniques Used: Expressing, FACS, Isolation, Ex Vivo, Flow Cytometry, Cytometry, Staining

Colonic CD4+αE+ lymphocytes express higher levels of Th1 cytokines relative to CD4+αE− lymphocytes. FACS-sorted CD4+αE+ and CD4+αE− T lymphocytes from colonic biopsies harvested from [A] control subjects [ n = 8] and [B] patients with active UC [ n = 10] were evaluated for gene expression of IFNγ, TNFα, IL-4 and IL-13. In a separate study cohort, ex-vivo stimulated T cells were evaluated by flow cytometry for intracellular [C] IFNγ [ n = 6 control subjects and n = 8 patients with active UC], and [D] TNFα protein expression [ n = 4 control subjects and n = 6 patients with active UC]. Representative two-colour FACS plots from control and UC patients are shown alongside summary Tukey box plots.
Figure Legend Snippet: Colonic CD4+αE+ lymphocytes express higher levels of Th1 cytokines relative to CD4+αE− lymphocytes. FACS-sorted CD4+αE+ and CD4+αE− T lymphocytes from colonic biopsies harvested from [A] control subjects [ n = 8] and [B] patients with active UC [ n = 10] were evaluated for gene expression of IFNγ, TNFα, IL-4 and IL-13. In a separate study cohort, ex-vivo stimulated T cells were evaluated by flow cytometry for intracellular [C] IFNγ [ n = 6 control subjects and n = 8 patients with active UC], and [D] TNFα protein expression [ n = 4 control subjects and n = 6 patients with active UC]. Representative two-colour FACS plots from control and UC patients are shown alongside summary Tukey box plots.

Techniques Used: FACS, Expressing, Ex Vivo, Flow Cytometry, Cytometry

6) Product Images from "Interleukin-10 Deficiency Impairs Reparative Properties of Bone Marrow-Derived Endothelial Progenitor Cell Exosomes"

Article Title: Interleukin-10 Deficiency Impairs Reparative Properties of Bone Marrow-Derived Endothelial Progenitor Cell Exosomes

Journal: Tissue Engineering. Part A

doi: 10.1089/ten.tea.2017.0084

Characterization of EPCs from C57BL/6J mouse bone marrow: (A) FACS of bone marrow cells; Lin, CD31, and Scar1 markers are checked. (B) The morphology of isolated EPCs cultured in vitro on day 7. (10 × , scale bar: 100 μm). EPC, endothelial progenitor cell.
Figure Legend Snippet: Characterization of EPCs from C57BL/6J mouse bone marrow: (A) FACS of bone marrow cells; Lin, CD31, and Scar1 markers are checked. (B) The morphology of isolated EPCs cultured in vitro on day 7. (10 × , scale bar: 100 μm). EPC, endothelial progenitor cell.

Techniques Used: FACS, Isolation, Cell Culture, In Vitro

7) Product Images from "Sex steroid blockade enhances thymopoiesis by modulating Notch signaling"

Article Title: Sex steroid blockade enhances thymopoiesis by modulating Notch signaling

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20131289

Notch ligand DLL4 regulates thymopoiesis in a dosage-sensitive manner. (A and B) Lymphoid differentiation of sorted LSK after 12 d of culture with IL-7, FLT3-ligand, SCF, and scalar concentrations of rDLL4. (A) Absolute numbers of DN2 cells. (B) DN cell proportions, gated on Lin − CD45 + . A and B represent the mean + SEM of one of two representative experiments performed in triplicate. (C) Dll4 expression in FACS-sorted cTEC from 6-wk-old Foxn1-cre::Dll4 +/fl mice and Foxn1-cre::Dll4 +/+ mice. (D) Total thymic cellularity of 5–6-wk-old Foxn1-cre::Dll4 +/fl mice ( n = 7) and Foxn1-cre::Dll4 +/+ mice ( n = 5). (E) Total thymic cellularity of K14-cre::Dll4 +/fl mice ( n = 7) and K14-cre::Dll4 +/+ mice ( n = 2). Data represents the mean + SEM of one experiment. ^, P = 0.0556. Data represent the mean + SEM of two independent experiments unless otherwise specified. *, P ≤ 0.05, unpaired Mann-Whitney U test.
Figure Legend Snippet: Notch ligand DLL4 regulates thymopoiesis in a dosage-sensitive manner. (A and B) Lymphoid differentiation of sorted LSK after 12 d of culture with IL-7, FLT3-ligand, SCF, and scalar concentrations of rDLL4. (A) Absolute numbers of DN2 cells. (B) DN cell proportions, gated on Lin − CD45 + . A and B represent the mean + SEM of one of two representative experiments performed in triplicate. (C) Dll4 expression in FACS-sorted cTEC from 6-wk-old Foxn1-cre::Dll4 +/fl mice and Foxn1-cre::Dll4 +/+ mice. (D) Total thymic cellularity of 5–6-wk-old Foxn1-cre::Dll4 +/fl mice ( n = 7) and Foxn1-cre::Dll4 +/+ mice ( n = 5). (E) Total thymic cellularity of K14-cre::Dll4 +/fl mice ( n = 7) and K14-cre::Dll4 +/+ mice ( n = 2). Data represents the mean + SEM of one experiment. ^, P = 0.0556. Data represent the mean + SEM of two independent experiments unless otherwise specified. *, P ≤ 0.05, unpaired Mann-Whitney U test.

Techniques Used: Expressing, FACS, Mouse Assay, MANN-WHITNEY

8) Product Images from "Embryonic Neocortical Microglia Express Toll-Like Receptor 9 and Respond to Plasmid DNA Injected into the Ventricle: Technical Considerations Regarding Microglial Distribution in Electroporated Brain Walls"

Article Title: Embryonic Neocortical Microglia Express Toll-Like Receptor 9 and Respond to Plasmid DNA Injected into the Ventricle: Technical Considerations Regarding Microglial Distribution in Electroporated Brain Walls

Journal: eNeuro

doi: 10.1523/ENEURO.0312-18.2018

Intraventricular administration of TLR9 antagonist decreases microglial infiltration induced by plasmid DNA injection. A , Relative expression of TLR9 (normalized against GAPDH ) in FACS-isolated CX3CR1 - and CX3CR1 + cells derived from the cerebral wall of E14 CX3CR1-GFP mice. Data represent mean ± SD ( n = 4 samples obtained from independent experiments; p = 0.0286, Mann–Whitney U test). B , Experimental design for ODN 2088 treatment. ODN 2088 was injected together with plasmid DNA into the lateral ventricle of E12 CX3CR1-GFP mice, and after 2 d (E14) the embryonic brains were fixed. C , Immunofluorescence with anti-GFP antibody, showing the distribution of microglia in the pallium and choroid plexus. Yellow arrowheads indicate microglia aberrantly accumulated on the apical surface of the pallium or in the choroid plexus. Cyan arrowheads show microglia which were almost homogenously distributed in the cerebral wall. Scale bar, 100 µm. D , E , Graphs indicate the number of CX3CR1-GFP + cells in each 40 µm bin of the pallium ( D ) and density of microglia directly adhered to the choroid plexus ( E ), comparing control, only ODN 2088-treated, plasmid DNA-injected, and plasmid DNA + ODN 2088 coinjected brains. F , Graph showing the total number of pallial microglia within 240 µm from the apical surface. G , Double-immunofluorescence for GFP (CX3CR1) and RFP (Lyn-mCherry) in the cortex of IUE E14 brain treated with ODN 2088. Microglia exhibited a normal distribution pattern in the Lyn-mCherry expressing region where IUE succeeded ( Movies 1 ). Scale bar, 100 µm. For statistical analyses in D – F , n = 16 samples obtained from eight embryos (2 sections, each) were quantified. Two or three littermates per dam were subjected to a series of tests. Data represent mean ± SD. *** p
Figure Legend Snippet: Intraventricular administration of TLR9 antagonist decreases microglial infiltration induced by plasmid DNA injection. A , Relative expression of TLR9 (normalized against GAPDH ) in FACS-isolated CX3CR1 - and CX3CR1 + cells derived from the cerebral wall of E14 CX3CR1-GFP mice. Data represent mean ± SD ( n = 4 samples obtained from independent experiments; p = 0.0286, Mann–Whitney U test). B , Experimental design for ODN 2088 treatment. ODN 2088 was injected together with plasmid DNA into the lateral ventricle of E12 CX3CR1-GFP mice, and after 2 d (E14) the embryonic brains were fixed. C , Immunofluorescence with anti-GFP antibody, showing the distribution of microglia in the pallium and choroid plexus. Yellow arrowheads indicate microglia aberrantly accumulated on the apical surface of the pallium or in the choroid plexus. Cyan arrowheads show microglia which were almost homogenously distributed in the cerebral wall. Scale bar, 100 µm. D , E , Graphs indicate the number of CX3CR1-GFP + cells in each 40 µm bin of the pallium ( D ) and density of microglia directly adhered to the choroid plexus ( E ), comparing control, only ODN 2088-treated, plasmid DNA-injected, and plasmid DNA + ODN 2088 coinjected brains. F , Graph showing the total number of pallial microglia within 240 µm from the apical surface. G , Double-immunofluorescence for GFP (CX3CR1) and RFP (Lyn-mCherry) in the cortex of IUE E14 brain treated with ODN 2088. Microglia exhibited a normal distribution pattern in the Lyn-mCherry expressing region where IUE succeeded ( Movies 1 ). Scale bar, 100 µm. For statistical analyses in D – F , n = 16 samples obtained from eight embryos (2 sections, each) were quantified. Two or three littermates per dam were subjected to a series of tests. Data represent mean ± SD. *** p

Techniques Used: Plasmid Preparation, Injection, Expressing, FACS, Isolation, Derivative Assay, Mouse Assay, MANN-WHITNEY, Immunofluorescence

Endotoxins trigger microglial aberrant accumulation. A , Relative expression of TLR4 (normalized against GAPDH ) in FACS-isolated CX3CR1 − and CX3CR1 + cells derived from the cerebral wall of E14 CX3CR1-GFP mice. Data represent mean ± SD ( n = 4 samples obtained from independent experiments; p = 0.0286, Mann–Whitney U test). B , Immunofluorescence with anti-GFP antibody, showing the distribution of microglia in the pallium and choroid plexus in brains injected with the indicated amount of LPS (2.5 ng, 250 pg, 25 pg, and 2.5 pg). Yellow arrowheads indicate microglia accumulated near the apical surface of the pallium and on the choroid plexus. Scale bar, 100 µm. C , D , Graphs depicting the number of pallial microglia positioned in each bin ( C ) and density of microglia adhered to the choroid plexus ( D ) in brains treated with various amounts of LPS. E , The total number of pallial microglia within 240 µm from the apical surface. For statistical analyses in C – E , n = 10 samples obtained from five embryos (2 sections, each) were quantified. One or Two littermates per dam were subjected to a series of tests. Data represent mean ± SD. *** p
Figure Legend Snippet: Endotoxins trigger microglial aberrant accumulation. A , Relative expression of TLR4 (normalized against GAPDH ) in FACS-isolated CX3CR1 − and CX3CR1 + cells derived from the cerebral wall of E14 CX3CR1-GFP mice. Data represent mean ± SD ( n = 4 samples obtained from independent experiments; p = 0.0286, Mann–Whitney U test). B , Immunofluorescence with anti-GFP antibody, showing the distribution of microglia in the pallium and choroid plexus in brains injected with the indicated amount of LPS (2.5 ng, 250 pg, 25 pg, and 2.5 pg). Yellow arrowheads indicate microglia accumulated near the apical surface of the pallium and on the choroid plexus. Scale bar, 100 µm. C , D , Graphs depicting the number of pallial microglia positioned in each bin ( C ) and density of microglia adhered to the choroid plexus ( D ) in brains treated with various amounts of LPS. E , The total number of pallial microglia within 240 µm from the apical surface. For statistical analyses in C – E , n = 10 samples obtained from five embryos (2 sections, each) were quantified. One or Two littermates per dam were subjected to a series of tests. Data represent mean ± SD. *** p

Techniques Used: Expressing, FACS, Isolation, Derivative Assay, Mouse Assay, MANN-WHITNEY, Immunofluorescence, Injection

9) Product Images from "Transcriptional activities of human elongation factor-1α and cytomegalovirus promoter in transgenic dogs generated by somatic cell nuclear transfer"

Article Title: Transcriptional activities of human elongation factor-1α and cytomegalovirus promoter in transgenic dogs generated by somatic cell nuclear transfer

Journal: PLoS ONE

doi: 10.1371/journal.pone.0233784

Analysis of transgenic dogs with EGFP controlled by CMV promoter sequence. (A) Representative image of all born puppies under ultraviolet light exposure, to assess EGFP expression, BTF-963, - 964, -965, -966, -967, and -968, from left to right in order. Claws of three puppies, BTF-965,—967, and -968, showed a detectable EGFP signal, but the signal was undetectable in the other three pupies. (B) PCR analysis to detect whole CMV-EGFP or EGFP construct in genomic DNA of each born dog. GAPDH is the loading control. (C) Detection of GFP signals in fibroblasts isolated from each transgenic dog using fluorescence microscopy and (D) FACS analysis. Scale bars indicate 50 μm. (E) Immunohistochemical analysis showing EGFP expression in brain and muscle tissues from BTF-967 dog. RFP is a negative control of the immunohistochemical reaction.
Figure Legend Snippet: Analysis of transgenic dogs with EGFP controlled by CMV promoter sequence. (A) Representative image of all born puppies under ultraviolet light exposure, to assess EGFP expression, BTF-963, - 964, -965, -966, -967, and -968, from left to right in order. Claws of three puppies, BTF-965,—967, and -968, showed a detectable EGFP signal, but the signal was undetectable in the other three pupies. (B) PCR analysis to detect whole CMV-EGFP or EGFP construct in genomic DNA of each born dog. GAPDH is the loading control. (C) Detection of GFP signals in fibroblasts isolated from each transgenic dog using fluorescence microscopy and (D) FACS analysis. Scale bars indicate 50 μm. (E) Immunohistochemical analysis showing EGFP expression in brain and muscle tissues from BTF-967 dog. RFP is a negative control of the immunohistochemical reaction.

Techniques Used: Transgenic Assay, Sequencing, Expressing, Polymerase Chain Reaction, Construct, Isolation, Fluorescence, Microscopy, FACS, Immunohistochemistry, Negative Control

10) Product Images from "PEDF is a novel oligodendrogenic morphogen acting on the adult SVZ and corpus callosum"

Article Title: PEDF is a novel oligodendrogenic morphogen acting on the adult SVZ and corpus callosum

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

doi: 10.1523/JNEUROSCI.0628-12.2012

PEDF exerts oligodendrogenic effect on adult SVZ GFAP+ neural precursors (A) Experimental flow chart. GFAP:GFP+NG2− and GFAP:GFP+NG2+ single cells were isolated by FACS from primary neurospheres derived from the SVZ of adult GFAP:GFP mice. Secondary neurospheres were then produced from these two FACS-purified populations in the absence or presence of PEDF (50ng/ml). Assays were then performed as indicated. (B–C) qRT-PCR analysis showing that PEDF elevated expression levels of oligodendroglial transcription factors and PDGFrα in both GFP+NG2− and GFP+NG2+ precursor-derived neurospheres. (D) Western blot analysis of Olig1, Olig2, Sox10, and PDGFrα in control and PEDF-treated wild type secondary neurospheres. (E–G) Control and PEDF-treated secondary neurospheres derived from FACS purified GFP+NG2− or GFP+NG2+ subsets were plated in differentiation medium without mitogens or PEDF for 3 days before immunostaining for NG2, O4, or Tuj1. (E and G) Greater numbers of NG2+ and O4+ oligodendroglial cells were produced from both GFP+NG2− and GFP+NG2+ cell-derived neurospheres with PEDF treatment. (F and G) PEDF treatment resulted in diminished neuronal differentiation of GFP+NG2− and GFP+NG2+ cell-derived neurospheres. (G) Representative images of cells differentiated from control or PEDF-treated GFP+NG2− cell-derived neurospheres. Results are means +/− SEM of three or four independent experiments. Scale bar = 25μm. (* P
Figure Legend Snippet: PEDF exerts oligodendrogenic effect on adult SVZ GFAP+ neural precursors (A) Experimental flow chart. GFAP:GFP+NG2− and GFAP:GFP+NG2+ single cells were isolated by FACS from primary neurospheres derived from the SVZ of adult GFAP:GFP mice. Secondary neurospheres were then produced from these two FACS-purified populations in the absence or presence of PEDF (50ng/ml). Assays were then performed as indicated. (B–C) qRT-PCR analysis showing that PEDF elevated expression levels of oligodendroglial transcription factors and PDGFrα in both GFP+NG2− and GFP+NG2+ precursor-derived neurospheres. (D) Western blot analysis of Olig1, Olig2, Sox10, and PDGFrα in control and PEDF-treated wild type secondary neurospheres. (E–G) Control and PEDF-treated secondary neurospheres derived from FACS purified GFP+NG2− or GFP+NG2+ subsets were plated in differentiation medium without mitogens or PEDF for 3 days before immunostaining for NG2, O4, or Tuj1. (E and G) Greater numbers of NG2+ and O4+ oligodendroglial cells were produced from both GFP+NG2− and GFP+NG2+ cell-derived neurospheres with PEDF treatment. (F and G) PEDF treatment resulted in diminished neuronal differentiation of GFP+NG2− and GFP+NG2+ cell-derived neurospheres. (G) Representative images of cells differentiated from control or PEDF-treated GFP+NG2− cell-derived neurospheres. Results are means +/− SEM of three or four independent experiments. Scale bar = 25μm. (* P

Techniques Used: Flow Cytometry, Isolation, FACS, Derivative Assay, Mouse Assay, Produced, Purification, Quantitative RT-PCR, Expressing, Western Blot, Immunostaining

Characterization of control and PEDF-treated neurospheres derived from the SVZ of adult wild type or GFAP:GFP transgenic mice (A) Experimental flow chart. Secondary neurospheres derived from the SVZ of either adult wild type or GFAP:GFP transgenic mice were grown in the absence or presence of PEDF (50ng/ml) for 5 days, and then assayed as indicated. (B) RT-PCR analysis for expression of PEDF receptor in SVZ neurospheres. (C–D) Wild type control and PEDF-treated secondary neurosphere cells were immunostained for nestin, NG2, O4, or Tuj1. Immunostaining images (C) and quantification data (D), showing that PEDF significantly increased the numbers of NG2+ and O4+ cells. (E) Quantification of BrdU incorporation and Ki67 labeling in wild type control and PEDF-treated secondary neurospheres. PEDF did not alter proliferation of neurosphere cells. (F) FACS plots for GFP+ and GFP− cell fractions dissociated from adult SVZ GFAP:GFP neurospheres. GFP+ selection was set using wild type SVZ neurospheres as a negative control. (G) GFAP immunostaining acutely done on plated GFP+ and GFP− cells FACS-purified from GFAP:GFP neurospheres. (H) Graphic representation of flow cytometric quantification of percentages of NG2+ cells among GFP+ cells in control and PEDF-treated secondary GFAP:GFP neurospheres, showing that PEDF promoted NG2 induction in GFP+ cells. (I) Quantification of BrdU incorporation among GFP+NG2+ cells FACS-purified from control and PEDF-treated secondary GFAP:GFP neurospheres, indicating that PEDF-mediated NG2 induction in GFAP:GFP+ precursors was not due to selective proliferation of GFP+NG2+ cells. ND = not determined. Results are means +/− SEM of three or four independent experiments. Scale bar = 30μm. (* P
Figure Legend Snippet: Characterization of control and PEDF-treated neurospheres derived from the SVZ of adult wild type or GFAP:GFP transgenic mice (A) Experimental flow chart. Secondary neurospheres derived from the SVZ of either adult wild type or GFAP:GFP transgenic mice were grown in the absence or presence of PEDF (50ng/ml) for 5 days, and then assayed as indicated. (B) RT-PCR analysis for expression of PEDF receptor in SVZ neurospheres. (C–D) Wild type control and PEDF-treated secondary neurosphere cells were immunostained for nestin, NG2, O4, or Tuj1. Immunostaining images (C) and quantification data (D), showing that PEDF significantly increased the numbers of NG2+ and O4+ cells. (E) Quantification of BrdU incorporation and Ki67 labeling in wild type control and PEDF-treated secondary neurospheres. PEDF did not alter proliferation of neurosphere cells. (F) FACS plots for GFP+ and GFP− cell fractions dissociated from adult SVZ GFAP:GFP neurospheres. GFP+ selection was set using wild type SVZ neurospheres as a negative control. (G) GFAP immunostaining acutely done on plated GFP+ and GFP− cells FACS-purified from GFAP:GFP neurospheres. (H) Graphic representation of flow cytometric quantification of percentages of NG2+ cells among GFP+ cells in control and PEDF-treated secondary GFAP:GFP neurospheres, showing that PEDF promoted NG2 induction in GFP+ cells. (I) Quantification of BrdU incorporation among GFP+NG2+ cells FACS-purified from control and PEDF-treated secondary GFAP:GFP neurospheres, indicating that PEDF-mediated NG2 induction in GFAP:GFP+ precursors was not due to selective proliferation of GFP+NG2+ cells. ND = not determined. Results are means +/− SEM of three or four independent experiments. Scale bar = 30μm. (* P

