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  • 99
    Thermo Fisher foxp3 transcription factor
    Flow-cytometry analysis of immune cell populations. a Flow-cytometry analysis of CD8 + cells (left), CD4 + cells (middle), and <t>Foxp3</t> + cells (Treg, right) of all cells or gated cells in tumor on day 7 ( n = 6). b Flow-cytometry analysis of IA/IE + CD11c + cells (DC, top) and F4/80 + CD11b + cells (MΦ, bottom) of all cells in tumor on day 7 ( n = 6). c Flow-cytometry analysis of CD8 + cells (left), CD4 + cells (middle), and Foxp3 + cells (right) of all cells or gated cells in spleen on day 7 ( n = 6). d Flow-cytometry analysis of IA/IE + CD11c + cells (top) and F4/80 + CD11b + cells (bottom) of all cells in spleen on day 7 ( n = 6). Data represent mean ± SEM; * P
    Foxp3 Transcription Factor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5753 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher foxp3 staining buffer set
    Increased Proportion of IL-17-Producing T Cells in the SI LP and spleen of anti-NMDAR encephalitis FMT mice ( n = 8). a Representative staining of IL-17A + CD4 T cell subsets and statistical analysis of the percentages in spleen and SI LP, gated on TCRβ + CD4 + . b Representative staining of <t>FOXP3</t> + CD4 T cell subsets and statistical analysis of the percentages in spleen and SI LP, gated on TCRβ + CD4 + . Each dot represents a mouse and this is one of three different experiments performed with similar results in each experiment. (* P
    Foxp3 Staining Buffer Set, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6461 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Becton Dickinson transcription factor buffer set
    Increased Proportion of IL-17-Producing T Cells in the SI LP and spleen of anti-NMDAR encephalitis FMT mice ( n = 8). a Representative staining of IL-17A + CD4 T cell subsets and statistical analysis of the percentages in spleen and SI LP, gated on TCRβ + CD4 + . b Representative staining of <t>FOXP3</t> + CD4 T cell subsets and statistical analysis of the percentages in spleen and SI LP, gated on TCRβ + CD4 + . Each dot represents a mouse and this is one of three different experiments performed with similar results in each experiment. (* P
    Transcription Factor Buffer Set, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 992 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega transcription buffer
    Increased Proportion of IL-17-Producing T Cells in the SI LP and spleen of anti-NMDAR encephalitis FMT mice ( n = 8). a Representative staining of IL-17A + CD4 T cell subsets and statistical analysis of the percentages in spleen and SI LP, gated on TCRβ + CD4 + . b Representative staining of <t>FOXP3</t> + CD4 T cell subsets and statistical analysis of the percentages in spleen and SI LP, gated on TCRβ + CD4 + . Each dot represents a mouse and this is one of three different experiments performed with similar results in each experiment. (* P
    Transcription Buffer, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 1238 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    BioLegend true nuclear transcription factor buffer set
    Increased Proportion of IL-17-Producing T Cells in the SI LP and spleen of anti-NMDAR encephalitis FMT mice ( n = 8). a Representative staining of IL-17A + CD4 T cell subsets and statistical analysis of the percentages in spleen and SI LP, gated on TCRβ + CD4 + . b Representative staining of <t>FOXP3</t> + CD4 T cell subsets and statistical analysis of the percentages in spleen and SI LP, gated on TCRβ + CD4 + . Each dot represents a mouse and this is one of three different experiments performed with similar results in each experiment. (* P
    True Nuclear Transcription Factor Buffer Set, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 582 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher foxp3 transcription factor staining buffer
    High dietary salt intake enhances bone loss by modulating Th17-Treg cell balance. Cells from bone marrow (BM) of normal, low salt diet (LSD) and high salt diet mice groups (HSD) were isolated at the end of experiment, labelled and analysed by flow cytometry for percentage of <t>Foxp3</t> + and Rorγt + , IL-10 + Foxp3 + and Rorγt + IL-17 + cells. Gating was first performed for CD4 + and later analysed for the expression of CD4 + Foxp3 + (Tregs), CD4 + Rorγt + (Th17), CD4 + IL-10 + (Tregs) and CD4 + IL-17 + (Th17). ( A ) Average percentage of CD4 + Foxp3 + T cells ( B ) Average percentage of CD8 + Foxp3 + T cells ( C ) Average percentage of CD4 + Rorγt + T cells. ( D ) Average percentage of Foxp3 + IL-10 + (Treg). ( E ) Average percentage of Rorγt + IL-17 + (Th17). The results were evaluated by using ANOVA with subsequent comparisons by Student t test for paired or nonpaired data, as appropriate. Analysis was performed using Sigma plot software (Systat Software, Inc., Germany). Values are reported as mean ± SEM (n = 10) and similar results were obtained in three independent experiments. Statistical significance was defined as p ≤ 0.05 (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001) with respect to low salt diet group.