Techniques Used: Derivative Assay, Transgenic Assay, Mouse Assay, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunostaining, BrdU Incorporation Assay, Labeling, FACS, Selection, Negative Control, Purification

11) Product Images from "Improving T-cell expansion and function for adoptive T-cell therapy using ex vivo treatment with PI3Kδ inhibitors and VIP antagonists"

Article Title: Improving T-cell expansion and function for adoptive T-cell therapy using ex vivo treatment with PI3Kδ inhibitors and VIP antagonists

Journal: Blood Advances

doi: 10.1182/bloodadvances.2017011254

Depletion of CD27 − CD28 − cells improves the expansion of T cells from heavily pretreated DLBCL patients. PBMCs from pretreated DLBCL patients were rested overnight followed by FACS sorting to separate CD27 − CD28 − cells from the remaining populations. Cells were then stimulated with anti-CD3/CD28 beads with IL-2 for 14 days. (A) Comparison of normal T-cell expansion from healthy donors, untreated DLBCL patients, and treated DLBCL patients. (B) Flow plots showing the sorting strategy and postsort purity. (C) T-cell viability at the end of the expansion period as assessed by Sytox blue staining. (D) Quantitation of cell viabilities for the indicated populations on day 14 of expansion (n = 4 DLBCL patient donors). (E) Cell counts of cultures consisting of the indicated T-cell populations from 4 patient sorts. ** P
Figure Legend Snippet: Depletion of CD27 − CD28 − cells improves the expansion of T cells from heavily pretreated DLBCL patients. PBMCs from pretreated DLBCL patients were rested overnight followed by FACS sorting to separate CD27 − CD28 − cells from the remaining populations. Cells were then stimulated with anti-CD3/CD28 beads with IL-2 for 14 days. (A) Comparison of normal T-cell expansion from healthy donors, untreated DLBCL patients, and treated DLBCL patients. (B) Flow plots showing the sorting strategy and postsort purity. (C) T-cell viability at the end of the expansion period as assessed by Sytox blue staining. (D) Quantitation of cell viabilities for the indicated populations on day 14 of expansion (n = 4 DLBCL patient donors). (E) Cell counts of cultures consisting of the indicated T-cell populations from 4 patient sorts. ** P

Techniques Used: FACS, Flow Cytometry, Staining, Quantitation Assay

12) Product Images from "Soluble vascular endothelial growth factor receptor-3 suppresses lymphangiogenesis and lymphatic metastasis in bladder cancer"

Article Title: Soluble vascular endothelial growth factor receptor-3 suppresses lymphangiogenesis and lymphatic metastasis in bladder cancer

Journal: Molecular Cancer

doi: 10.1186/1476-4598-10-36

CD11b + or CD68 + TAM abundantly express VEGF-C/D and VEGFR-3 . 1 × 10 6 MBT-2 cells were injected into the urinary bladders of 8-10-week old female C3H mice. 4 weeks after the tumor cell injection, single cell suspension of OUBC was prepared. ( A ) CD11b + cells and CD11b - cells were enriched by FACS with ~95% purity. ( B ) Comparison of mRNA expressions of VEGF-C/D in CD11b + cells. Data are presented as relative fold to CD11b - cells after standardization with hypoxanthine-guanine phosphoribosyl transferase (HPRT). Graph shows mean ± SD; n = 3 for each group. *p
Figure Legend Snippet: CD11b + or CD68 + TAM abundantly express VEGF-C/D and VEGFR-3 . 1 × 10 6 MBT-2 cells were injected into the urinary bladders of 8-10-week old female C3H mice. 4 weeks after the tumor cell injection, single cell suspension of OUBC was prepared. ( A ) CD11b + cells and CD11b - cells were enriched by FACS with ~95% purity. ( B ) Comparison of mRNA expressions of VEGF-C/D in CD11b + cells. Data are presented as relative fold to CD11b - cells after standardization with hypoxanthine-guanine phosphoribosyl transferase (HPRT). Graph shows mean ± SD; n = 3 for each group. *p

Techniques Used: Injection, Mouse Assay, FACS

13) Product Images from "RNA:DNA hybrids are a novel molecular pattern sensed by TLR9"

Article Title: RNA:DNA hybrids are a novel molecular pattern sensed by TLR9

Journal: The EMBO Journal

doi: 10.1002/embj.201386117

TLR9 is activated by intact RNA-DNA hybrids. A, B The cytokine response (IL-6 (A), IFN-α (B)) of FLDCs to R:D60 is distinct from that of its single-stranded nucleic acid components. Day 8-FLDC cultures were FACS-sorted into pDC and cDC populations and transfected with R:D60 or equimolar amounts of ssRNA60 and ssDNA60. Data shown are the mean of three independent experiments ± s.e.m. C Cytokine induction by R:D60 but not ssRNA60 is TLR9-dependent. FLDCs derived from Tlr9 −/− and C57BL/6 mice were transfected as described for (A) and (B). Data shown are from two independent experiments ± s.d. D, E The cytokine response of FLDCs to R:D60 is not due to immunostimulatory contaminants remaining after FPLC. Nucleic acid hybridisation reactions were generated as outlined in Fig 1 A omitting one of the two oligonucleotides and fractionated using identical FPLC conditions to those used to purify annealed RNA:DNA hybrids. Upper panel: nucleic acids from mock hybridization reactions (minus ssRNA (D), minus ssDNA (E)) eluted later than the established elution position of R:D60 (arrow). The fractions (12.9 ml – 13.7 ml) corresponding to those that would usually contain the hybrid (“R:D fraction”) and fractions containing the nucleic acids at the indicated peaks, were concentrated by ethanol precipitation. Lower panels: 200 ng of FPLC-purified R:D60, 200 ng of hybridisation reaction column input (labelled “minus ssRNA” and “minus ssDNA” respectively), along with equal volumes of each fraction, were analysed by native PAGE. F Immunostimulatory contaminants are absent from the R:D60 fraction. FLDCs transfected with FPLC-purified R:D60 and the equivalent volume of R:D fractions from the hybridisations shown in (D) and (E) using Lipofectamine 2000. Supernatant concentrations of IL-6 (upper panel) were quantified 18 h post-transfection by ELISA and Ifna1 transcript levels (lower panel) normalised to Actb expression quantified 6 h post-transfection by qRT-PCR (fold mRNA induction from medium alone samples). Data shown are the mean of three experiments ± s.e.m. (IL-6, ** P = 0.0028) and PCR triplicates ± s.d. ( Ifna1 , ** P = 0.0001).
Figure Legend Snippet: TLR9 is activated by intact RNA-DNA hybrids. A, B The cytokine response (IL-6 (A), IFN-α (B)) of FLDCs to R:D60 is distinct from that of its single-stranded nucleic acid components. Day 8-FLDC cultures were FACS-sorted into pDC and cDC populations and transfected with R:D60 or equimolar amounts of ssRNA60 and ssDNA60. Data shown are the mean of three independent experiments ± s.e.m. C Cytokine induction by R:D60 but not ssRNA60 is TLR9-dependent. FLDCs derived from Tlr9 −/− and C57BL/6 mice were transfected as described for (A) and (B). Data shown are from two independent experiments ± s.d. D, E The cytokine response of FLDCs to R:D60 is not due to immunostimulatory contaminants remaining after FPLC. Nucleic acid hybridisation reactions were generated as outlined in Fig 1 A omitting one of the two oligonucleotides and fractionated using identical FPLC conditions to those used to purify annealed RNA:DNA hybrids. Upper panel: nucleic acids from mock hybridization reactions (minus ssRNA (D), minus ssDNA (E)) eluted later than the established elution position of R:D60 (arrow). The fractions (12.9 ml – 13.7 ml) corresponding to those that would usually contain the hybrid (“R:D fraction”) and fractions containing the nucleic acids at the indicated peaks, were concentrated by ethanol precipitation. Lower panels: 200 ng of FPLC-purified R:D60, 200 ng of hybridisation reaction column input (labelled “minus ssRNA” and “minus ssDNA” respectively), along with equal volumes of each fraction, were analysed by native PAGE. F Immunostimulatory contaminants are absent from the R:D60 fraction. FLDCs transfected with FPLC-purified R:D60 and the equivalent volume of R:D fractions from the hybridisations shown in (D) and (E) using Lipofectamine 2000. Supernatant concentrations of IL-6 (upper panel) were quantified 18 h post-transfection by ELISA and Ifna1 transcript levels (lower panel) normalised to Actb expression quantified 6 h post-transfection by qRT-PCR (fold mRNA induction from medium alone samples). Data shown are the mean of three experiments ± s.e.m. (IL-6, ** P = 0.0028) and PCR triplicates ± s.d. ( Ifna1 , ** P = 0.0001).

Techniques Used: FACS, Transfection, Derivative Assay, Mouse Assay, Fast Protein Liquid Chromatography, Hybridization, Generated, Ethanol Precipitation, Purification, Clear Native PAGE, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Polymerase Chain Reaction

14) Product Images from "Restoration of MYC-repressed targets mediates the negative effects of GM-CSF on RUNX1-ETO leukemogenicity"

Article Title: Restoration of MYC-repressed targets mediates the negative effects of GM-CSF on RUNX1-ETO leukemogenicity

Journal: Leukemia

doi: 10.1038/leu.2016.167

Gene expression profiling of murine RE HSPCs treated with GM (A) Diagram of experimental methods for gene expression profiling of murine RE HSPCs treated with GM. Lineage negative (Lin − ) cells were transduced with empty vector control (MIG) or MIG-RE (RE) retrovirus. The following day, cells were washed and cultured in StemSpan SFEM media with or without 10 ng/mL GM for 24 hours. Lin − /c-Kit + /GFP + cells were isolated by FACS and used for microarray analysis. (B) Venn diagram displaying the number of unique and overlapping differentially expressed genes after each perturbation. GM-treated control MIG HSPCs were compared to untreated MIG HSPCs (MIG+GM, left Venn diagram). GM-treated RE HSPCs were compared to untreated RE HSPCs (RE+GM, left Venn diagram). RE cells were compared to control MIG cells (RE, right Venn diagram). GM-treated RE cells were compared to untreated RE cells (RE+GM, right Venn diagram). A 2-fold differential gene expression cutoff was applied. (C) Gene set enrichment analysis (GSEA) of the RE+GM gene expression signature with human myelopoiesis, monocyte, and neutrophil gene sets generated from publicly available data from Ferrari et al 23 . The enrichment score (ES), nominal enrichment score (NES), nominal p -value (NOM p -val), and false discovery rate (FDR) for each gene set are shown.
Figure Legend Snippet: Gene expression profiling of murine RE HSPCs treated with GM (A) Diagram of experimental methods for gene expression profiling of murine RE HSPCs treated with GM. Lineage negative (Lin − ) cells were transduced with empty vector control (MIG) or MIG-RE (RE) retrovirus. The following day, cells were washed and cultured in StemSpan SFEM media with or without 10 ng/mL GM for 24 hours. Lin − /c-Kit + /GFP + cells were isolated by FACS and used for microarray analysis. (B) Venn diagram displaying the number of unique and overlapping differentially expressed genes after each perturbation. GM-treated control MIG HSPCs were compared to untreated MIG HSPCs (MIG+GM, left Venn diagram). GM-treated RE HSPCs were compared to untreated RE HSPCs (RE+GM, left Venn diagram). RE cells were compared to control MIG cells (RE, right Venn diagram). GM-treated RE cells were compared to untreated RE cells (RE+GM, right Venn diagram). A 2-fold differential gene expression cutoff was applied. (C) Gene set enrichment analysis (GSEA) of the RE+GM gene expression signature with human myelopoiesis, monocyte, and neutrophil gene sets generated from publicly available data from Ferrari et al 23 . The enrichment score (ES), nominal enrichment score (NES), nominal p -value (NOM p -val), and false discovery rate (FDR) for each gene set are shown.

Techniques Used: Expressing, Transduction, Plasmid Preparation, Cell Culture, Isolation, FACS, Microarray, Generated

15) Product Images from "Bone Marrow-Derived Microglia Infiltrate into the Paraventricular Nucleus of Chronic Psychological Stress-Loaded Mice"

Article Title: Bone Marrow-Derived Microglia Infiltrate into the Paraventricular Nucleus of Chronic Psychological Stress-Loaded Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0081744

Isolation of bone marrow-derived microglia and resident microglia from hypothalamic tissue and comparison of expression of various molecules in chronic PS-loaded and sham-treated mice. (A) Representative FACS chart in chronic PS-loaded mice with whole body radiation and the number of isolated GFP - CD45 low (resident microglia) and GFP + CD45 low (bone marrow-derived microglia) from mice with whole body radiation and the radiation with head protection ( n = 4−6). Total events in FACS were 20000. Data are expressed as mean ± sem. * P
Figure Legend Snippet: Isolation of bone marrow-derived microglia and resident microglia from hypothalamic tissue and comparison of expression of various molecules in chronic PS-loaded and sham-treated mice. (A) Representative FACS chart in chronic PS-loaded mice with whole body radiation and the number of isolated GFP - CD45 low (resident microglia) and GFP + CD45 low (bone marrow-derived microglia) from mice with whole body radiation and the radiation with head protection ( n = 4−6). Total events in FACS were 20000. Data are expressed as mean ± sem. * P

Techniques Used: Isolation, Derivative Assay, Expressing, Mouse Assay, FACS

16) Product Images from "A distinct metabolic state arises during the emergence of 2‐cell‐like cells"

Article Title: A distinct metabolic state arises during the emergence of 2‐cell‐like cells

Journal: EMBO Reports

doi: 10.15252/embr.201948354

Zscan4 + cells exhibit higher glucose uptake than both naïve and primed ES cells Experimental design. ES cells were cultured in serum/LIF conditions over feeders for at least 5 days in the absence of 2i, and subsequently FACS‐sorted into naïve pluripotent, primed pluripotent or Zscan4 + cells. Glucose uptake rates were measured thereafter. Reporter constructs employed to identify all three distinct populations are represented on the left. An EGFP reporter driven by the Rex1 endogenous promoter was used to distinguish between Rex1‐high (naïve pluripotent) and Rex1‐low (primed pluripotent) cells, and a tdTomato cassette expressed downstream of an ectopic Zscan4c promoter was used to mark Zscan4 + cells. Representative sorting gates used for the isolation of naïve pluripotent, primed pluripotent or Zscan4 + cells. Zscan4 + cells were defined as those positive for Zscan4c::tdTomato reporter (left), irrespective of their Rex1‐EGFP fluorescence level. Naïve pluripotent and primed pluripotent stem cells were gated based on the bimodality of the Rex1‐EGFP distribution (right). Glucose uptake rates in Zscan4 + cells (red), naïve pluripotent stem cells (dark blue) and primed pluripotent stem cells (light blue) were measured using a luciferase‐based assay across three independent biological replicates. Measurements are represented relative to the levels of Zscan4 + cells. Boxes indicate the range between the first and third quartile, the band specifies the median, and the whiskers span the range of the data while extending no further than 1.5 times the interquartile range. Individual dots indicate the measurements obtained in each technical replicate. * P
Figure Legend Snippet: Zscan4 + cells exhibit higher glucose uptake than both naïve and primed ES cells Experimental design. ES cells were cultured in serum/LIF conditions over feeders for at least 5 days in the absence of 2i, and subsequently FACS‐sorted into naïve pluripotent, primed pluripotent or Zscan4 + cells. Glucose uptake rates were measured thereafter. Reporter constructs employed to identify all three distinct populations are represented on the left. An EGFP reporter driven by the Rex1 endogenous promoter was used to distinguish between Rex1‐high (naïve pluripotent) and Rex1‐low (primed pluripotent) cells, and a tdTomato cassette expressed downstream of an ectopic Zscan4c promoter was used to mark Zscan4 + cells. Representative sorting gates used for the isolation of naïve pluripotent, primed pluripotent or Zscan4 + cells. Zscan4 + cells were defined as those positive for Zscan4c::tdTomato reporter (left), irrespective of their Rex1‐EGFP fluorescence level. Naïve pluripotent and primed pluripotent stem cells were gated based on the bimodality of the Rex1‐EGFP distribution (right). Glucose uptake rates in Zscan4 + cells (red), naïve pluripotent stem cells (dark blue) and primed pluripotent stem cells (light blue) were measured using a luciferase‐based assay across three independent biological replicates. Measurements are represented relative to the levels of Zscan4 + cells. Boxes indicate the range between the first and third quartile, the band specifies the median, and the whiskers span the range of the data while extending no further than 1.5 times the interquartile range. Individual dots indicate the measurements obtained in each technical replicate. * P

Techniques Used: Cell Culture, FACS, Construct, Isolation, Fluorescence, Luciferase

Controls and set‐up for Seahorse and ROS measurements Representative sorting gates used for the isolation of ES, Zscan4 + and 2‐cell‐like cells used throughout this study. Feeder cells were removed on the basis of their lack of far‐red fluorescence, which is higher in ES cells because of the presence of an H2B‐iRFP cassette. ESCs were defined as double negative for both the Zscan4 (Zscan4c::mCherry − ) and the MERV‐L reporters (2C::tbGFP − ). Zscan4 + cells were defined as positive for the Zscan4 reporter but negative for the MERV‐L reporter, and 2‐cell‐like cells were defined as positive for both reporters. Brightfield microscopy images indicating the confluency of the three populations shortly after plating in the Seahorse extracellular flux analyser plates. Representative images, in two magnifications, for the three independent biological replicates presented in Fig 1 C and D are shown. Representative sorting gate for ES and 2‐cell‐like cells (left) used for the FACS‐assisted ROS measurements (right). Fluorescence intensity distributions for ES cells (blue) and 2‐cell‐like cells (green) in control (centre) and CellROX‐treated samples (right) are shown.
Figure Legend Snippet: Controls and set‐up for Seahorse and ROS measurements Representative sorting gates used for the isolation of ES, Zscan4 + and 2‐cell‐like cells used throughout this study. Feeder cells were removed on the basis of their lack of far‐red fluorescence, which is higher in ES cells because of the presence of an H2B‐iRFP cassette. ESCs were defined as double negative for both the Zscan4 (Zscan4c::mCherry − ) and the MERV‐L reporters (2C::tbGFP − ). Zscan4 + cells were defined as positive for the Zscan4 reporter but negative for the MERV‐L reporter, and 2‐cell‐like cells were defined as positive for both reporters. Brightfield microscopy images indicating the confluency of the three populations shortly after plating in the Seahorse extracellular flux analyser plates. Representative images, in two magnifications, for the three independent biological replicates presented in Fig 1 C and D are shown. Representative sorting gate for ES and 2‐cell‐like cells (left) used for the FACS‐assisted ROS measurements (right). Fluorescence intensity distributions for ES cells (blue) and 2‐cell‐like cells (green) in control (centre) and CellROX‐treated samples (right) are shown.

Techniques Used: Isolation, Fluorescence, Microscopy, FACS

Increased glucose uptake supports higher flux into the pentose phosphate pathway in Zscan4 + cells ATP content in ES (blue), Zscan4 + (red) and 2‐cell‐like cells (green) across four independent biological replicates. Extracellular acidification rate of ES (blue), Zscan4 + (red) and 2‐cell‐like cells (green) across three independent biological replicates performed on the Seahorse extracellular flux analyser. Glucose uptake rates in Zscan4 + (red) and 2‐cell‐like cells (green) were measured using a luciferase‐based assay across four independent biological replicates and are represented relative to those of control ES cells (blue). Schematic representation of measured fluxes (left). In order to ascertain whether the increased glucose uptake observed leads to higher flux into the hexosamine biosynthesis pathway (HBP) or the pentose phosphate pathway (PPP), one enzyme of each pathway was disrupted through siRNA‐mediated knockdown (right). Experimental design. ESC cultures were transfected with siRNAs targeting Gnpnat1 (HBP), G6pdx (PPP) or a negative control siRNA (Neg). After 48 h of culture, cells were FACS‐sorted into a 96‐well plate based on their fluorescent reporters and glucose uptake rates were measured using a luciferase‐based assay. Glucose uptake rates upon knockdown of Gnpnat1 or a G6pdx were measured in ES, Zscan4 + and 2‐cell‐like cells. Measurements were quantified relative to the glucose uptake rate of ESCs transfected with a negative control siRNA. Shown are the mean ± s.d. of the indicated number of independent cell cultures, performed across 2 or more independent biological replicates each. Glucose‐6‐phosphate dehydrogenase activity was measured in ES (blue), Zscan4 + (red) and 2‐cell‐like cell (green) lysates using a fluorometric assay. Measurements were obtained from three independent biological replicates, performed in three technical replicates each. n.s.—not significant; one‐way ANOVA. Data information: In panels (A–C and G), boxes indicate the range between the first and third quartile, the band depicts the median, and the whiskers span the range of the data while extending no further than 1.5 times the interquartile range. Individual dots indicate the measurements obtained in each individual technical replicate. Source data are available online for this figure.
Figure Legend Snippet: Increased glucose uptake supports higher flux into the pentose phosphate pathway in Zscan4 + cells ATP content in ES (blue), Zscan4 + (red) and 2‐cell‐like cells (green) across four independent biological replicates. Extracellular acidification rate of ES (blue), Zscan4 + (red) and 2‐cell‐like cells (green) across three independent biological replicates performed on the Seahorse extracellular flux analyser. Glucose uptake rates in Zscan4 + (red) and 2‐cell‐like cells (green) were measured using a luciferase‐based assay across four independent biological replicates and are represented relative to those of control ES cells (blue). Schematic representation of measured fluxes (left). In order to ascertain whether the increased glucose uptake observed leads to higher flux into the hexosamine biosynthesis pathway (HBP) or the pentose phosphate pathway (PPP), one enzyme of each pathway was disrupted through siRNA‐mediated knockdown (right). Experimental design. ESC cultures were transfected with siRNAs targeting Gnpnat1 (HBP), G6pdx (PPP) or a negative control siRNA (Neg). After 48 h of culture, cells were FACS‐sorted into a 96‐well plate based on their fluorescent reporters and glucose uptake rates were measured using a luciferase‐based assay. Glucose uptake rates upon knockdown of Gnpnat1 or a G6pdx were measured in ES, Zscan4 + and 2‐cell‐like cells. Measurements were quantified relative to the glucose uptake rate of ESCs transfected with a negative control siRNA. Shown are the mean ± s.d. of the indicated number of independent cell cultures, performed across 2 or more independent biological replicates each. Glucose‐6‐phosphate dehydrogenase activity was measured in ES (blue), Zscan4 + (red) and 2‐cell‐like cell (green) lysates using a fluorometric assay. Measurements were obtained from three independent biological replicates, performed in three technical replicates each. n.s.—not significant; one‐way ANOVA. Data information: In panels (A–C and G), boxes indicate the range between the first and third quartile, the band depicts the median, and the whiskers span the range of the data while extending no further than 1.5 times the interquartile range. Individual dots indicate the measurements obtained in each individual technical replicate. Source data are available online for this figure.

Techniques Used: Luciferase, Transfection, Negative Control, FACS, Activity Assay

Early‐embryonic‐like cells exhibit decreased mitochondrial respiration Heatmap showing changes in RNA expression levels for various enzymes and regulators of central carbon metabolism in endogenous and in CAF1‐knockdown‐induced (through p60 or p150 KD) 2‐cell‐like cells. Fold‐changes relative to ESCs were calculated based on bulk RNA‐seq data 15 . Schematic representation of 2‐cell‐like cell emergence from ES cells, which transit through the intermediate Zscan4 + state before becoming 2‐cell‐like cells. Reporter constructs used to identify the three cell populations are shown. Oxygen consumption rate of ES (blue line) and 2‐cell‐like cells (green line). Assay medium was formulated to recapitulate standard ES cell culture conditions and contained glucose, l ‐glutamine and pyruvate. Basal, maximal (FCCP‐induced) and non‐mitochondrial (rotenone and antimycin A‐mediated) respiratory rates are indicated. A representative graph of three independent biological replicates performed on the Seahorse extracellular flux analyser is shown. Due to the low number of 2‐cell‐like cells available, compared to ESCs, one technical replicate of the former was analysed per biological replicate, while three or more technical replicates were performed for the latter. Accordingly, mean ± s.d. of technical replicates is shown for ESCs. Basal oxygen consumption rate of ES (blue), Zscan4 + (red) and 2‐cell‐like cells (green) across three independent biological replicates performed on the Seahorse extracellular flux analyser. Assay medium was formulated to recapitulate standard ES cell culture conditions and contained glucose, l ‐glutamine and pyruvate. Boxes indicate the range between the first and third quartile, the band specifies the median, and the whiskers span the range of the data while extending no further than 1.5 times the interquartile range. Individual dots indicate the measurements obtained in each of the individual technical replicates. Oxygen consumption rate of ES (blue line), Zscan4 + (red line) and 2‐cell‐like cells (green line) in glucose‐free media and upon acute injection of sodium pyruvate or sodium l ‐lactate. Note that l ‐glutamine—but not glucose or pyruvate—was initially present in the assay medium. Maximal (FCCP‐induced) and non‐mitochondrial (rotenone and antimycin A‐mediated) respiratory rates following pyruvate or lactate treatment are also indicated. A graph including data from three independent biological replicates is presented, and the mean ± s.d. of technical replicates is shown. Representative electron micrographs of mitochondria from ES ( n = 49 sections) and 2‐cell‐like cells ( n = 57 sections) generated across two independent technical and biological replicates. Scale bar, 1 μm. Representative images from two independent biological and technical replicates are shown. 162 and 99 mitochondria were analysed for 2‐cell‐like and ES cells, respectively. Representative single section of CellROX‐DeepRed fluorescence in ES and 2‐cell‐like cells (green arrow) obtained using live‐cell microscopy. Scale bar, 10 μm. Representative images from three independent biological replicates are shown. FACS‐assisted quantification of CellROX‐DeepRed fluorescence intensity in ES and 2‐cell‐like cells. Measurements were obtained from two independent biological replicates. *** P
Figure Legend Snippet: Early‐embryonic‐like cells exhibit decreased mitochondrial respiration Heatmap showing changes in RNA expression levels for various enzymes and regulators of central carbon metabolism in endogenous and in CAF1‐knockdown‐induced (through p60 or p150 KD) 2‐cell‐like cells. Fold‐changes relative to ESCs were calculated based on bulk RNA‐seq data 15 . Schematic representation of 2‐cell‐like cell emergence from ES cells, which transit through the intermediate Zscan4 + state before becoming 2‐cell‐like cells. Reporter constructs used to identify the three cell populations are shown. Oxygen consumption rate of ES (blue line) and 2‐cell‐like cells (green line). Assay medium was formulated to recapitulate standard ES cell culture conditions and contained glucose, l ‐glutamine and pyruvate. Basal, maximal (FCCP‐induced) and non‐mitochondrial (rotenone and antimycin A‐mediated) respiratory rates are indicated. A representative graph of three independent biological replicates performed on the Seahorse extracellular flux analyser is shown. Due to the low number of 2‐cell‐like cells available, compared to ESCs, one technical replicate of the former was analysed per biological replicate, while three or more technical replicates were performed for the latter. Accordingly, mean ± s.d. of technical replicates is shown for ESCs. Basal oxygen consumption rate of ES (blue), Zscan4 + (red) and 2‐cell‐like cells (green) across three independent biological replicates performed on the Seahorse extracellular flux analyser. Assay medium was formulated to recapitulate standard ES cell culture conditions and contained glucose, l ‐glutamine and pyruvate. Boxes indicate the range between the first and third quartile, the band specifies the median, and the whiskers span the range of the data while extending no further than 1.5 times the interquartile range. Individual dots indicate the measurements obtained in each of the individual technical replicates. Oxygen consumption rate of ES (blue line), Zscan4 + (red line) and 2‐cell‐like cells (green line) in glucose‐free media and upon acute injection of sodium pyruvate or sodium l ‐lactate. Note that l ‐glutamine—but not glucose or pyruvate—was initially present in the assay medium. Maximal (FCCP‐induced) and non‐mitochondrial (rotenone and antimycin A‐mediated) respiratory rates following pyruvate or lactate treatment are also indicated. A graph including data from three independent biological replicates is presented, and the mean ± s.d. of technical replicates is shown. Representative electron micrographs of mitochondria from ES ( n = 49 sections) and 2‐cell‐like cells ( n = 57 sections) generated across two independent technical and biological replicates. Scale bar, 1 μm. Representative images from two independent biological and technical replicates are shown. 162 and 99 mitochondria were analysed for 2‐cell‐like and ES cells, respectively. Representative single section of CellROX‐DeepRed fluorescence in ES and 2‐cell‐like cells (green arrow) obtained using live‐cell microscopy. Scale bar, 10 μm. Representative images from three independent biological replicates are shown. FACS‐assisted quantification of CellROX‐DeepRed fluorescence intensity in ES and 2‐cell‐like cells. Measurements were obtained from two independent biological replicates. *** P

Techniques Used: RNA Expression, RNA Sequencing Assay, Construct, Cell Culture, Injection, Generated, Fluorescence, Microscopy, FACS

Sodium acetate induces Zscan4 + and 2‐cell ‐like cells Immunofluorescence staining for OCT4, ZSCAN4 and 2C::tbGFP in control and acetate‐treated ESC cultures. Green arrows indicate 2‐cell‐like cells, and inlets highlight their DAPI structure. Scale bar, 20 μm. RT–qPCR of the indicated genes in ESC cultures treated with sodium acetate for 24 h. Shown are the mean ± s.d. of three independent cell cultures, performed in two technical replicates. Percentage of 2‐cell‐like cells obtained upon transfection of control or Dux‐targeting siRNAs in control conditions or in combination with sodium acetate treatment. Measurements were obtained from two independent cell cultures, performed across three independent biological replicates. Boxes indicate the range between the first and third quartile, the band depicts the median, and the whiskers span the range of the data while extending no further than 1.5 times the interquartile range. Individual dots indicate the measurements obtained in each technical replicate. Percentage of 2‐cell‐like cells obtained upon transfection siRNAs targeting the indicated chromatin factors in control conditions or in combination with sodium acetate treatment. Shown are the mean ± s.d. of four independent cell cultures, performed across two independent biological replicates. Experimental design. ESC cultures were FACS‐sorted to remove Zscan4 + and 2‐cell‐like cells and plated in a glass bottom 96‐well plate. Cells were then imaged for 96 h in the presence of sodium acetate (32 mM), sodium l ‐lactate (32 mM) or in control conditions. Proportion of Zscan4 + cells at various timepoints during the time‐lapse experiment. Shown are the mean ± s.d. of three independent experiments. Source data are available online for this figure.
Figure Legend Snippet: Sodium acetate induces Zscan4 + and 2‐cell ‐like cells Immunofluorescence staining for OCT4, ZSCAN4 and 2C::tbGFP in control and acetate‐treated ESC cultures. Green arrows indicate 2‐cell‐like cells, and inlets highlight their DAPI structure. Scale bar, 20 μm. RT–qPCR of the indicated genes in ESC cultures treated with sodium acetate for 24 h. Shown are the mean ± s.d. of three independent cell cultures, performed in two technical replicates. Percentage of 2‐cell‐like cells obtained upon transfection of control or Dux‐targeting siRNAs in control conditions or in combination with sodium acetate treatment. Measurements were obtained from two independent cell cultures, performed across three independent biological replicates. Boxes indicate the range between the first and third quartile, the band depicts the median, and the whiskers span the range of the data while extending no further than 1.5 times the interquartile range. Individual dots indicate the measurements obtained in each technical replicate. Percentage of 2‐cell‐like cells obtained upon transfection siRNAs targeting the indicated chromatin factors in control conditions or in combination with sodium acetate treatment. Shown are the mean ± s.d. of four independent cell cultures, performed across two independent biological replicates. Experimental design. ESC cultures were FACS‐sorted to remove Zscan4 + and 2‐cell‐like cells and plated in a glass bottom 96‐well plate. Cells were then imaged for 96 h in the presence of sodium acetate (32 mM), sodium l ‐lactate (32 mM) or in control conditions. Proportion of Zscan4 + cells at various timepoints during the time‐lapse experiment. Shown are the mean ± s.d. of three independent experiments. Source data are available online for this figure.

Techniques Used: Immunofluorescence, Staining, Quantitative RT-PCR, Transfection, FACS

17) Product Images from "Oral Escherichia coli Colonization Factor Antigen I (CFA/I) Fimbriae Ameliorate Arthritis via IL-35, not IL-27"

Article Title: Oral Escherichia coli Colonization Factor Antigen I (CFA/I) Fimbriae Ameliorate Arthritis via IL-35, not IL-27

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1302018

Oral CFA/I fimbriae do not stimulate, rather suppress IL-27 expression by CD4 + and CD11c + cells. CIA was induced, and mice were treated with CFA/I fimbriae as described above. Flow cytometry was performed on day 40 post-challenge. A , Representative FACS
Figure Legend Snippet: Oral CFA/I fimbriae do not stimulate, rather suppress IL-27 expression by CD4 + and CD11c + cells. CIA was induced, and mice were treated with CFA/I fimbriae as described above. Flow cytometry was performed on day 40 post-challenge. A , Representative FACS

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

18) Product Images from "Environmental stimuli shape microglial plasticity in glioma"

Article Title: Environmental stimuli shape microglial plasticity in glioma

Journal: eLife

doi: 10.7554/eLife.33415

Representative FACS analysis to verify CD45+/Ly6c + cell depletion in the brains of GL261-bearing mice housed in EE or SE, upon clodronate liposome treatment; and F4/80+/Ly6c + cells in the spleen. On top, scheme of liposome administration.
Figure Legend Snippet: Representative FACS analysis to verify CD45+/Ly6c + cell depletion in the brains of GL261-bearing mice housed in EE or SE, upon clodronate liposome treatment; and F4/80+/Ly6c + cells in the spleen. On top, scheme of liposome administration.

Techniques Used: FACS, Mouse Assay

19) Product Images from "DNA-damage-induced differentiation of leukaemic cells as an anti-cancer barrier"

Article Title: DNA-damage-induced differentiation of leukaemic cells as an anti-cancer barrier

Journal: Nature

doi: 10.1038/nature13483

Deletion of MLL4 in haematopoietic stem cells a , MLL4f/f mice (see methods) were crossed with the interferon-inducible transgene MxCre to obtain MLL4f/f MxCre and WTMxCre mice. Animals were then treated with intraperitoneal (ip) injections of 300 μg of polyIC five times every other day and analysed 3 weeks after the last polyIC injection (generating WT and MLL4 −/− mice). b , The MLL4 wild-type locus (WT) and the floxed exons locus before and after Cre excision. Exons are represented in numbered boxes. The loxP sites (red rectangles) and the MLL4 PCR primers (black arrows) are indicated. c , PCR analysis for conditional MLL4 knockout mice (exons 16, 17, 18, 19). Genomic DNA from sorted HSCs derived from WT and MLL4 −/− mice (PolyIC treated as in a ) and from mouse embryonic fibroblasts (MEFs) derived from MLL4 f/f cells infected with a retrovirus expressing Cre or empty vector (EV) were analysed by PCR. The wild-type (258 base pairs (bp)) and floxed band (320 bp) were amplified with primers A and B, and the knockout band (390 bp) was amplified with primers A and C in different reactions. One of two independent genotyping experiments is shown. d , qPCR quantification of deletion efficiency in conditional knockout Cre-expressing cells. e , Whole bone-marrow cellularity 3 weeks after polyIC treatment of WTMxCre and MLL4 f/f MxCre mice (referred to as WT and MLL4 −/− respectively). f , Representative FACS profiles pre-gated on live cells showing LSKs, LT-HSCs and common lymphoid progenitors, and quantification of these bone-marrow populations ( g – i ) as well as B cells and myeloid cells ( j ) in the bone marrow. All bar graphs show mean ± s.d. of at least three independent experiments.
Figure Legend Snippet: Deletion of MLL4 in haematopoietic stem cells a , MLL4f/f mice (see methods) were crossed with the interferon-inducible transgene MxCre to obtain MLL4f/f MxCre and WTMxCre mice. Animals were then treated with intraperitoneal (ip) injections of 300 μg of polyIC five times every other day and analysed 3 weeks after the last polyIC injection (generating WT and MLL4 −/− mice). b , The MLL4 wild-type locus (WT) and the floxed exons locus before and after Cre excision. Exons are represented in numbered boxes. The loxP sites (red rectangles) and the MLL4 PCR primers (black arrows) are indicated. c , PCR analysis for conditional MLL4 knockout mice (exons 16, 17, 18, 19). Genomic DNA from sorted HSCs derived from WT and MLL4 −/− mice (PolyIC treated as in a ) and from mouse embryonic fibroblasts (MEFs) derived from MLL4 f/f cells infected with a retrovirus expressing Cre or empty vector (EV) were analysed by PCR. The wild-type (258 base pairs (bp)) and floxed band (320 bp) were amplified with primers A and B, and the knockout band (390 bp) was amplified with primers A and C in different reactions. One of two independent genotyping experiments is shown. d , qPCR quantification of deletion efficiency in conditional knockout Cre-expressing cells. e , Whole bone-marrow cellularity 3 weeks after polyIC treatment of WTMxCre and MLL4 f/f MxCre mice (referred to as WT and MLL4 −/− respectively). f , Representative FACS profiles pre-gated on live cells showing LSKs, LT-HSCs and common lymphoid progenitors, and quantification of these bone-marrow populations ( g – i ) as well as B cells and myeloid cells ( j ) in the bone marrow. All bar graphs show mean ± s.d. of at least three independent experiments.