    Foxp3 Transcription Factor Staining Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 362 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher transcription buffer
    High dietary salt intake enhances bone loss by modulating Th17-Treg cell balance. Cells from bone marrow (BM) of normal, low salt diet (LSD) and high salt diet mice groups (HSD) were isolated at the end of experiment, labelled and analysed by flow cytometry for percentage of <t>Foxp3</t> + and Rorγt + , IL-10 + Foxp3 + and Rorγt + IL-17 + cells. Gating was first performed for CD4 + and later analysed for the expression of CD4 + Foxp3 + (Tregs), CD4 + Rorγt + (Th17), CD4 + IL-10 + (Tregs) and CD4 + IL-17 + (Th17). ( A ) Average percentage of CD4 + Foxp3 + T cells ( B ) Average percentage of CD8 + Foxp3 + T cells ( C ) Average percentage of CD4 + Rorγt + T cells. ( D ) Average percentage of Foxp3 + IL-10 + (Treg). ( E ) Average percentage of Rorγt + IL-17 + (Th17). The results were evaluated by using ANOVA with subsequent comparisons by Student t test for paired or nonpaired data, as appropriate. Analysis was performed using Sigma plot software (Systat Software, Inc., Germany). Values are reported as mean ± SEM (n = 10) and similar results were obtained in three independent experiments. Statistical significance was defined as p ≤ 0.05 (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001) with respect to low salt diet group.
    Transcription Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1036 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher reverse transcription buffer
    High dietary salt intake enhances bone loss by modulating Th17-Treg cell balance. Cells from bone marrow (BM) of normal, low salt diet (LSD) and high salt diet mice groups (HSD) were isolated at the end of experiment, labelled and analysed by flow cytometry for percentage of <t>Foxp3</t> + and Rorγt + , IL-10 + Foxp3 + and Rorγt + IL-17 + cells. Gating was first performed for CD4 + and later analysed for the expression of CD4 + Foxp3 + (Tregs), CD4 + Rorγt + (Th17), CD4 + IL-10 + (Tregs) and CD4 + IL-17 + (Th17). ( A ) Average percentage of CD4 + Foxp3 + T cells ( B ) Average percentage of CD8 + Foxp3 + T cells ( C ) Average percentage of CD4 + Rorγt + T cells. ( D ) Average percentage of Foxp3 + IL-10 + (Treg). ( E ) Average percentage of Rorγt + IL-17 + (Th17). The results were evaluated by using ANOVA with subsequent comparisons by Student t test for paired or nonpaired data, as appropriate. Analysis was performed using Sigma plot software (Systat Software, Inc., Germany). Values are reported as mean ± SEM (n = 10) and similar results were obtained in three independent experiments. Statistical significance was defined as p ≤ 0.05 (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001) with respect to low salt diet group.