Techniques Used: Mouse Assay, Injection, Polymerase Chain Reaction, Knock-Out, Derivative Assay, Infection, Expressing, Plasmid Preparation, Amplification, Real-time Polymerase Chain Reaction, FACS

MLL4 is required for MLL–AF9 transformation in vivo and in vitro a , WT and MLL4 −/− bone-marrow cells were transformed with MLL–AF9 and injected into irradiated recipients (650 rad) or maintained in culture for in vitro experiments. b , Non-polyIC-treated WTMxCre and MLL4 f/f MxCre bone-marrow cells were transformed with MLL–AF9. Cells were subsequently infected with retrovirus containing Cre-recombinase (CRE-Tomato) or injected into mice that were administered polyIC 1 week later. c , PCR analysis of genomic DNA shows the extent of MLL4 deletion in MLL–AF9-infected cells. MLL4f/f and MLL4f/f Cre-infected MEFs were used as a control. Genotyping was performed once. d , Spleens from mice 29 days after injection with WT MLL–AF9 or MLL4 −/− MLL–AF9 cells, and spleen from non-injected littermates (WT) (see also ). Photographs were taken in one experiment. e , Normalized colony counts scored 11 days after culture of WT or MLL4 −/− whole bone marrow (non-transformed) in semi-solid media in the presence of IL3, IL6 and SCF. f , Representative FACS plots showing AnnexinV versus GFP staining in MLL–AF9 WT or MLL4 −/− cells cultured in semi-solid media (as in a ). g , Histogram of GFP expression 10 days after MLL–AF9 transformation. h . Data are shown normalized to WT counts (dotted line) in three independent experiments. i , The morphological changes observed in MLL4-deficient MLL–AF9 cells are accompanied by increased expression of the myeloid markers Mac1 (right) and Gr1 (left). j , WT or MLL4 f/f bone-marrow cells were transformed with MLL–AF9 and subsequently MLL4 was excised by retroviral expression of CRE as in b . Five days later, MLL4 mRNA levels were measured by qPCR. k . Data are shown normalized to WT counts (dotted line) in three independent experiments. l – o , WT bone-marrow cells were transformed with MLL–AF9 and subsequently infected with retroviruses expressing CRE (as in b ). Blast colony counts ( l ), frequency of blasts evaluated by May-Grünwald-Giemsa stained cytospins ( m , n ) and frequency of apoptotic cells determined by morphology ( o ) were calculated after culture in semi-solid media supplemented with SCF, IL3 and IL6. Images of cytospins were acquired once. p , WT MLL–AF9 cells were stably infected with a retrovirus encoding an shRNA to target and silence MLL4 expression. shRNA depletion of MLL4 mRNA was measured by qPCR and normalized to levels in non-target control shRNA-infected cells. q , Frequency of cells identified as blasts in the cytospin samples from MLL4 shRNA-infected cells compared with control shRNA-infected cells. Fig. 2a–c
Figure Legend Snippet: MLL4 is required for MLL–AF9 transformation in vivo and in vitro a , WT and MLL4 −/− bone-marrow cells were transformed with MLL–AF9 and injected into irradiated recipients (650 rad) or maintained in culture for in vitro experiments. b , Non-polyIC-treated WTMxCre and MLL4 f/f MxCre bone-marrow cells were transformed with MLL–AF9. Cells were subsequently infected with retrovirus containing Cre-recombinase (CRE-Tomato) or injected into mice that were administered polyIC 1 week later. c , PCR analysis of genomic DNA shows the extent of MLL4 deletion in MLL–AF9-infected cells. MLL4f/f and MLL4f/f Cre-infected MEFs were used as a control. Genotyping was performed once. d , Spleens from mice 29 days after injection with WT MLL–AF9 or MLL4 −/− MLL–AF9 cells, and spleen from non-injected littermates (WT) (see also ). Photographs were taken in one experiment. e , Normalized colony counts scored 11 days after culture of WT or MLL4 −/− whole bone marrow (non-transformed) in semi-solid media in the presence of IL3, IL6 and SCF. f , Representative FACS plots showing AnnexinV versus GFP staining in MLL–AF9 WT or MLL4 −/− cells cultured in semi-solid media (as in a ). g , Histogram of GFP expression 10 days after MLL–AF9 transformation. h . Data are shown normalized to WT counts (dotted line) in three independent experiments. i , The morphological changes observed in MLL4-deficient MLL–AF9 cells are accompanied by increased expression of the myeloid markers Mac1 (right) and Gr1 (left). j , WT or MLL4 f/f bone-marrow cells were transformed with MLL–AF9 and subsequently MLL4 was excised by retroviral expression of CRE as in b . Five days later, MLL4 mRNA levels were measured by qPCR. k . Data are shown normalized to WT counts (dotted line) in three independent experiments. l – o , WT bone-marrow cells were transformed with MLL–AF9 and subsequently infected with retroviruses expressing CRE (as in b ). Blast colony counts ( l ), frequency of blasts evaluated by May-Grünwald-Giemsa stained cytospins ( m , n ) and frequency of apoptotic cells determined by morphology ( o ) were calculated after culture in semi-solid media supplemented with SCF, IL3 and IL6. Images of cytospins were acquired once. p , WT MLL–AF9 cells were stably infected with a retrovirus encoding an shRNA to target and silence MLL4 expression. shRNA depletion of MLL4 mRNA was measured by qPCR and normalized to levels in non-target control shRNA-infected cells. q , Frequency of cells identified as blasts in the cytospin samples from MLL4 shRNA-infected cells compared with control shRNA-infected cells. Fig. 2a–c

Techniques Used: Transformation Assay, In Vivo, In Vitro, Injection, Irradiation, Infection, Mouse Assay, Polymerase Chain Reaction, FACS, Staining, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Stable Transfection, shRNA

MLL4 regulates the expression of genes in the glutathione- and FoxO-dependent pathways a , Representative FACs plots showing Ki-67 versus DAPI profiles in LSK CD34 lo cells (left panel) and LSK cells (right panel). b , Summary of the cell-cycle profiles as in a for three independent mice per group. c , Schematic of division patterns of HSCs. d , Tie2 expression in bone-marrow CD150 + CD48 − CD41 − Flt3 − CD34 − LSK cells from WT or MLL4 −/− mice. e , Canonical pathways and biological processes over-represented within the 1,000 most downregulated genes in MLL4 f/f MxCre ( MLL4 −/− ) relative to WT MxCre (WT) sorted LSKs. f , GSEA shows enrichment of glutathione detoxification pathway in WT MxCre (WT) relative to MLL4 f/f MxCre ( MLL4 −/− ) LSKs (FDR
Figure Legend Snippet: MLL4 regulates the expression of genes in the glutathione- and FoxO-dependent pathways a , Representative FACs plots showing Ki-67 versus DAPI profiles in LSK CD34 lo cells (left panel) and LSK cells (right panel). b , Summary of the cell-cycle profiles as in a for three independent mice per group. c , Schematic of division patterns of HSCs. d , Tie2 expression in bone-marrow CD150 + CD48 − CD41 − Flt3 − CD34 − LSK cells from WT or MLL4 −/− mice. e , Canonical pathways and biological processes over-represented within the 1,000 most downregulated genes in MLL4 f/f MxCre ( MLL4 −/− ) relative to WT MxCre (WT) sorted LSKs. f , GSEA shows enrichment of glutathione detoxification pathway in WT MxCre (WT) relative to MLL4 f/f MxCre ( MLL4 −/− ) LSKs (FDR

Techniques Used: Expressing, FACS, Mouse Assay

Extramedullary haematopoiesis in the absence of MLL4 a , Representative FACS plot of LSKs separated based on CD34 and subsequently analysed by cell surface expression of CD150 (Slamf1). b , Pie charts summarize data from three independent mice of each genotype (χ 2 test, P > 0.8). c , Frequency of cells determined by FACS analysis of Lin − Sca1 + c-Kit − separated based on CD34 and FcγRII/III. CMPs, common myeloid progenitors; MEPs, megakaryocyte-erythroid progenitors; GMPs, granulocyte macrophage progenitors. Mean ± s.d. of three mice per group is shown. d , Quantification of colony numbers generated by WT and MLL4 −/− myeloid progenitors (LSK) in serial colony forming assays; y axis, number of colonies; x axis, serial assay, primary to quaternary. e – g , Splenomegaly and increased numbers of myeloid and erythroid cells in the spleens of MLL4 −/− mice 3 weeks after polyIC treatment. Image of spleen representative of more than three independent experiments. h , Haematoxylin and eosin staining of spleen (upper panel, ×20; middle panel; ×40; lower panel, magnified picture of the middle panel to visualize details). Black arrows show presence of erythrocytes in MLL4-deficient spleens. Images were acquired in one experiment.
Figure Legend Snippet: Extramedullary haematopoiesis in the absence of MLL4 a , Representative FACS plot of LSKs separated based on CD34 and subsequently analysed by cell surface expression of CD150 (Slamf1). b , Pie charts summarize data from three independent mice of each genotype (χ 2 test, P > 0.8). c , Frequency of cells determined by FACS analysis of Lin − Sca1 + c-Kit − separated based on CD34 and FcγRII/III. CMPs, common myeloid progenitors; MEPs, megakaryocyte-erythroid progenitors; GMPs, granulocyte macrophage progenitors. Mean ± s.d. of three mice per group is shown. d , Quantification of colony numbers generated by WT and MLL4 −/− myeloid progenitors (LSK) in serial colony forming assays; y axis, number of colonies; x axis, serial assay, primary to quaternary. e – g , Splenomegaly and increased numbers of myeloid and erythroid cells in the spleens of MLL4 −/− mice 3 weeks after polyIC treatment. Image of spleen representative of more than three independent experiments. h , Haematoxylin and eosin staining of spleen (upper panel, ×20; middle panel; ×40; lower panel, magnified picture of the middle panel to visualize details). Black arrows show presence of erythrocytes in MLL4-deficient spleens. Images were acquired in one experiment.

Techniques Used: FACS, Expressing, Mouse Assay, Generated, Staining

20) Product Images from "The fate and lifespan of human monocyte subsets in steady state and systemic inflammation"

Article Title: The fate and lifespan of human monocyte subsets in steady state and systemic inflammation

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20170355

Development of intermediate and nonclassical human monocytes from classical monocytes . (a) Classical human monocyte LIN − HLA-DR + CD14 + CD16 − cells were sorted from healthy blood by FACS. (b) 1.5 × 10 6 sorted classical monocytes were grafted i.v. into the humanized MISTRG mouse. Grafted cells could be readily identified by expression of the human isoform of CD45 compared with recipient leukocytes expressing mouse CD45. (c) Flow cytometry analysis identified human CD45 + circulating monocytes from MISTRG recipients following adoptive transfer of human CD14 + CD16 − classical monocytes at 10 min and 24, 72, and 96 h after infusion. Results are representative of three analyzed mice per time point.
Figure Legend Snippet: Development of intermediate and nonclassical human monocytes from classical monocytes . (a) Classical human monocyte LIN − HLA-DR + CD14 + CD16 − cells were sorted from healthy blood by FACS. (b) 1.5 × 10 6 sorted classical monocytes were grafted i.v. into the humanized MISTRG mouse. Grafted cells could be readily identified by expression of the human isoform of CD45 compared with recipient leukocytes expressing mouse CD45. (c) Flow cytometry analysis identified human CD45 + circulating monocytes from MISTRG recipients following adoptive transfer of human CD14 + CD16 − classical monocytes at 10 min and 24, 72, and 96 h after infusion. Results are representative of three analyzed mice per time point.

Techniques Used: FACS, Expressing, Flow Cytometry, Cytometry, Adoptive Transfer Assay, Mouse Assay

21) Product Images from "Type I interferons affect the metabolic fitness of CD8+ T cells from patients with systemic lupus erythematosus"

Article Title: Type I interferons affect the metabolic fitness of CD8+ T cells from patients with systemic lupus erythematosus

Journal: Nature Communications

doi: 10.1038/s41467-021-22312-y

Metabolic changes triggered by 7-day IFNα exposure and T cell activation. a , b Purified CD8 + T cells from healthy donors treated with or without αCD3/CD28 beads in the presence or absence of 1000U/ml IFNα for 7 days. Mitochondria-encoded gene expression ( n = 5–7) ( a ) and changes in mitotracker stainings ( n = 6–7) ( b ) are shown. c , d PBMCs from healthy donors treated with or without αCD3/CD28 beads in the presence or absence of 1000U/ml IFNα for 7 days. CD8 + T cells were FACs-sorted and analysed using the extracellular flux assay. c Graphs showing the basal and maximal OCR levels under the different experimental conditions as indicated. Spare respiratory capacity (SRC) was normalized to basal level of each individual ( n = 7–8). d Oxidative response of FACs-sorted CD8 + T cells upon re-stimulation with anti-CD3/CD28 beads or PMA/I injected during the extracellular flux assay. A representative graph (left panel) and levels (right panels) at different time points normalised to basal level of each individual are shown ( n = 6). a – d Data presented as mean ± S.E.M. Each symbol represents one donor. a – c Two-tailed Wilcoxon matched-pairs signed rank test was utilized, d two-way ANOVA; only significant differences are indicated; MFI mean fluorescence intensity, MTG MitoTracker green, MTDR membrane potential dependent-Mitotracker Deep Read, MT-ND3 mitochondrially encoded NADH:Ubiquinone oxidoreductase core subunit 3, MT-CYTB mitochondrially encoded cytochrome B, PMA/I phorbol12-myristate13-acetate/Ionomycin. Source data for this figure are provided as a Source Data file.
Figure Legend Snippet: Metabolic changes triggered by 7-day IFNα exposure and T cell activation. a , b Purified CD8 + T cells from healthy donors treated with or without αCD3/CD28 beads in the presence or absence of 1000U/ml IFNα for 7 days. Mitochondria-encoded gene expression ( n = 5–7) ( a ) and changes in mitotracker stainings ( n = 6–7) ( b ) are shown. c , d PBMCs from healthy donors treated with or without αCD3/CD28 beads in the presence or absence of 1000U/ml IFNα for 7 days. CD8 + T cells were FACs-sorted and analysed using the extracellular flux assay. c Graphs showing the basal and maximal OCR levels under the different experimental conditions as indicated. Spare respiratory capacity (SRC) was normalized to basal level of each individual ( n = 7–8). d Oxidative response of FACs-sorted CD8 + T cells upon re-stimulation with anti-CD3/CD28 beads or PMA/I injected during the extracellular flux assay. A representative graph (left panel) and levels (right panels) at different time points normalised to basal level of each individual are shown ( n = 6). a – d Data presented as mean ± S.E.M. Each symbol represents one donor. a – c Two-tailed Wilcoxon matched-pairs signed rank test was utilized, d two-way ANOVA; only significant differences are indicated; MFI mean fluorescence intensity, MTG MitoTracker green, MTDR membrane potential dependent-Mitotracker Deep Read, MT-ND3 mitochondrially encoded NADH:Ubiquinone oxidoreductase core subunit 3, MT-CYTB mitochondrially encoded cytochrome B, PMA/I phorbol12-myristate13-acetate/Ionomycin. Source data for this figure are provided as a Source Data file.

Techniques Used: Activation Assay, Purification, Expressing, FACS, XF Assay, Injection, Two Tailed Test, Fluorescence

22) Product Images from "Neurons generated by direct conversion of fibroblasts reproduce synaptic phenotype caused by autism-associated neuroligin-3 mutation"

Article Title: Neurons generated by direct conversion of fibroblasts reproduce synaptic phenotype caused by autism-associated neuroligin-3 mutation

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

doi: 10.1073/pnas.1316240110

Generation of iN cells from MEFs isolated from WT and Nlgn3 R704C-mutant mice. ( A ) Schematic representation of the experimental design. MEFs were seeded on day 0 and transduced with the BAM transcription factors on day 1, and transgene expression was induced by the addition of doxycycline to the medium on day 2. Olfactory bulb neurons were cultured from newborn WT mice on day 5, tau-EGFP–positive iN cells were isolated by FACS sorting and placed on top of the primary neurons on day 6, and electrophysiological and imaging experiments were performed on days 22–24. ( B ) Representative image of R704C-mutant iN cells stained 14 d after transduction of MEFs for the pan-neuronal marker Tuj1. ( C ) Representative quantitative RT-PCR analysis of Nlgn3 expression in MEFs, mouse brain, and WT and R704C-mutant iN cells that were analyzed without FACS sorting 20 d posttransduction. ( D ) ( Left ) FACS analysis illustrating the efficiency of the conversion of MEFs into iN cells (defined as tau-EGFP–positive cells) in R704C-mutant and WT cells. ( Right ) The bar graph shows the average abundance of tau-EGFP–positive cells ( n = 5 experiments). ( E and F ) Immunofluorescence analysis of iN cells (cultured on glia only). At day 20, iN cells were stained for DAPI (blue) and MAP2 (red) ( E ) and for tau-GFP (green), synapsin (red), and DAPI (blue) ( F ).
Figure Legend Snippet: Generation of iN cells from MEFs isolated from WT and Nlgn3 R704C-mutant mice. ( A ) Schematic representation of the experimental design. MEFs were seeded on day 0 and transduced with the BAM transcription factors on day 1, and transgene expression was induced by the addition of doxycycline to the medium on day 2. Olfactory bulb neurons were cultured from newborn WT mice on day 5, tau-EGFP–positive iN cells were isolated by FACS sorting and placed on top of the primary neurons on day 6, and electrophysiological and imaging experiments were performed on days 22–24. ( B ) Representative image of R704C-mutant iN cells stained 14 d after transduction of MEFs for the pan-neuronal marker Tuj1. ( C ) Representative quantitative RT-PCR analysis of Nlgn3 expression in MEFs, mouse brain, and WT and R704C-mutant iN cells that were analyzed without FACS sorting 20 d posttransduction. ( D ) ( Left ) FACS analysis illustrating the efficiency of the conversion of MEFs into iN cells (defined as tau-EGFP–positive cells) in R704C-mutant and WT cells. ( Right ) The bar graph shows the average abundance of tau-EGFP–positive cells ( n = 5 experiments). ( E and F ) Immunofluorescence analysis of iN cells (cultured on glia only). At day 20, iN cells were stained for DAPI (blue) and MAP2 (red) ( E ) and for tau-GFP (green), synapsin (red), and DAPI (blue) ( F ).

Techniques Used: Isolation, Mutagenesis, Mouse Assay, Transduction, Expressing, Cell Culture, FACS, Imaging, Staining, Marker, Quantitative RT-PCR, Immunofluorescence

23) Product Images from "V2-Directed Vaccine-like Antibodies from HIV-1 Infection Identify an Additional K169-Binding Light Chain Motif with Broad ADCC Activity"

Article Title: V2-Directed Vaccine-like Antibodies from HIV-1 Infection Identify an Additional K169-Binding Light Chain Motif with Broad ADCC Activity

Journal: Cell reports

doi: 10.1016/j.celrep.2018.11.058

Isolation of V2p-Binding Antibodies from an HIV-1-Infected Donor (A)Hierarchical clustering of 15 HIV-1 + plasma samples and four anti-V2 mAbs (labeled on the right) based on their relative reactivity with V2 peptides or scaffolded V1V2 antigens (labeled on the top and in the key) in an ELISA. Plasma samples and mAbs with broadly neutralizing activity are indicated with an asterisk on the left-hand side. Binding intensity was recorded as a relative percentage of maximum OD, where 0%−40% was colored yellow; 40%−80%, orange; and 80%−100%, red. Samples were grouped either with mAbs CH58 and CH59 (based on strong binding to peptides), with PG9 (based on binding to monomeric V1V2 proteins), or with CH01 (based on no/limited binding to the peptides or proteins). (b) ELISA binding of CAP228 longitudinal plasma to CAP45 wild-type (blue) and K169E mutant (cyan) cyclical V2 peptides. Absorbance readings are plotted on the y axis against time on the x axis. The 134 and 174 wpi time points at which mAbs were isolated from stored PBMC samples are indicated with the vertical dotted lines. (C) Fluorescence-activated cell sorting (FACS) plot showing the CAP228 memory B cells (in blue) that bind BV421-labeled wild-type (y axis), but not AF647-labeled K169E mutant (x axis) V2 peptide antigens, and were therefore sorted for PCR amplification. ). (E) Properties of the CAP228 antibodies, defined by the International Immunogenetics Database (IMGT), compared to vaccine-elicited mAbs CH58 and CH59. CDRH3 lengths are also given for Kabat numbering in parentheses. Shared immunoglobulin germline genes are shaded, and the negatively charged amino acids in each CDRL2 are shown in red.
Figure Legend Snippet: Isolation of V2p-Binding Antibodies from an HIV-1-Infected Donor (A)Hierarchical clustering of 15 HIV-1 + plasma samples and four anti-V2 mAbs (labeled on the right) based on their relative reactivity with V2 peptides or scaffolded V1V2 antigens (labeled on the top and in the key) in an ELISA. Plasma samples and mAbs with broadly neutralizing activity are indicated with an asterisk on the left-hand side. Binding intensity was recorded as a relative percentage of maximum OD, where 0%−40% was colored yellow; 40%−80%, orange; and 80%−100%, red. Samples were grouped either with mAbs CH58 and CH59 (based on strong binding to peptides), with PG9 (based on binding to monomeric V1V2 proteins), or with CH01 (based on no/limited binding to the peptides or proteins). (b) ELISA binding of CAP228 longitudinal plasma to CAP45 wild-type (blue) and K169E mutant (cyan) cyclical V2 peptides. Absorbance readings are plotted on the y axis against time on the x axis. The 134 and 174 wpi time points at which mAbs were isolated from stored PBMC samples are indicated with the vertical dotted lines. (C) Fluorescence-activated cell sorting (FACS) plot showing the CAP228 memory B cells (in blue) that bind BV421-labeled wild-type (y axis), but not AF647-labeled K169E mutant (x axis) V2 peptide antigens, and were therefore sorted for PCR amplification. ). (E) Properties of the CAP228 antibodies, defined by the International Immunogenetics Database (IMGT), compared to vaccine-elicited mAbs CH58 and CH59. CDRH3 lengths are also given for Kabat numbering in parentheses. Shared immunoglobulin germline genes are shaded, and the negatively charged amino acids in each CDRL2 are shown in red.

Techniques Used: Isolation, Binding Assay, Infection, Labeling, Enzyme-linked Immunosorbent Assay, Activity Assay, Mutagenesis, Fluorescence, FACS, Polymerase Chain Reaction, Amplification

24) Product Images from "An integrated multi-omic single cell atlas to redefine human B cell memory"

Article Title: An integrated multi-omic single cell atlas to redefine human B cell memory

Journal: bioRxiv

doi: 10.1101/801530

CD45RB marks human memory B cells and identifies a unique early memory population A) Computational workflow for UMAP generation. UMAP coordinates were generated using conserved molecules as input on a random subset of donor-pooled cells from each of the twelve mass cytometry panels of the screen. B) UMAP plots colored by individually-scaled expression of the indicated molecule. Molecules are organized and colored by function or by their identity as a conserved molecule (right column). Molecules in each row are highly correlated (Pearson method, ± r > 0.3) with the conserved molecule in their row. The canonical memory marker (CD27) and putative memory marker (CD45RB) are boxed. Arrows indicate CD45RB+ CD27- cells. C) UMAP plot colored by canonical B cell subset. Circles indicate islands of cells with memory-like phenotypes based on UMAP co-localization and high-dimensional expression profiles. Arrows are the same as in B) and indicate cells with memory-like phenotypes that are not identified as memory cells by the canonical gating scheme. D) CD38 lo B cells (non-transitional B cells) are plotted on a biaxial and colored by their canonical B cell gate. Percent of CD38 lo B cells in each quadrant is quantified (red text). E) Percent of cells in each quadrant from D) by canonical B cell subset. High percentage of ungated cells in quadrant II is boxed and bolded. F) Experimental workflow for immune repertoire analysis. Peripheral blood was drawn from healthy, human donors (n=2), density gradient centrifugation was performed, and cell preparations were magnetically depleted of non-B cells. Singlet Viable CD45+ lin- CD19+ CD38 lo B cells were FACS-sorted from the four quadrants of CD27 x CD45RB, gDNA was extracted, and the IgH locus was sequenced by NGS. G) Mutation frequency of the IgH loci outside of the CDR3 region for sorted populations, colored by mutation type. Data are pooled from donors. All pairwise comparisons of populations were significantly different (Wilcoxon rank sum test with Bonferroni correction, p
Figure Legend Snippet: CD45RB marks human memory B cells and identifies a unique early memory population A) Computational workflow for UMAP generation. UMAP coordinates were generated using conserved molecules as input on a random subset of donor-pooled cells from each of the twelve mass cytometry panels of the screen. B) UMAP plots colored by individually-scaled expression of the indicated molecule. Molecules are organized and colored by function or by their identity as a conserved molecule (right column). Molecules in each row are highly correlated (Pearson method, ± r > 0.3) with the conserved molecule in their row. The canonical memory marker (CD27) and putative memory marker (CD45RB) are boxed. Arrows indicate CD45RB+ CD27- cells. C) UMAP plot colored by canonical B cell subset. Circles indicate islands of cells with memory-like phenotypes based on UMAP co-localization and high-dimensional expression profiles. Arrows are the same as in B) and indicate cells with memory-like phenotypes that are not identified as memory cells by the canonical gating scheme. D) CD38 lo B cells (non-transitional B cells) are plotted on a biaxial and colored by their canonical B cell gate. Percent of CD38 lo B cells in each quadrant is quantified (red text). E) Percent of cells in each quadrant from D) by canonical B cell subset. High percentage of ungated cells in quadrant II is boxed and bolded. F) Experimental workflow for immune repertoire analysis. Peripheral blood was drawn from healthy, human donors (n=2), density gradient centrifugation was performed, and cell preparations were magnetically depleted of non-B cells. Singlet Viable CD45+ lin- CD19+ CD38 lo B cells were FACS-sorted from the four quadrants of CD27 x CD45RB, gDNA was extracted, and the IgH locus was sequenced by NGS. G) Mutation frequency of the IgH loci outside of the CDR3 region for sorted populations, colored by mutation type. Data are pooled from donors. All pairwise comparisons of populations were significantly different (Wilcoxon rank sum test with Bonferroni correction, p

Techniques Used: Generated, Mass Cytometry, Expressing, Marker, Gradient Centrifugation, FACS, Next-Generation Sequencing, Mutagenesis

25) Product Images from "Simulation of Drug Release from PLGA Particles In Vivo"

Article Title: Simulation of Drug Release from PLGA Particles In Vivo

Journal: Journal of Drug Delivery

doi: 10.1155/2013/513950

(a) Dose response relationship between Hoechst 33342 and fluorescence intensity in the presence or absence of 50 μ M verapamil in IEC-6 cells, (b) Hoechst 33342 dose response for fluorescence intensity in IU-937 cells, and (c) FACS analysis of IEC-6 cells incubated with 0, 10, 100, or 1000 ng/mL Hoechst 33342. Note that the small peak (indicated by an arrow) is also observed at 100 ng/mL of dye. (d) FACS analysis of U-937 cells incubated with 0, 10, 100, or 1000 ng/mL Hoechst 33342.
Figure Legend Snippet: (a) Dose response relationship between Hoechst 33342 and fluorescence intensity in the presence or absence of 50 μ M verapamil in IEC-6 cells, (b) Hoechst 33342 dose response for fluorescence intensity in IU-937 cells, and (c) FACS analysis of IEC-6 cells incubated with 0, 10, 100, or 1000 ng/mL Hoechst 33342. Note that the small peak (indicated by an arrow) is also observed at 100 ng/mL of dye. (d) FACS analysis of U-937 cells incubated with 0, 10, 100, or 1000 ng/mL Hoechst 33342.

Techniques Used: Fluorescence, FACS, Incubation

26) Product Images from "An Immunocompetent Mouse Model for MLL/AF9 Leukemia Reveals the Potential of Spontaneous Cytotoxic T-Cell Response to an Antigen Expressed in Leukemia Cells"

Article Title: An Immunocompetent Mouse Model for MLL/AF9 Leukemia Reveals the Potential of Spontaneous Cytotoxic T-Cell Response to an Antigen Expressed in Leukemia Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0144594

Spontaneous regression of leukemia was observed in the presence, but not in the absence, of adaptive immunity. (A) Flow-cytometry analyses of BM cells of non-irradiated wild-type recipients 7 days after transplantation with different numbers (3 × 10 3 , 3 × 10 4 , or 3 × 10 5 ) of MLL/AF9-OVA leukemia cells. (B) FACS analysis of BM from non-irradiated wild-type or Rag2 -/- mice transplanted with 3 × 10 4 MLL/AF9-OVA leukemia cells. Mice were analyzed 3 weeks after transplant. (C) Kaplan–Meier curves for overall survival of non-irradiated wild-type (n = 7) or Rag2 -/- (n = 3) recipients transplanted with 3 × 10 4 MLL/AF9-OVA leukemia cells. (D) Percentages of GFP + leukemia cells in BM after transplantation into non-irradiated wild-type mice were examined every week. Each dot and line corresponds to a recipient mouse. The results of four mice in which leukemia spontaneously regressed (Exp. 3 in Table 1 ) are shown. (E) FACS analysis of BM from non-irradiated wild-type or Rag2 -/- mice transplanted with 3 × 10 4 of MLL/AF9 leukemia cells (OVA-). Mice were analyzed 3 weeks after transplant.
Figure Legend Snippet: Spontaneous regression of leukemia was observed in the presence, but not in the absence, of adaptive immunity. (A) Flow-cytometry analyses of BM cells of non-irradiated wild-type recipients 7 days after transplantation with different numbers (3 × 10 3 , 3 × 10 4 , or 3 × 10 5 ) of MLL/AF9-OVA leukemia cells. (B) FACS analysis of BM from non-irradiated wild-type or Rag2 -/- mice transplanted with 3 × 10 4 MLL/AF9-OVA leukemia cells. Mice were analyzed 3 weeks after transplant. (C) Kaplan–Meier curves for overall survival of non-irradiated wild-type (n = 7) or Rag2 -/- (n = 3) recipients transplanted with 3 × 10 4 MLL/AF9-OVA leukemia cells. (D) Percentages of GFP + leukemia cells in BM after transplantation into non-irradiated wild-type mice were examined every week. Each dot and line corresponds to a recipient mouse. The results of four mice in which leukemia spontaneously regressed (Exp. 3 in Table 1 ) are shown. (E) FACS analysis of BM from non-irradiated wild-type or Rag2 -/- mice transplanted with 3 × 10 4 of MLL/AF9 leukemia cells (OVA-). Mice were analyzed 3 weeks after transplant.

Techniques Used: Flow Cytometry, Cytometry, Irradiation, Transplantation Assay, FACS, Mouse Assay

Establishment of MLL/AF9 leukemia cells expressing a model tumor antigen. (A) Scheme for establishment of MLL/AF9-HPC-OVA cells. (B) FACS analysis of MLL/AF9-HPC-OVA cells. Blue lines represent MLL/AF9-HPC cells. (C) 51 Cr-release assay using activated CD8 + T cells from the OT-1 mouse as effector cells. MLL/AF9-HPC-OVA or MLL/AF9-HPC cells were used as targets. E:T ratio denotes effector-per-target ratio. (D) Kaplan–Meier curves for overall survival of wild-type mice that received 1 × 10 6 MLL/AF9-HPC-OVA cells in the third transplant (n = 5). Recipient mice were not irradiated. Results from the first and second transplants are shown in S1 Fig . (E) May–Giemsa staining of MLL/AF9-OVA leukemia cells that developed in non-irradiated recipients (Magnification: 400×). (F) Flow-cytometry analysis of GFP + BM cells from mice with leukemia. Blue lines represent MLL/AF9-HPC cells.
Figure Legend Snippet: Establishment of MLL/AF9 leukemia cells expressing a model tumor antigen. (A) Scheme for establishment of MLL/AF9-HPC-OVA cells. (B) FACS analysis of MLL/AF9-HPC-OVA cells. Blue lines represent MLL/AF9-HPC cells. (C) 51 Cr-release assay using activated CD8 + T cells from the OT-1 mouse as effector cells. MLL/AF9-HPC-OVA or MLL/AF9-HPC cells were used as targets. E:T ratio denotes effector-per-target ratio. (D) Kaplan–Meier curves for overall survival of wild-type mice that received 1 × 10 6 MLL/AF9-HPC-OVA cells in the third transplant (n = 5). Recipient mice were not irradiated. Results from the first and second transplants are shown in S1 Fig . (E) May–Giemsa staining of MLL/AF9-OVA leukemia cells that developed in non-irradiated recipients (Magnification: 400×). (F) Flow-cytometry analysis of GFP + BM cells from mice with leukemia. Blue lines represent MLL/AF9-HPC cells.

Techniques Used: Expressing, FACS, Release Assay, Mouse Assay, Irradiation, Staining, Flow Cytometry, Cytometry

27) Product Images from "Human CD8+ CD57- TEMRA cells: Too young to be called "old""

Article Title: Human CD8+ CD57- TEMRA cells: Too young to be called "old"

Journal: PLoS ONE

doi: 10.1371/journal.pone.0177405

Phenotypic and functional characterization of CMV tetramer+ cells. (A) CD8+ CMV tetramer+ T cells were FACS sorted from the peripheral blood of 3 patients after allogeneic SCT and in vitro expanded on autologous feeder cells. Depicted is the T EM and T EMRA subset distribution within CD8+ CMV HLA/tetramer+ T cells (left) and CD57+ distribution within CD8+ CMV HLA/tetramer+ T EMRA cells (right) in the peripheral blood compared to after in vitro expansion of FACS sorted CD8+ CMV tetramer+ T cells. Y-axis: % subset distribution within CD8+ CMV HLA/tetramer+ T cells and CD8+ CMV HLA/tetramer+ T EMRA cells. Error bars indicate standard deviation. (B) Sorting strategy for viable in vitro expanded CD8+ CMV HLA/tetramer+ CD8+ T cells for CD45RA and CD57 allowing functional analysis. (C) Absolute telomere length directly after sorting. (D) BrdU uptake 4 days after stimulation with CD14+ monocytes loaded with increasing concentrations of the relevant HLA/CMV peptide. (E) INF-γ release in the supernatant from the BrdU uptake assay. (F) Specific lysis of CFSE labelled PHA blasts loaded with increasing concentrations of the relevant HLA/CMV peptide. Significance was calculated using Mann-Whitney-U test. * indicates p
Figure Legend Snippet: Phenotypic and functional characterization of CMV tetramer+ cells. (A) CD8+ CMV tetramer+ T cells were FACS sorted from the peripheral blood of 3 patients after allogeneic SCT and in vitro expanded on autologous feeder cells. Depicted is the T EM and T EMRA subset distribution within CD8+ CMV HLA/tetramer+ T cells (left) and CD57+ distribution within CD8+ CMV HLA/tetramer+ T EMRA cells (right) in the peripheral blood compared to after in vitro expansion of FACS sorted CD8+ CMV tetramer+ T cells. Y-axis: % subset distribution within CD8+ CMV HLA/tetramer+ T cells and CD8+ CMV HLA/tetramer+ T EMRA cells. Error bars indicate standard deviation. (B) Sorting strategy for viable in vitro expanded CD8+ CMV HLA/tetramer+ CD8+ T cells for CD45RA and CD57 allowing functional analysis. (C) Absolute telomere length directly after sorting. (D) BrdU uptake 4 days after stimulation with CD14+ monocytes loaded with increasing concentrations of the relevant HLA/CMV peptide. (E) INF-γ release in the supernatant from the BrdU uptake assay. (F) Specific lysis of CFSE labelled PHA blasts loaded with increasing concentrations of the relevant HLA/CMV peptide. Significance was calculated using Mann-Whitney-U test. * indicates p

Techniques Used: Functional Assay, FACS, In Vitro, Standard Deviation, Lysis, MANN-WHITNEY

28) Product Images from "CD56 regulates human NK cell cytotoxicity through Pyk2"

Article Title: CD56 regulates human NK cell cytotoxicity through Pyk2

Journal: bioRxiv

doi: 10.1101/2020.03.19.998427

CD56-deficient primary NK cells retain lytic function. Primary NK cells were isolated and allowed to rest overnight in the presence of low-dose IL-15 prior to delivery of CD56 CRISPR-Cas9. Cells were further expanded in the presence of 25 ng/ml IL-15 for 15 days and cytotoxicity against K562 targets was measured. A) Representative FACS plot of CD56-deficient (blue) or control primary cells (red) after 15 days of Il-15 expansion. Shown also is the fluorescence minus one control (yellow). B) K562 target cell lysis by primary NK cells shown in (A). C) Control or CD56-deficient NK cells from 3 healthy donors were incubated for 1 week after CD56 CRISPR-Cas9 delivery in 25 ng/mL IL-15 then cells were isolated by FACS and cultured for an additional 8 days and the MFI of CD56 was measured by flow cytometry. D) Specific lysis of K562 target cells by isolated and expanded CD56 bright NK cells from the 3 healthy donors shown in (C). E) Primary NK cells were incubated and expanded for 14 days in the presence of 50 ng/ml IL-15 then cytotoxicity against K562 target cells was tested in the presence or absence of Pyk2 inhibitor PF431396. Freshly isolated, non-expanded NK cells were used as a control and similarly treated with PF431396. F) WT or CD56-KO NK92 cells were incubated for 7 days in the presence of 50 ng/ml IL-15 then cytotoxicity was tested in the presence or absence of PF431396. Shown is one representative experiment from 3 independent biological repeats. Error bars indicate technical repeats ( 3 ), SEM.
Figure Legend Snippet: CD56-deficient primary NK cells retain lytic function. Primary NK cells were isolated and allowed to rest overnight in the presence of low-dose IL-15 prior to delivery of CD56 CRISPR-Cas9. Cells were further expanded in the presence of 25 ng/ml IL-15 for 15 days and cytotoxicity against K562 targets was measured. A) Representative FACS plot of CD56-deficient (blue) or control primary cells (red) after 15 days of Il-15 expansion. Shown also is the fluorescence minus one control (yellow). B) K562 target cell lysis by primary NK cells shown in (A). C) Control or CD56-deficient NK cells from 3 healthy donors were incubated for 1 week after CD56 CRISPR-Cas9 delivery in 25 ng/mL IL-15 then cells were isolated by FACS and cultured for an additional 8 days and the MFI of CD56 was measured by flow cytometry. D) Specific lysis of K562 target cells by isolated and expanded CD56 bright NK cells from the 3 healthy donors shown in (C). E) Primary NK cells were incubated and expanded for 14 days in the presence of 50 ng/ml IL-15 then cytotoxicity against K562 target cells was tested in the presence or absence of Pyk2 inhibitor PF431396. Freshly isolated, non-expanded NK cells were used as a control and similarly treated with PF431396. F) WT or CD56-KO NK92 cells were incubated for 7 days in the presence of 50 ng/ml IL-15 then cytotoxicity was tested in the presence or absence of PF431396. Shown is one representative experiment from 3 independent biological repeats. Error bars indicate technical repeats ( 3 ), SEM.

Techniques Used: Isolation, CRISPR, FACS, Fluorescence, Lysis, Incubation, Cell Culture, Flow Cytometry

29) Product Images from "Visualization of stem cell activity in pancreatic cancer expansion by direct lineage tracing with live imaging"

Article Title: Visualization of stem cell activity in pancreatic cancer expansion by direct lineage tracing with live imaging

Journal: eLife

doi: 10.7554/eLife.55117

Dclk1 + pancreatic ductal adenocarcinoma (PDAC) cells have remarkable spheroid- and tumor-forming potential. ( A ) Scheme of FACS and 3D culture of Dclk1 + and Dclk1 − PDAC cells from DKPF mice. ( B ) FACS-sorting of PDAC cells on the basis of Epcam and Dclk1 expression in DKPF mice. ( C and D ) Representative images of tumor spheroids derived from sorted Dclk1 − ( C ) and Dclk1 + ( D ) PDAC cells at day 7. ( E ) Size of spheroids were compared (mean ± SEM; Dclk1 + , n = 5; Dclk1 − , n = 5; n : number of mice). Statistical significance of the differences is indicated as ***p
Figure Legend Snippet: Dclk1 + pancreatic ductal adenocarcinoma (PDAC) cells have remarkable spheroid- and tumor-forming potential. ( A ) Scheme of FACS and 3D culture of Dclk1 + and Dclk1 − PDAC cells from DKPF mice. ( B ) FACS-sorting of PDAC cells on the basis of Epcam and Dclk1 expression in DKPF mice. ( C and D ) Representative images of tumor spheroids derived from sorted Dclk1 − ( C ) and Dclk1 + ( D ) PDAC cells at day 7. ( E ) Size of spheroids were compared (mean ± SEM; Dclk1 + , n = 5; Dclk1 − , n = 5; n : number of mice). Statistical significance of the differences is indicated as ***p

Techniques Used: FACS, Mouse Assay, Expressing, Derivative Assay

30) Product Images from "Transcriptional fingerprints of antigen-presenting cell subsets in the human vaginal mucosa and skin reflect tissue-specific immune microenvironments"

Article Title: Transcriptional fingerprints of antigen-presenting cell subsets in the human vaginal mucosa and skin reflect tissue-specific immune microenvironments

Journal: Genome Medicine

doi: 10.1186/s13073-014-0098-y

Surface receptor expression on vaginal antigen-presenting cells. (a) Transcriptional levels of CD209 (DC-SIGN), OLR1 (LOX-1), CLEC10A (DC-ASGPR) and CLEC4A (DCIR) in the four vaginal APC populations. Bar charts represent the mean ± standard deviation of batch-corrected expression data. (b) Fluorescence-activated cell sorting (FACS) analysis of CD209, DC-ASGPR, LOX-1, DCIR, DEC-205 and CD40 expression on the surface of vaginal APC subsets. Vaginal cell suspensions were stained with the indicated antibodies and gated as described in Figure 1 b. Gray histograms represent isotype controls. Data are representative of 10 donors. (c) Frozen tissue sections were stained for (i) DC-SIGN, DC-ASGPR or LOX-1 (green), CD1c (light blue); and CD14 (red); or (ii) DCIR (red), CD1c (light blue) and CD14 (green); or (iii) DEC-205 (green), CD1c (light blue) and CD207 (red) (DAPI (dark blue), ×20, the horizontal bar represents 100 μm). Data are representative of four to eight independent experiments (left panel) or combined (right panel). Isotype controls are presented in Additional file 18 .
Figure Legend Snippet: Surface receptor expression on vaginal antigen-presenting cells. (a) Transcriptional levels of CD209 (DC-SIGN), OLR1 (LOX-1), CLEC10A (DC-ASGPR) and CLEC4A (DCIR) in the four vaginal APC populations. Bar charts represent the mean ± standard deviation of batch-corrected expression data. (b) Fluorescence-activated cell sorting (FACS) analysis of CD209, DC-ASGPR, LOX-1, DCIR, DEC-205 and CD40 expression on the surface of vaginal APC subsets. Vaginal cell suspensions were stained with the indicated antibodies and gated as described in Figure 1 b. Gray histograms represent isotype controls. Data are representative of 10 donors. (c) Frozen tissue sections were stained for (i) DC-SIGN, DC-ASGPR or LOX-1 (green), CD1c (light blue); and CD14 (red); or (ii) DCIR (red), CD1c (light blue) and CD14 (green); or (iii) DEC-205 (green), CD1c (light blue) and CD207 (red) (DAPI (dark blue), ×20, the horizontal bar represents 100 μm). Data are representative of four to eight independent experiments (left panel) or combined (right panel). Isotype controls are presented in Additional file 18 .

Techniques Used: Expressing, Standard Deviation, Fluorescence, FACS, Staining

Isolation protocols for the eight antigen-presenting cell populations studied. (a) Workflow representing the protocol for isolation of APCs from skin, vagina and blood. (b) Gating strategy for FACS sorting of vaginal APC subsets (upper panels), skin DCs (middle panels) or blood mDCs (lower panels). mDC, myeloid dendritic cell; FCS, fetal calf serum; PBS, phosphate-buffered saline; s, skin; v, vaginal.
Figure Legend Snippet: Isolation protocols for the eight antigen-presenting cell populations studied. (a) Workflow representing the protocol for isolation of APCs from skin, vagina and blood. (b) Gating strategy for FACS sorting of vaginal APC subsets (upper panels), skin DCs (middle panels) or blood mDCs (lower panels). mDC, myeloid dendritic cell; FCS, fetal calf serum; PBS, phosphate-buffered saline; s, skin; v, vaginal.

Techniques Used: Isolation, FACS

31) Product Images from "Phenotypic and functional characteristics of HLA-DR+ neutrophils in Brazilians with cutaneous leishmaniasis"

Article Title: Phenotypic and functional characteristics of HLA-DR+ neutrophils in Brazilians with cutaneous leishmaniasis

Journal: Journal of Leukocyte Biology

doi: 10.1189/jlb.4A0915-442RR

Sorted HLA-DR + PMN from healthy donors have morphologic and biochemical characteristics of neutrophils. (A and B) Micrographs showing cytospun, FACS-isolated HLA-DR − or HLA-DR + PMN (Diff Quik stain). (C) Quantification of band neutrophils in FACS-isolated HLA-DR + vs. HLA-DR − fractions from healthy blood donors ( n = 4; paired Student’s t test). (D) Quantification of the total percentage of band and metamyelocyte (Meta) neutrophil forms in blood smear differential counts from patients with CL compared with endemic healthy controls (unpaired students t test). (E) Correlation plot between the percentage of HLA-DR + PMN vs. the percentage of bands plus metamyelocytes in peripheral blood smears, showing a lack of correlation between the abundance of immature neutrophil forms and the abundance of HLA-DR + PMN in patients with CL ( P = 0.732). (F and G) Representative flow cytometry plots showing gating on granulocytes (62.1%) and monocytes (5.2%) in the whole blood of a patient with CL (F), and gating on HLA-DR + and HLA-DR − CD66b + neutrophils in a patient with CL (G). (H) Histogram showing intracellular staining of HLA-DR + PMNs, HLA-DR − PMNs, and monocytes for MPO. Plots are compared with the fluorescence minus one (FMO) staining control lacking MPO Abs. (I) Quantification of G-MFI of HLA-DR − PMNs, HLA-DR + PMNs, and monocytes in patients with CL (statistical analysis, 1-way ANOVA with Tukey’s posttest analysis).
Figure Legend Snippet: Sorted HLA-DR + PMN from healthy donors have morphologic and biochemical characteristics of neutrophils. (A and B) Micrographs showing cytospun, FACS-isolated HLA-DR − or HLA-DR + PMN (Diff Quik stain). (C) Quantification of band neutrophils in FACS-isolated HLA-DR + vs. HLA-DR − fractions from healthy blood donors ( n = 4; paired Student’s t test). (D) Quantification of the total percentage of band and metamyelocyte (Meta) neutrophil forms in blood smear differential counts from patients with CL compared with endemic healthy controls (unpaired students t test). (E) Correlation plot between the percentage of HLA-DR + PMN vs. the percentage of bands plus metamyelocytes in peripheral blood smears, showing a lack of correlation between the abundance of immature neutrophil forms and the abundance of HLA-DR + PMN in patients with CL ( P = 0.732). (F and G) Representative flow cytometry plots showing gating on granulocytes (62.1%) and monocytes (5.2%) in the whole blood of a patient with CL (F), and gating on HLA-DR + and HLA-DR − CD66b + neutrophils in a patient with CL (G). (H) Histogram showing intracellular staining of HLA-DR + PMNs, HLA-DR − PMNs, and monocytes for MPO. Plots are compared with the fluorescence minus one (FMO) staining control lacking MPO Abs. (I) Quantification of G-MFI of HLA-DR − PMNs, HLA-DR + PMNs, and monocytes in patients with CL (statistical analysis, 1-way ANOVA with Tukey’s posttest analysis).

Techniques Used: FACS, Isolation, Diff-Quik, Staining, Flow Cytometry, Cytometry, Fluorescence

32) Product Images from "Azacitidine mitigates GvHD via differential effects on the proliferation of T effectors and nTregs in vivo"

Article Title: Azacitidine mitigates GvHD via differential effects on the proliferation of T effectors and nTregs in vivo

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1502399

p21-/- attenuates the antiproliferative effects of AzaC (a) p21 mRNA is significantly upregulated in CD4+ and CD8+ Teff following treatment with AzaC. Teffs were isolated from the spleens of B6. Foxp3 GFP × B6.CAG DSRED and nTregs were isolated from B6. Foxp3 GFP . Cells were co-cultured at a 1:10 ratio of nTregs to Teffs for 2 days in the presence of anti-CD3/CD28 beads (bead:cell 1:1; Invitrogen) and Xcyte medium supplemented with L-glutamine (4 mM), penicillin (100 U/mL), streptomycin (100 μg/mL), and human recombinant IL-2 (hIL-2; 500 U/mL). The activated T cells were cultured with AzaC (1 μM) or PBS for an additional 2 days. Cells were sorted using FACS Aria II (BD) to isolate nTregs (CD4+DSRED-FOXP3GFP+), CD4+ Teffs (CD4+DSRed+FOXP3GFP-), and CD8+ Teffs (CD8+DSRed+FOXP3GFP-) prior to RNA extraction. QPCR was performed on the Applied Biosystems StepOnePlus Real-Time System using pre-designed TaqMan® Gene Expression Assays (18S RNA Mm03928990 and p21 Mm04205640). Relative fold changes in expression were determined using the ΔΔCT method. AzaC treatment resulted in a 3.4 fold increase of p21 expression in CD4+ Teffs (FACS sorted to remove AzaC converted Tregs) (AzaC vs. PBS p
Figure Legend Snippet: p21-/- attenuates the antiproliferative effects of AzaC (a) p21 mRNA is significantly upregulated in CD4+ and CD8+ Teff following treatment with AzaC. Teffs were isolated from the spleens of B6. Foxp3 GFP × B6.CAG DSRED and nTregs were isolated from B6. Foxp3 GFP . Cells were co-cultured at a 1:10 ratio of nTregs to Teffs for 2 days in the presence of anti-CD3/CD28 beads (bead:cell 1:1; Invitrogen) and Xcyte medium supplemented with L-glutamine (4 mM), penicillin (100 U/mL), streptomycin (100 μg/mL), and human recombinant IL-2 (hIL-2; 500 U/mL). The activated T cells were cultured with AzaC (1 μM) or PBS for an additional 2 days. Cells were sorted using FACS Aria II (BD) to isolate nTregs (CD4+DSRED-FOXP3GFP+), CD4+ Teffs (CD4+DSRed+FOXP3GFP-), and CD8+ Teffs (CD8+DSRed+FOXP3GFP-) prior to RNA extraction. QPCR was performed on the Applied Biosystems StepOnePlus Real-Time System using pre-designed TaqMan® Gene Expression Assays (18S RNA Mm03928990 and p21 Mm04205640). Relative fold changes in expression were determined using the ΔΔCT method. AzaC treatment resulted in a 3.4 fold increase of p21 expression in CD4+ Teffs (FACS sorted to remove AzaC converted Tregs) (AzaC vs. PBS p

Techniques Used: Isolation, Cell Culture, Recombinant, FACS, RNA Extraction, Real-time Polymerase Chain Reaction, Expressing

33) Product Images from "Glycosylation Status of CD43 Protein Is Associated with Resistance of Leukemia Cells to CTL-Mediated Cytolysis"

Article Title: Glycosylation Status of CD43 Protein Is Associated with Resistance of Leukemia Cells to CTL-Mediated Cytolysis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0152326

Glycosylation status of CD43 on leukemia cells is associated with sensitivity to CTL-mediated cytolysis. (A) Gating strategies for FACS-sorting the R54 high and R54 low subpopulations of OVA-expressing MLL/AF9 leukemia cells. (B) FACS analysis of intracellular IFN-γ in OT-1 T cells after co-culture with either R54 high or R54 low MLL/AF9 leukemia cells. IFN-γ expression in CD8 + T cells is shown. (C) 51 Cr cytotoxicity assay with OT-1 T cells, using either R54 high or R54 low leukemia cells as targets. (D) FACS analysis of OVA-IRES-GFP expression levels in MLL/AF9-OVA leukemia clones derived from c-kit + BM cells of the wild type or CD43 -/- mouse (E) 51 Cr cytotoxicity assay with OT-1 T cells, using either the wild type or CD43 -/- leukemia cells as targets (F) 51 Cr cytotoxicity assay with OT-1 T cells, using leukemia cells with or without sialidase treatment (E/T ratio = 1).
Figure Legend Snippet: Glycosylation status of CD43 on leukemia cells is associated with sensitivity to CTL-mediated cytolysis. (A) Gating strategies for FACS-sorting the R54 high and R54 low subpopulations of OVA-expressing MLL/AF9 leukemia cells. (B) FACS analysis of intracellular IFN-γ in OT-1 T cells after co-culture with either R54 high or R54 low MLL/AF9 leukemia cells. IFN-γ expression in CD8 + T cells is shown. (C) 51 Cr cytotoxicity assay with OT-1 T cells, using either R54 high or R54 low leukemia cells as targets. (D) FACS analysis of OVA-IRES-GFP expression levels in MLL/AF9-OVA leukemia clones derived from c-kit + BM cells of the wild type or CD43 -/- mouse (E) 51 Cr cytotoxicity assay with OT-1 T cells, using either the wild type or CD43 -/- leukemia cells as targets (F) 51 Cr cytotoxicity assay with OT-1 T cells, using leukemia cells with or without sialidase treatment (E/T ratio = 1).

Techniques Used: CTL Assay, FACS, Expressing, Co-Culture Assay, Cytotoxicity Assay, Clone Assay, Derivative Assay

34) Product Images from "Mechanisms of Tumor-Induced Lymphovascular Niche Formation in Draining Lymph Nodes"

Article Title: Mechanisms of Tumor-Induced Lymphovascular Niche Formation in Draining Lymph Nodes

Journal: Cell Reports

doi: 10.1016/j.celrep.2018.12.002

Integrin αIIb Is Upregulated on LECs in Tumor-Draining LNs and Mediates Adhesion of LN LECs to Fibrinogen In Vitro (A) Representative images and quantification of integrin αIIb immunofluorescence stainings. Each dot represents one image; n = 8 LNs/group. (B) FACS analysis of integrin-αIIb-positive LECs in 4T1 tumor-draining LNs. n = 3 LNs/group. (C) Representative images of integrin αIIb expression in the subcapsular sinus of a human melanoma-draining LN. (D) FACS analysis of integrin αIIb expression on primary murine LN LECs in vitro after stimulation for 24 hr. Points represent biological replicates (n = 2–3). (E) FACS analysis of integrin αIIb expression by LN LECs in vivo in 4T1-bearing mice on day 10 after tumor implantation in response to VEGFR-3 and IFN-γ blockage. n = 3–4 mice/group. (F) Matrix adhesion assay of primary murine LN LECs under integrin αIIbβ3 blockage in vitro . Points represent biological replicates (n = 10). (G) LYVE-1/fibrinogen immunofluorescence stainings of naive and tumor-draining LNs. Statistical significance was determined by the unpaired Student’s t test (A, B, D [each group compared to the unstimulated control] and F) and one-way ANOVA (E). Data are shown as mean with SD and differences were considered statistically significant when p
Figure Legend Snippet: Integrin αIIb Is Upregulated on LECs in Tumor-Draining LNs and Mediates Adhesion of LN LECs to Fibrinogen In Vitro (A) Representative images and quantification of integrin αIIb immunofluorescence stainings. Each dot represents one image; n = 8 LNs/group. (B) FACS analysis of integrin-αIIb-positive LECs in 4T1 tumor-draining LNs. n = 3 LNs/group. (C) Representative images of integrin αIIb expression in the subcapsular sinus of a human melanoma-draining LN. (D) FACS analysis of integrin αIIb expression on primary murine LN LECs in vitro after stimulation for 24 hr. Points represent biological replicates (n = 2–3). (E) FACS analysis of integrin αIIb expression by LN LECs in vivo in 4T1-bearing mice on day 10 after tumor implantation in response to VEGFR-3 and IFN-γ blockage. n = 3–4 mice/group. (F) Matrix adhesion assay of primary murine LN LECs under integrin αIIbβ3 blockage in vitro . Points represent biological replicates (n = 10). (G) LYVE-1/fibrinogen immunofluorescence stainings of naive and tumor-draining LNs. Statistical significance was determined by the unpaired Student’s t test (A, B, D [each group compared to the unstimulated control] and F) and one-way ANOVA (E). Data are shown as mean with SD and differences were considered statistically significant when p

Techniques Used: In Vitro, Immunofluorescence, FACS, Expressing, In Vivo, Mouse Assay, Tumor Implantation, Cell Adhesion Assay

Lymphatic Network Expansion in Tumor-Draining LNs Is Mediated by LEC Sprouting and Proliferation (A) 4T1 primary tumor growth and ex vivo inguinal LN weight. (B and C) Maximum intensity projections of 3D light-sheet-microscope images of whole LNs stained for the lymphatic marker LYVE-1 (B) at different times or (C) 10 days after 4T1 injection. (D) FACS quantification of LECs in naive and 4T1 tumor-draining LNs at day 10. (E and F) Immunofluorescence staining of 4T1 tumor-draining LNs for the lymphatic markers Prox1 and LYVE-1 and the proliferation marker Ki67. (E) Representative image of a tumor-draining LN with arrowheads indicating proliferating LECs. (F) Schematic of the analyzed LN areas (left) and quantification of LEC proliferation in these areas (right). Statistical significance was determined by (A) two-way ANOVA, (D) unpaired Student’s t test, or (F) one-way ANOVA. Data are shown as mean with SD and differences were considered statistically significant when p
Figure Legend Snippet: Lymphatic Network Expansion in Tumor-Draining LNs Is Mediated by LEC Sprouting and Proliferation (A) 4T1 primary tumor growth and ex vivo inguinal LN weight. (B and C) Maximum intensity projections of 3D light-sheet-microscope images of whole LNs stained for the lymphatic marker LYVE-1 (B) at different times or (C) 10 days after 4T1 injection. (D) FACS quantification of LECs in naive and 4T1 tumor-draining LNs at day 10. (E and F) Immunofluorescence staining of 4T1 tumor-draining LNs for the lymphatic markers Prox1 and LYVE-1 and the proliferation marker Ki67. (E) Representative image of a tumor-draining LN with arrowheads indicating proliferating LECs. (F) Schematic of the analyzed LN areas (left) and quantification of LEC proliferation in these areas (right). Statistical significance was determined by (A) two-way ANOVA, (D) unpaired Student’s t test, or (F) one-way ANOVA. Data are shown as mean with SD and differences were considered statistically significant when p

Techniques Used: Ex Vivo, Microscopy, Staining, Marker, Injection, FACS, Immunofluorescence

RNA Sequencing Reveals a Distinct Expression Profile of LECs from Tumor-Draining LNs (A) Schematic workflow for RNA sequencing of LN LECs. (B) Representative FACS plot showing the gating of LN LECs. (C) Principal-component analysis (PCA) of the sequenced LEC samples based on all genes (left) and differentially expressed genes (right). (D) Venn diagrams with the number of differentially expressed genes in LECs from tumor-draining LNs compared to naive in both tumor models. See also Figure S3 and Table S1 .
Figure Legend Snippet: RNA Sequencing Reveals a Distinct Expression Profile of LECs from Tumor-Draining LNs (A) Schematic workflow for RNA sequencing of LN LECs. (B) Representative FACS plot showing the gating of LN LECs. (C) Principal-component analysis (PCA) of the sequenced LEC samples based on all genes (left) and differentially expressed genes (right). (D) Venn diagrams with the number of differentially expressed genes in LECs from tumor-draining LNs compared to naive in both tumor models. See also Figure S3 and Table S1 .

Techniques Used: RNA Sequencing Assay, Expressing, FACS

35) Product Images from "Up-Regulation of Multiple CD8+ T Cell Exhaustion Pathways is Associated to Recurrent Ocular Herpes Simplex Virus Type 1 Infection"

Article Title: Up-Regulation of Multiple CD8+ T Cell Exhaustion Pathways is Associated to Recurrent Ocular Herpes Simplex Virus Type 1 Infection

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.2000131

In vitro blockade of LAG-3 and/or PD-1 immune checkpoints and reversion of functional exhaustion of HSV-1 VP11/12 220–228 -specific CD8 + T cells from SYMP individuals. ( A ) Representative FACS plots ( left panels ) and average percentages ( right panels ) of HSV-1 VP11/12 220–228 -specific CD8 + T cells expressing PD-1 + , LAG-3 + and LAG-3 + /PD-1 + from 5 ASYMP and 5 SYMP individuals, following in vitro blockade of the LAG-3 and/or PD-1 immune checkpoints. ( B ) Levels of expression and co-expression of LAG-3 and PD-1 on HSV-1 VP11/12 220–228 -specific CD8 + T cells following in vitro blockade of LAG-3 and/or PD-1 immune checkpoints. ( C ) Representative FACS plots ( left panels ) and average percentages ( right panels ) of functional HSV-1 VP11/12 220–228 -specific IFN-γ + CD8 + and CD107 a/b+ CD8 + T cells from 5 ASYMP and 5 SYMP individuals, following in vitro blockade of LAG-3 and/or PD-1 immune checkpoints. Each staining was performed in duplicate. The indicated P values, determined using unpaired t test, compared the frequency and function of CD8 + T cells, following LAG-3 and/or PD-1 blocking mAbs with isotype control mAb.
Figure Legend Snippet: In vitro blockade of LAG-3 and/or PD-1 immune checkpoints and reversion of functional exhaustion of HSV-1 VP11/12 220–228 -specific CD8 + T cells from SYMP individuals. ( A ) Representative FACS plots ( left panels ) and average percentages ( right panels ) of HSV-1 VP11/12 220–228 -specific CD8 + T cells expressing PD-1 + , LAG-3 + and LAG-3 + /PD-1 + from 5 ASYMP and 5 SYMP individuals, following in vitro blockade of the LAG-3 and/or PD-1 immune checkpoints. ( B ) Levels of expression and co-expression of LAG-3 and PD-1 on HSV-1 VP11/12 220–228 -specific CD8 + T cells following in vitro blockade of LAG-3 and/or PD-1 immune checkpoints. ( C ) Representative FACS plots ( left panels ) and average percentages ( right panels ) of functional HSV-1 VP11/12 220–228 -specific IFN-γ + CD8 + and CD107 a/b+ CD8 + T cells from 5 ASYMP and 5 SYMP individuals, following in vitro blockade of LAG-3 and/or PD-1 immune checkpoints. Each staining was performed in duplicate. The indicated P values, determined using unpaired t test, compared the frequency and function of CD8 + T cells, following LAG-3 and/or PD-1 blocking mAbs with isotype control mAb.

Techniques Used: In Vitro, Functional Assay, FACS, Expressing, Staining, Blocking Assay

Differential expression of genes for major exhaustion pathways in HSV-specific CD8 + T cells from HSV-1 seropositive symptomatic vs. asymptomatic individuals: ( A ) Experimental design and validation of differentially expressed genes in CD8 + T cells sharing the same HSV-1 epitope-specificities, from SYMP and ASYMP individuals. CD8 + T cells specific to HLA-A*0201-restricted HSV-1 VP11/12 220–228 epitope (RLNELLAYV) were sorted from HLA-A*0201-positive SYMP and ASYMP individuals. Total RNA was extracted from each clone of epitope-specific CD8 + T cells, and whole transcriptome analysis was performed using bulk RNA sequencing and NanoString digital barcoding technology to compare the expression levels of 26,000 genes. ( B ) Representative FACS plot of the frequencies of HSV-1 VP11/12 220–228 -specific CD8 + T cells in HSV-1 seropositive SYMP vs. ASYMP individuals and in seronegative healthy individuals ( HSV (–) ) ( upper panels ). Average percentages of HSV-1 VP11/12 220–228 -specific CD8 + T cells in 10 SYMP vs. 10 ASYMP individuals ( lower panels ). PBMCs from 5 HSV (–) seronegative individuals were used as control of the tetramer staining ( C ) Bulk RNA sequencing heat map showing differentially expressed immune genes (DEGs) in VP11/12 220–228 epitope-specific CD8 + T cells from sex- and age-matched HSV-1 seropositive SYMP vs. ASYMP individuals. ( D ) Average expression of eight major exhaustion receptor genes detected through RNA sequencing in VP11/12 220–228 -specific CD8 + T cells from ASYMP and SYMP individuals ( E ) Expression of twelve exhaustion-related genes detected using NanoString technology in CD8 + T cells, that shared the same gB 561–567 , VP11/12 220–228 , UL43 302–310 and UL44 400–408 epitopes specificities, sorted from 10 SYMP vs. ASYMP individuals. The indicated P values, determined using unpaired t test, compared differential gene expression between SYMP and ASYMP individuals.
Figure Legend Snippet: Differential expression of genes for major exhaustion pathways in HSV-specific CD8 + T cells from HSV-1 seropositive symptomatic vs. asymptomatic individuals: ( A ) Experimental design and validation of differentially expressed genes in CD8 + T cells sharing the same HSV-1 epitope-specificities, from SYMP and ASYMP individuals. CD8 + T cells specific to HLA-A*0201-restricted HSV-1 VP11/12 220–228 epitope (RLNELLAYV) were sorted from HLA-A*0201-positive SYMP and ASYMP individuals. Total RNA was extracted from each clone of epitope-specific CD8 + T cells, and whole transcriptome analysis was performed using bulk RNA sequencing and NanoString digital barcoding technology to compare the expression levels of 26,000 genes. ( B ) Representative FACS plot of the frequencies of HSV-1 VP11/12 220–228 -specific CD8 + T cells in HSV-1 seropositive SYMP vs. ASYMP individuals and in seronegative healthy individuals ( HSV (–) ) ( upper panels ). Average percentages of HSV-1 VP11/12 220–228 -specific CD8 + T cells in 10 SYMP vs. 10 ASYMP individuals ( lower panels ). PBMCs from 5 HSV (–) seronegative individuals were used as control of the tetramer staining ( C ) Bulk RNA sequencing heat map showing differentially expressed immune genes (DEGs) in VP11/12 220–228 epitope-specific CD8 + T cells from sex- and age-matched HSV-1 seropositive SYMP vs. ASYMP individuals. ( D ) Average expression of eight major exhaustion receptor genes detected through RNA sequencing in VP11/12 220–228 -specific CD8 + T cells from ASYMP and SYMP individuals ( E ) Expression of twelve exhaustion-related genes detected using NanoString technology in CD8 + T cells, that shared the same gB 561–567 , VP11/12 220–228 , UL43 302–310 and UL44 400–408 epitopes specificities, sorted from 10 SYMP vs. ASYMP individuals. The indicated P values, determined using unpaired t test, compared differential gene expression between SYMP and ASYMP individuals.

Techniques Used: Expressing, RNA Sequencing Assay, FACS, Staining

Frequency and function of HSV-1 VP11/12 220–228 epitope-specific CD8 + T cells, co-expressing LAG-3 with PD-1, TIGIT and TIM-3 receptors, in SYMP vs. ASYMP individuals. ( A ) Representative FACS plots of the frequencies of HSV-1 VP11/12 220–228 -specific CD8 + T cells ( upper row ) or control EBV BMFL-1 280–288 -specific CD8 + T cells (lower row) co-expressing LAG-3/PD-1, LAG-3/TIGIT and LAG-3/TIM-3 in SYMP and ASYMP individuals. On the right : FACS plots representing the isotype controls staining. ( B ) Average percentages ( upper row ) and absolute numbers ( lower row ) of HSV-1 VP11/12 220–228 -specific CD8 + T cells ( left panels ) and control EBV BMLF-1 280–288 -specific CD8 + T cells ( right panels ) co-expressing LAG-3/PD-1, LAG-3/TIGIT and LAG-3/TIM-3 in SYMP and ASYMP individuals. Representative FACS plots of the HSV-1 (VP11/12 220–228 ) and EBV (BMFL-1 280–288 ) tetramer staining are displayed on the top of each left and right panel, respectively. ( C ) Representative FACS plots ( left panels ) and average ( right panels ) frequencies of functional HSV-1 VP11/12 220–228 -specific CD8 + T cells expressing IFN-γ, CD107 a/b and Ki-67 from SYMP vs. ASYMP individuals. ( D ) Representative FACS plots ( top panels ) and average percentages ( lower panels ) of functional HSV-1 VP11/12 220–228 -specific IFN-γ + CD8 + T cells gated on the LAG-3 – , LAG-3 + , LAG-3 + /PD-1 + , LAG-3 + /TIGIT + and LAG-3 + /TIM-3 + T cell populations. Each staining was performed in duplicate. The indicated P values, determined using unpaired t test, compared expression of exhaustion receptors between SYMP and ASYMP individuals (B and C) and between cell populations (D).
Figure Legend Snippet: Frequency and function of HSV-1 VP11/12 220–228 epitope-specific CD8 + T cells, co-expressing LAG-3 with PD-1, TIGIT and TIM-3 receptors, in SYMP vs. ASYMP individuals. ( A ) Representative FACS plots of the frequencies of HSV-1 VP11/12 220–228 -specific CD8 + T cells ( upper row ) or control EBV BMFL-1 280–288 -specific CD8 + T cells (lower row) co-expressing LAG-3/PD-1, LAG-3/TIGIT and LAG-3/TIM-3 in SYMP and ASYMP individuals. On the right : FACS plots representing the isotype controls staining. ( B ) Average percentages ( upper row ) and absolute numbers ( lower row ) of HSV-1 VP11/12 220–228 -specific CD8 + T cells ( left panels ) and control EBV BMLF-1 280–288 -specific CD8 + T cells ( right panels ) co-expressing LAG-3/PD-1, LAG-3/TIGIT and LAG-3/TIM-3 in SYMP and ASYMP individuals. Representative FACS plots of the HSV-1 (VP11/12 220–228 ) and EBV (BMFL-1 280–288 ) tetramer staining are displayed on the top of each left and right panel, respectively. ( C ) Representative FACS plots ( left panels ) and average ( right panels ) frequencies of functional HSV-1 VP11/12 220–228 -specific CD8 + T cells expressing IFN-γ, CD107 a/b and Ki-67 from SYMP vs. ASYMP individuals. ( D ) Representative FACS plots ( top panels ) and average percentages ( lower panels ) of functional HSV-1 VP11/12 220–228 -specific IFN-γ + CD8 + T cells gated on the LAG-3 – , LAG-3 + , LAG-3 + /PD-1 + , LAG-3 + /TIGIT + and LAG-3 + /TIM-3 + T cell populations. Each staining was performed in duplicate. The indicated P values, determined using unpaired t test, compared expression of exhaustion receptors between SYMP and ASYMP individuals (B and C) and between cell populations (D).

Techniques Used: Expressing, FACS, Staining, Functional Assay

Effect of in vivo blockade of PD-1 and/or LAG-3 immune checkpoints on recurrent ocular herpes infection and disease. ( A ) Schematic representation of HSV-1 ocular infection, α-PD-1 and/or α-LAG-3 blockade treatment, UV-B induced reactivation, virological and immunological analyses in HSV-1 infected HLA-A*0201 Tg mice ( n = 40; 10 per condition of blockade treatment). ( B ) Cumulative recurrent corneal herpetic lesions ( left panel ) and cumulative number of eyes with recurrent corneal lesions ( left panel ) detected 30 days post UV-B exposure. The severity of corneal herpetic lesions was scored on a scale of 0 to 4. ( C ) Average scores of recurrent corneal disease detected 30 days post UV-B exposure. ( D ) Average titers of the virus shed in the cornea of treated and untreated HSV-1 infected HLA-A*0201 Tg mice. ( E ) Representative images of recurrent corneal herpetic disease taken from treated and untreated HSV-1 infected HLA-A*0201 Tg mice 20 days post UV-B induced reactivation. ( F and G ) Representative FACS plots ( left panels ) and average percentages and numbers ( right panels ) of functional HSV-1 VP11/12 220–228 -specific IFN-γ + CD8 + , CD107 a/b+ CD8 + and Ki67 + CD8 + T cells detected in the cornea ( F ) and TG ( G ) of treated and untreated HSV-1 latently infected HLA-A*0201 Tg mice. Results are representative of two independent experiments. The indicated P values, calculated using the unpaired t -test, show statistical significance between mAb treated and untreated control groups.
Figure Legend Snippet: Effect of in vivo blockade of PD-1 and/or LAG-3 immune checkpoints on recurrent ocular herpes infection and disease. ( A ) Schematic representation of HSV-1 ocular infection, α-PD-1 and/or α-LAG-3 blockade treatment, UV-B induced reactivation, virological and immunological analyses in HSV-1 infected HLA-A*0201 Tg mice ( n = 40; 10 per condition of blockade treatment). ( B ) Cumulative recurrent corneal herpetic lesions ( left panel ) and cumulative number of eyes with recurrent corneal lesions ( left panel ) detected 30 days post UV-B exposure. The severity of corneal herpetic lesions was scored on a scale of 0 to 4. ( C ) Average scores of recurrent corneal disease detected 30 days post UV-B exposure. ( D ) Average titers of the virus shed in the cornea of treated and untreated HSV-1 infected HLA-A*0201 Tg mice. ( E ) Representative images of recurrent corneal herpetic disease taken from treated and untreated HSV-1 infected HLA-A*0201 Tg mice 20 days post UV-B induced reactivation. ( F and G ) Representative FACS plots ( left panels ) and average percentages and numbers ( right panels ) of functional HSV-1 VP11/12 220–228 -specific IFN-γ + CD8 + , CD107 a/b+ CD8 + and Ki67 + CD8 + T cells detected in the cornea ( F ) and TG ( G ) of treated and untreated HSV-1 latently infected HLA-A*0201 Tg mice. Results are representative of two independent experiments. The indicated P values, calculated using the unpaired t -test, show statistical significance between mAb treated and untreated control groups.

Techniques Used: In Vivo, Infection, Mouse Assay, FACS, Functional Assay

Cell surface expression of exhaustion receptors by HSV VP11/12 220–228 epitope-specific CD8 + T cells from symptomatic vs. asymptomatic individuals: ( A ) Experimental design for differentially expressed exhaustion molecules detected by FACS on the surface of CD8 + T cells sharing the same HSV-1 epitope-specificities, from SYMP and ASYMP individuals. PBMCs from 10 SYMP and 10 ASYMP individuals were stained for CD3/CD8, VP11/12 220–228 . ( B ) Representative FACS plots and ( C ) average frequencies of HSV-1 VP11/12 220–228 -specific CD8 + T cells expressing PD-1, LAG-3, TIGIT, TIM-3, PSGL-1, GITR, BTLA and CTLA-4 receptors, detected from SYMP vs. ASYMP individuals. ( D ) Levels of expression of the eight exhaustion receptors on HSV-1 VP11/12 220–228 -specific CD8 + T cells from SYMP and ASYMP individuals, depicted as Mean fluorescent Intensity (MFI). Each staining was performed in duplicate. The data are expressed as means +/− the standard deviations (SD). The indicated P values, determined using unpaired t test, compared expression of exhaustion receptors between SYMP and ASYMP individuals.
Figure Legend Snippet: Cell surface expression of exhaustion receptors by HSV VP11/12 220–228 epitope-specific CD8 + T cells from symptomatic vs. asymptomatic individuals: ( A ) Experimental design for differentially expressed exhaustion molecules detected by FACS on the surface of CD8 + T cells sharing the same HSV-1 epitope-specificities, from SYMP and ASYMP individuals. PBMCs from 10 SYMP and 10 ASYMP individuals were stained for CD3/CD8, VP11/12 220–228 . ( B ) Representative FACS plots and ( C ) average frequencies of HSV-1 VP11/12 220–228 -specific CD8 + T cells expressing PD-1, LAG-3, TIGIT, TIM-3, PSGL-1, GITR, BTLA and CTLA-4 receptors, detected from SYMP vs. ASYMP individuals. ( D ) Levels of expression of the eight exhaustion receptors on HSV-1 VP11/12 220–228 -specific CD8 + T cells from SYMP and ASYMP individuals, depicted as Mean fluorescent Intensity (MFI). Each staining was performed in duplicate. The data are expressed as means +/− the standard deviations (SD). The indicated P values, determined using unpaired t test, compared expression of exhaustion receptors between SYMP and ASYMP individuals.

Techniques Used: Expressing, FACS, Staining

36) Product Images from "Modulation of macrophage polarization and lung cancer cell stemness by MUC1 and development of a related small-molecule inhibitor pterostilbene"

Article Title: Modulation of macrophage polarization and lung cancer cell stemness by MUC1 and development of a related small-molecule inhibitor pterostilbene

Journal: Oncotarget

doi: 10.18632/oncotarget.8101

Pterostilbene treatment prevented M2 polarization via down-regulation of Muc1/NF-κB signaling axis A. Pterostilbene treatment suppressed the Muc1 and NF-κB expression in M2 polarization of THP-1 cells. B . Muc1, NF-κB and VEGF mRNA expression levels in M2 polarization of THP-1 cells were significantly decreased after co-culturing with pterostilbene in H441 cells. C . FACS analysis indicated that in the presence of pterostilbene, the percentage of ALDH subpopulation cells in TAM co-cultured H441 was significantly decreased.
Figure Legend Snippet: Pterostilbene treatment prevented M2 polarization via down-regulation of Muc1/NF-κB signaling axis A. Pterostilbene treatment suppressed the Muc1 and NF-κB expression in M2 polarization of THP-1 cells. B . Muc1, NF-κB and VEGF mRNA expression levels in M2 polarization of THP-1 cells were significantly decreased after co-culturing with pterostilbene in H441 cells. C . FACS analysis indicated that in the presence of pterostilbene, the percentage of ALDH subpopulation cells in TAM co-cultured H441 was significantly decreased.

Techniques Used: Expressing, FACS, Cell Culture

37) Product Images from "Monocyte-Derived Signals Activate Human Natural Killer Cells in Response to Leishmania Parasites"

Article Title: Monocyte-Derived Signals Activate Human Natural Killer Cells in Response to Leishmania Parasites

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00024

Coculture of peripheral blood mononuclear cells (PBMCs) with Leishmania neither induced natural killer (NK) cell interferon (IFN)-γ response nor NK cell cytotoxicity. Human PBMCs were cocultured with Leishmania spp. promastigotes (multiplicity of infection 10) and/or interleukin (IL)-12 and IL-18 (10 ng/ml each) for 20 h. IFN-γ production was determined by (A,B) ELISA of cell culture supernatants or (B) flow cytometry of intracellular IFN-γ in NKp46 + CD3 − NK cells. (C) NK cell cytotoxicity was determined by measurement of specific lysis of 51 Cr-labeled K562 tumor cells in a chromium-release assay. (A) Mean ± SEM of 76/71/55/46/74 donors for the five stimulations. (B) Mean ± SEM of 17/14/15/8/17 donors for the five stimulations; FACS plots show results of one representative donor. (C) Mean ± SEM of six (PBMC) or nine (PBMC + IL-12/18) donors. * ,# p
Figure Legend Snippet: Coculture of peripheral blood mononuclear cells (PBMCs) with Leishmania neither induced natural killer (NK) cell interferon (IFN)-γ response nor NK cell cytotoxicity. Human PBMCs were cocultured with Leishmania spp. promastigotes (multiplicity of infection 10) and/or interleukin (IL)-12 and IL-18 (10 ng/ml each) for 20 h. IFN-γ production was determined by (A,B) ELISA of cell culture supernatants or (B) flow cytometry of intracellular IFN-γ in NKp46 + CD3 − NK cells. (C) NK cell cytotoxicity was determined by measurement of specific lysis of 51 Cr-labeled K562 tumor cells in a chromium-release assay. (A) Mean ± SEM of 76/71/55/46/74 donors for the five stimulations. (B) Mean ± SEM of 17/14/15/8/17 donors for the five stimulations; FACS plots show results of one representative donor. (C) Mean ± SEM of six (PBMC) or nine (PBMC + IL-12/18) donors. * ,# p

Techniques Used: Infection, Enzyme-linked Immunosorbent Assay, Cell Culture, Flow Cytometry, Cytometry, Lysis, Labeling, Release Assay, FACS

CD69 is upregulated on natural killer (NK) cells after contact with Leishmania . Human peripheral blood mononuclear cells were cocultured for 20 h with Leishmania promastigotes of different species (A) , strains (B) , amounts (C) , or integrity (D) , and with or without host cell/parasite contact (E) or interleukin (IL)-12 and IL-18 (10 ng/ml) (A,B) , followed by surface expression analysis of CD69 on NKp46 + CD3 − NK cells by flow cytometry. Unless otherwise indicated the multiplicity of infection (MOI) was 10. (A) Results of 129/117/107/83/21/88 blood samples for the six stimulations. Medians are indicated by red lines. FACS plots show results of NKp46 + CD3 − NK cells of one representative experiment. (B) Leishmania infantum strains isolated from human patients with visceral leishmaniasis (VL) (VL1, VL2) or CL or a dog [canine leishmaniasis (CanL)]. Mean ± SEM of 9/9/9/9/7/9 donors. (C) Mean ± SEM of 119/43/17/119 donors for the four stimulations. (D) Mean ± SEM of 22/22/18/18 (viable), 8/9/6 [paraformaldehyde (Pfa)-fixed], and 17/15/14 [freeze–thaw lysate (Ft-lysate)] donors. (E) Mean ± SEM of six donors. Abbreviation: TW, transwell. * ,# p
Figure Legend Snippet: CD69 is upregulated on natural killer (NK) cells after contact with Leishmania . Human peripheral blood mononuclear cells were cocultured for 20 h with Leishmania promastigotes of different species (A) , strains (B) , amounts (C) , or integrity (D) , and with or without host cell/parasite contact (E) or interleukin (IL)-12 and IL-18 (10 ng/ml) (A,B) , followed by surface expression analysis of CD69 on NKp46 + CD3 − NK cells by flow cytometry. Unless otherwise indicated the multiplicity of infection (MOI) was 10. (A) Results of 129/117/107/83/21/88 blood samples for the six stimulations. Medians are indicated by red lines. FACS plots show results of NKp46 + CD3 − NK cells of one representative experiment. (B) Leishmania infantum strains isolated from human patients with visceral leishmaniasis (VL) (VL1, VL2) or CL or a dog [canine leishmaniasis (CanL)]. Mean ± SEM of 9/9/9/9/7/9 donors. (C) Mean ± SEM of 119/43/17/119 donors for the four stimulations. (D) Mean ± SEM of 22/22/18/18 (viable), 8/9/6 [paraformaldehyde (Pfa)-fixed], and 17/15/14 [freeze–thaw lysate (Ft-lysate)] donors. (E) Mean ± SEM of six donors. Abbreviation: TW, transwell. * ,# p

Techniques Used: Expressing, Flow Cytometry, Cytometry, Infection, FACS, Isolation

38) Product Images from "Global gene expression profiles of canine macrophages and canine mammary cancer cells grown as a co-culture in vitro"

Article Title: Global gene expression profiles of canine macrophages and canine mammary cancer cells grown as a co-culture in vitro

Journal: BMC Veterinary Research

doi: 10.1186/1746-6148-8-16

FACS mononuclear blood cells and co-culture analysis and sorting . Cytograms and histograms obtained using FACS Aria II (Becton Dickinson, USA). ( A ) The cytogram of mononuclear cells obtained from canine blood after the Accuspin System-Histopaque 1077 centrifugation. The monocytes were gated based on the morphological criteria (SSC v/s FSC cytogram). ( B ) Histogram of CD64 stained leukocytes: CD64 negative (CD64-) leukocytes showed low FITC signal, whereas CD64 positive (CD64+) leukocytes showed high FITC signal. ( C ) The cytogram of CD64-positive cells showing antibodies specificity to monocytes. ( D ) Histogram of the fluorescence intensity of unstained macrophages grown as a single culture. ( E ) Histogram of the fluorescence intensity of CMTMR-stained cancer cells grown as the single culture. ( F ) Histogram and sorting gates of unstained macrophages and CMTMR-stained cancer cells grown as a co-culture for 72 hrs.
Figure Legend Snippet: FACS mononuclear blood cells and co-culture analysis and sorting . Cytograms and histograms obtained using FACS Aria II (Becton Dickinson, USA). ( A ) The cytogram of mononuclear cells obtained from canine blood after the Accuspin System-Histopaque 1077 centrifugation. The monocytes were gated based on the morphological criteria (SSC v/s FSC cytogram). ( B ) Histogram of CD64 stained leukocytes: CD64 negative (CD64-) leukocytes showed low FITC signal, whereas CD64 positive (CD64+) leukocytes showed high FITC signal. ( C ) The cytogram of CD64-positive cells showing antibodies specificity to monocytes. ( D ) Histogram of the fluorescence intensity of unstained macrophages grown as a single culture. ( E ) Histogram of the fluorescence intensity of CMTMR-stained cancer cells grown as the single culture. ( F ) Histogram and sorting gates of unstained macrophages and CMTMR-stained cancer cells grown as a co-culture for 72 hrs.

Techniques Used: FACS, Co-Culture Assay, Centrifugation, Staining, Fluorescence

39) Product Images from "Cardiovascular disease risk factors induce mesenchymal features and senescence in cardiac endothelial cells"

Article Title: Cardiovascular disease risk factors induce mesenchymal features and senescence in cardiac endothelial cells

Journal: bioRxiv

doi: 10.1101/2020.10.21.349472

Effects of exercise training, aging, obesity and pressure overload on cardiac endothelial cell number and vascular density. A-B. FACS analysis and quantification of mean fluorescence intensity (MFI) of the cardiac endothelial cells (CD31+CD140a-CD45-Ter119-DAPI-) in various mice models. C-D. Representative immunofluorescence images and quantification of CD31+ blood vessel area (%) in the heart. Scale bar, 100μm. Data is presented as mean ± SEM. Student’s t test was used, *p
Figure Legend Snippet: Effects of exercise training, aging, obesity and pressure overload on cardiac endothelial cell number and vascular density. A-B. FACS analysis and quantification of mean fluorescence intensity (MFI) of the cardiac endothelial cells (CD31+CD140a-CD45-Ter119-DAPI-) in various mice models. C-D. Representative immunofluorescence images and quantification of CD31+ blood vessel area (%) in the heart. Scale bar, 100μm. Data is presented as mean ± SEM. Student’s t test was used, *p

Techniques Used: FACS, Fluorescence, Mouse Assay, Immunofluorescence

FACS analysis of cardiac EC. A-D. Representative pseudocolor FACS plots showing the gating and percentage of cardiac ECs (CD31+ CD140a-CD45-Ter119-DAPI-) in the different treatment groups. In panel A-D, number of mice in each experimental group are indicated in the respective FACS plots.
Figure Legend Snippet: FACS analysis of cardiac EC. A-D. Representative pseudocolor FACS plots showing the gating and percentage of cardiac ECs (CD31+ CD140a-CD45-Ter119-DAPI-) in the different treatment groups. In panel A-D, number of mice in each experimental group are indicated in the respective FACS plots.

Techniques Used: FACS, Mouse Assay

40) Product Images from "Establishment of single-cell screening system for the rapid identification of transcriptional modulators involved in direct cell reprogramming"

Article Title: Establishment of single-cell screening system for the rapid identification of transcriptional modulators involved in direct cell reprogramming

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks732

Workflow of single-cell screening and Nested-single-cell-PCR (Nesc-PCR). ( A ) Lentviruses encoding multiple transcription ‘modulating’ factors (TMs) are pooled and transduced into human dermal fibroblasts for a period of 2 weeks. Because of the stochastic nature of virus infection, a variety of TM combinations are expressed at random per single cell. Cells expressing a defined set of TMs give rise to the expression of target-cell-specific markers (e.g. CD14 and HLA-DR) allowing them to be individually sorted using FACS. Subsequently, single cells are profiled using a Nesc-PCR gene expression analysis by reverse transcribing (RT) the exogenous transcripts with a virus-specific primer (primer 1) followed by 18 cycles of pre-amplification with gene-specific forward primers (primer 2) and primer 1. The cDNA products are then subjected to a microfluidic qPCR for the identification of exogenous transcripts using gene-specific sense (primer 2) and anti-sense (primer 3) primer pairs and fluorescence probes (yellow star) ( B ). ( C ) The single cells transduced with pooled lentivirus (18 factors) at 5 MOI and 10 MOI were sorted and profiled using Nesc-PCR. At 10 MOI, > 30% of single cells expressed 4 out of 18 TMs, while 20% of single cells expressed 2 out 18 TMs at 5 MOI.
Figure Legend Snippet: Workflow of single-cell screening and Nested-single-cell-PCR (Nesc-PCR). ( A ) Lentviruses encoding multiple transcription ‘modulating’ factors (TMs) are pooled and transduced into human dermal fibroblasts for a period of 2 weeks. Because of the stochastic nature of virus infection, a variety of TM combinations are expressed at random per single cell. Cells expressing a defined set of TMs give rise to the expression of target-cell-specific markers (e.g. CD14 and HLA-DR) allowing them to be individually sorted using FACS. Subsequently, single cells are profiled using a Nesc-PCR gene expression analysis by reverse transcribing (RT) the exogenous transcripts with a virus-specific primer (primer 1) followed by 18 cycles of pre-amplification with gene-specific forward primers (primer 2) and primer 1. The cDNA products are then subjected to a microfluidic qPCR for the identification of exogenous transcripts using gene-specific sense (primer 2) and anti-sense (primer 3) primer pairs and fluorescence probes (yellow star) ( B ). ( C ) The single cells transduced with pooled lentivirus (18 factors) at 5 MOI and 10 MOI were sorted and profiled using Nesc-PCR. At 10 MOI, > 30% of single cells expressed 4 out of 18 TMs, while 20% of single cells expressed 2 out 18 TMs at 5 MOI.

Techniques Used: Polymerase Chain Reaction, Infection, Expressing, FACS, Amplification, Real-time Polymerase Chain Reaction, Fluorescence, Transduction

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Article Title: Six-transmembrane epithelial antigen of the prostate 1 accelerates cell proliferation by targeting c-Myc in liver cancer cells
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Cell Culture:

Article Title: Six-transmembrane epithelial antigen of the prostate 1 accelerates cell proliferation by targeting c-Myc in liver cancer cells
Article Snippet: .. Liver cancer cells were seeded at a density of 3×105 cells/well into 6-well plates and cultured for 24 h. Subsequently, cells were transfected with control siRNA or an siRNA targeting human STEAP1, and incubated for 72 h. After incubation, floating cells in media were collected and adhesive cells were washed, fixed in ethanol, and stained with propidium iodide using a cell-cycle analysis kit (FxCycle PI/RNase Staining Solution; Thermo Fisher Scientific), followed by analysis on a BD FACS II (BD Biosciences) instrument using FACSDiva (BD Biosciences) as previously described ( ). ..

Transfection:

Article Title: Six-transmembrane epithelial antigen of the prostate 1 accelerates cell proliferation by targeting c-Myc in liver cancer cells
Article Snippet: .. Liver cancer cells were seeded at a density of 3×105 cells/well into 6-well plates and cultured for 24 h. Subsequently, cells were transfected with control siRNA or an siRNA targeting human STEAP1, and incubated for 72 h. After incubation, floating cells in media were collected and adhesive cells were washed, fixed in ethanol, and stained with propidium iodide using a cell-cycle analysis kit (FxCycle PI/RNase Staining Solution; Thermo Fisher Scientific), followed by analysis on a BD FACS II (BD Biosciences) instrument using FACSDiva (BD Biosciences) as previously described ( ). ..

Incubation:

Article Title: Six-transmembrane epithelial antigen of the prostate 1 accelerates cell proliferation by targeting c-Myc in liver cancer cells
Article Snippet: .. Liver cancer cells were seeded at a density of 3×105 cells/well into 6-well plates and cultured for 24 h. Subsequently, cells were transfected with control siRNA or an siRNA targeting human STEAP1, and incubated for 72 h. After incubation, floating cells in media were collected and adhesive cells were washed, fixed in ethanol, and stained with propidium iodide using a cell-cycle analysis kit (FxCycle PI/RNase Staining Solution; Thermo Fisher Scientific), followed by analysis on a BD FACS II (BD Biosciences) instrument using FACSDiva (BD Biosciences) as previously described ( ). ..

Staining:

Article Title: Six-transmembrane epithelial antigen of the prostate 1 accelerates cell proliferation by targeting c-Myc in liver cancer cells
Article Snippet: .. Liver cancer cells were seeded at a density of 3×105 cells/well into 6-well plates and cultured for 24 h. Subsequently, cells were transfected with control siRNA or an siRNA targeting human STEAP1, and incubated for 72 h. After incubation, floating cells in media were collected and adhesive cells were washed, fixed in ethanol, and stained with propidium iodide using a cell-cycle analysis kit (FxCycle PI/RNase Staining Solution; Thermo Fisher Scientific), followed by analysis on a BD FACS II (BD Biosciences) instrument using FACSDiva (BD Biosciences) as previously described ( ). ..

Cell Cycle Assay:

Article Title: Six-transmembrane epithelial antigen of the prostate 1 accelerates cell proliferation by targeting c-Myc in liver cancer cells
Article Snippet: .. Liver cancer cells were seeded at a density of 3×105 cells/well into 6-well plates and cultured for 24 h. Subsequently, cells were transfected with control siRNA or an siRNA targeting human STEAP1, and incubated for 72 h. After incubation, floating cells in media were collected and adhesive cells were washed, fixed in ethanol, and stained with propidium iodide using a cell-cycle analysis kit (FxCycle PI/RNase Staining Solution; Thermo Fisher Scientific), followed by analysis on a BD FACS II (BD Biosciences) instrument using FACSDiva (BD Biosciences) as previously described ( ). ..

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    Becton Dickinson bd facs aria ii
    Fluorescence analysis of 2-NBDG uptake by flow cytometry. <t>FACS</t> analysis of 2-NBDG uptake in differentiated L6 cells by plotting cell count against <t>FITC</t> revealed that 8%, 8.1% and 30% of cells uptake 2-NBDG in control, TBHP and Rosiglitazone treated cells respectively whereas 30.6%, 33.1%, 28%, 32% of cells uptake 2-NBDG, pretreated with two different concentrations (10 and 100 μM) of Naringin along with/without TBHP respectively. Each value represents mean ± SD (standard deviation) from triplicate measurements (n = 3) of three different experiments. Significance test between various groups was determined by using one way ANOVA followed by Duncan’s multiple range test. * P≤0.05 versus control.
    Bd Facs Aria Ii, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson facs aria ii flow cytometer
    Depicts mitochondrial reactive oxygen species (mtROS) content in study subjects. US-unstained (autofluorescence). (A) Representative <t>FACS</t> image of mitosox fluorescence intensity (B) bar graph represents delta mean fluorescence in controls (GP-I); prediabetic subjects (GP-II); NDT2DM (GP-III); and ADT2DM patients (GP-IV). Values are expressed in median and interquartile range ( n = 20). (* = vs. GP-I), ( # = vs. GP-II), ( $ = vs. GP-III), * p
    Facs Aria Ii Flow Cytometer, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson facs aria ii
    CD3 + <t>CD4</t> + cells were isolated from L-Tg-FIR and FIR mice and stained for Foxp3 expression. (A) Representative <t>FACS</t> plot depicts percent Foxp3 expression from WT and L-Tg splenocytes. (B) MFI of Foxp3 expression is plotted from different tissues taken from
    Facs Aria Ii, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fluorescence analysis of 2-NBDG uptake by flow cytometry. FACS analysis of 2-NBDG uptake in differentiated L6 cells by plotting cell count against FITC revealed that 8%, 8.1% and 30% of cells uptake 2-NBDG in control, TBHP and Rosiglitazone treated cells respectively whereas 30.6%, 33.1%, 28%, 32% of cells uptake 2-NBDG, pretreated with two different concentrations (10 and 100 μM) of Naringin along with/without TBHP respectively. Each value represents mean ± SD (standard deviation) from triplicate measurements (n = 3) of three different experiments. Significance test between various groups was determined by using one way ANOVA followed by Duncan’s multiple range test. * P≤0.05 versus control.

    Journal: PLoS ONE

    Article Title: Preconditioning L6 Muscle Cells with Naringin Ameliorates Oxidative Stress and Increases Glucose Uptake

    doi: 10.1371/journal.pone.0132429

    Figure Lengend Snippet: Fluorescence analysis of 2-NBDG uptake by flow cytometry. FACS analysis of 2-NBDG uptake in differentiated L6 cells by plotting cell count against FITC revealed that 8%, 8.1% and 30% of cells uptake 2-NBDG in control, TBHP and Rosiglitazone treated cells respectively whereas 30.6%, 33.1%, 28%, 32% of cells uptake 2-NBDG, pretreated with two different concentrations (10 and 100 μM) of Naringin along with/without TBHP respectively. Each value represents mean ± SD (standard deviation) from triplicate measurements (n = 3) of three different experiments. Significance test between various groups was determined by using one way ANOVA followed by Duncan’s multiple range test. * P≤0.05 versus control.

    Article Snippet: Samples were analyzed using BD FACS Aria II (BD Biosciences) at FITC range (excitation 490 nm, emission 525 nm band pass filter).

    Techniques: Fluorescence, Flow Cytometry, Cytometry, FACS, Cell Counting, Standard Deviation

    Depicts mitochondrial reactive oxygen species (mtROS) content in study subjects. US-unstained (autofluorescence). (A) Representative FACS image of mitosox fluorescence intensity (B) bar graph represents delta mean fluorescence in controls (GP-I); prediabetic subjects (GP-II); NDT2DM (GP-III); and ADT2DM patients (GP-IV). Values are expressed in median and interquartile range ( n = 20). (* = vs. GP-I), ( # = vs. GP-II), ( $ = vs. GP-III), * p

    Journal: Frontiers in Endocrinology

    Article Title: Alterations in Mitochondrial Oxidative Stress and Mitophagy in Subjects with Prediabetes and Type 2 Diabetes Mellitus

    doi: 10.3389/fendo.2017.00347

    Figure Lengend Snippet: Depicts mitochondrial reactive oxygen species (mtROS) content in study subjects. US-unstained (autofluorescence). (A) Representative FACS image of mitosox fluorescence intensity (B) bar graph represents delta mean fluorescence in controls (GP-I); prediabetic subjects (GP-II); NDT2DM (GP-III); and ADT2DM patients (GP-IV). Values are expressed in median and interquartile range ( n = 20). (* = vs. GP-I), ( # = vs. GP-II), ( $ = vs. GP-III), * p

    Article Snippet: Furthermore, mtROS levels were determined using Becton Dickinson FACS Aria II flow cytometer by DIVA software (Becton Dickinson, Franklin Lakes, NJ, USA).

    Techniques: FACS, Fluorescence

    CD3 + CD4 + cells were isolated from L-Tg-FIR and FIR mice and stained for Foxp3 expression. (A) Representative FACS plot depicts percent Foxp3 expression from WT and L-Tg splenocytes. (B) MFI of Foxp3 expression is plotted from different tissues taken from

    Journal: Inflammatory bowel diseases

    Article Title: Differential levels of Tl1a affect the expansion and function of regulatory T cells in modulating murine colitis

    doi: 10.1097/MIB.0000000000000653

    Figure Lengend Snippet: CD3 + CD4 + cells were isolated from L-Tg-FIR and FIR mice and stained for Foxp3 expression. (A) Representative FACS plot depicts percent Foxp3 expression from WT and L-Tg splenocytes. (B) MFI of Foxp3 expression is plotted from different tissues taken from

    Article Snippet: CD3+ CD4+ RFP+ GFPhigh and CD4+ RFP+ GFPlow cells were sorted with a BD FACS Aria II (BD Bioscience) using anti-CD3, anti-CD4 Abs (eBioscience), GFP and RFP expression. iTregs were generated from sorted naïve CD3+ CD4+ CD25− or RFP− lymphocytes from WT, L-Tg, GFPhigh or GFPlow populations.

    Techniques: Isolation, Mouse Assay, Staining, Expressing, FACS

    FGF1 is necessary for overnutrition-induced β-cell differentiation. A : RT-PCR analysis of fgf1 , insa , gcga , and amy2a expression in FACS-sorted β-cells. nc, no template control. B : Overnutrition-induced mild hyperglycemia in fgf1 mu1/mu1 fish. Wild-type and fgf1 mu1/mu1 larvae were cultured for 8 h in nutrient-free or 5% egg yolk solution at 6 dpf, and their total free glucose levels were determined immediately after. n = 10. Representative confocal projections of β-cells of 6-dpf ( C ) and 4-week-old ( E ) Tg(−1.2ins:H2B-mCherry) or fgf1 mu1/mu1 ;Tg(−1.2ins:H2B-mCherry) larvae cultured for 8 h in nutrient-free medium or 5% egg yolk. Scale bar indicates 10 µm in C and 20 μm in E . Quantification of β-cell number from 6-dpf larvae ( D ) or 4-week-old fish ( F ) suggested a loss of overnutrition-induced β-cell differentiation in fgf1 -deficient fish. n = 7–24 in D and n = 10 in F . * P

    Journal: Diabetes

    Article Title: FGF1 Mediates Overnutrition-Induced Compensatory β-Cell Differentiation

    doi: 10.2337/db15-0085

    Figure Lengend Snippet: FGF1 is necessary for overnutrition-induced β-cell differentiation. A : RT-PCR analysis of fgf1 , insa , gcga , and amy2a expression in FACS-sorted β-cells. nc, no template control. B : Overnutrition-induced mild hyperglycemia in fgf1 mu1/mu1 fish. Wild-type and fgf1 mu1/mu1 larvae were cultured for 8 h in nutrient-free or 5% egg yolk solution at 6 dpf, and their total free glucose levels were determined immediately after. n = 10. Representative confocal projections of β-cells of 6-dpf ( C ) and 4-week-old ( E ) Tg(−1.2ins:H2B-mCherry) or fgf1 mu1/mu1 ;Tg(−1.2ins:H2B-mCherry) larvae cultured for 8 h in nutrient-free medium or 5% egg yolk. Scale bar indicates 10 µm in C and 20 μm in E . Quantification of β-cell number from 6-dpf larvae ( D ) or 4-week-old fish ( F ) suggested a loss of overnutrition-induced β-cell differentiation in fgf1 -deficient fish. n = 7–24 in D and n = 10 in F . * P

    Article Snippet: The mCherry-positive β-cells were collected using the BD FACS Aria II in the Vanderbilt University Medical Center (VUMC) Flow Cytometry Core.

    Techniques: Cell Differentiation, Reverse Transcription Polymerase Chain Reaction, Expressing, FACS, Fluorescence In Situ Hybridization, Cell Culture