    Reverse Transcription Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 508 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher transcription factor staining buffer set
    High dietary salt intake enhances bone loss by modulating Th17-Treg cell balance. Cells from bone marrow (BM) of normal, low salt diet (LSD) and high salt diet mice groups (HSD) were isolated at the end of experiment, labelled and analysed by flow cytometry for percentage of <t>Foxp3</t> + and Rorγt + , IL-10 + Foxp3 + and Rorγt + IL-17 + cells. Gating was first performed for CD4 + and later analysed for the expression of CD4 + Foxp3 + (Tregs), CD4 + Rorγt + (Th17), CD4 + IL-10 + (Tregs) and CD4 + IL-17 + (Th17). ( A ) Average percentage of CD4 + Foxp3 + T cells ( B ) Average percentage of CD8 + Foxp3 + T cells ( C ) Average percentage of CD4 + Rorγt + T cells. ( D ) Average percentage of Foxp3 + IL-10 + (Treg). ( E ) Average percentage of Rorγt + IL-17 + (Th17). The results were evaluated by using ANOVA with subsequent comparisons by Student t test for paired or nonpaired data, as appropriate. Analysis was performed using Sigma plot software (Systat Software, Inc., Germany). Values are reported as mean ± SEM (n = 10) and similar results were obtained in three independent experiments. Statistical significance was defined as p ≤ 0.05 (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001) with respect to low salt diet group.
    Transcription Factor Staining Buffer Set, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 271 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher foxp3 transcription factor buffer kit
    Gamma-secretase inhibitors alter Th1 and Th17 responses but do not inhibit EAE. B6 mice were immunized with MOG35-55 to induce active EAE. Beginning on the day after immunization, the mice were randomized and treated with DMSO or GSI every other day for 18 days. A. Clinical course of EAE. B. Peak clinical score of DMSO and GSI-treated mice. C. Cumulative EAE scores for DMSO and GSI-treated animals. T cell cytokine expression in CNS-infiltrating CD4+ T cells from DMSO or GSI-treated mice following the Peak of EAE. On day 17 post-immunization, CNS cells were isolated and intracellular cytokine staining performed. D. Expression of IL-17 and IFNγ. E. Expression of GM-CSF and IFNγ. Distribution of the percentages of cells expressing IFNγ (F), IL-17 (G), GM-CSF (H) or <t>FoxP3</t> (I) among CNS-infiltrating CD4+ T cells. J-L, Presence of cytokine expression in the spleens of EAE mice on day 17 post-immunization. Distribution of the percentages of cells expressing IFNγ (J), IL-17 (K), GM-CSF (L). Symbols indicate the percentage of cells from individual mice. Also plotted are the mean and SEM for each treatment group. Open circles indicate DMSO treatment, filled squares indicate treatment with GSI. Error bars represent SEM. Asterisks indicate significant differences (* p
    Foxp3 Transcription Factor Buffer Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 82 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Becton Dickinson transcription factor phospho buffer set
    Gamma-secretase inhibitors alter Th1 and Th17 responses but do not inhibit EAE. B6 mice were immunized with MOG35-55 to induce active EAE. Beginning on the day after immunization, the mice were randomized and treated with DMSO or GSI every other day for 18 days. A. Clinical course of EAE. B. Peak clinical score of DMSO and GSI-treated mice. C. Cumulative EAE scores for DMSO and GSI-treated animals. T cell cytokine expression in CNS-infiltrating CD4+ T cells from DMSO or GSI-treated mice following the Peak of EAE. On day 17 post-immunization, CNS cells were isolated and intracellular cytokine staining performed. D. Expression of IL-17 and IFNγ. E. Expression of GM-CSF and IFNγ. Distribution of the percentages of cells expressing IFNγ (F), IL-17 (G), GM-CSF (H) or <t>FoxP3</t> (I) among CNS-infiltrating CD4+ T cells. J-L, Presence of cytokine expression in the spleens of EAE mice on day 17 post-immunization. Distribution of the percentages of cells expressing IFNγ (J), IL-17 (K), GM-CSF (L). Symbols indicate the percentage of cells from individual mice. Also plotted are the mean and SEM for each treatment group. Open circles indicate DMSO treatment, filled squares indicate treatment with GSI. Error bars represent SEM. Asterisks indicate significant differences (* p
    Transcription Factor Phospho Buffer Set, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Flow-cytometry analysis of immune cell populations. a Flow-cytometry analysis of CD8 + cells (left), CD4 + cells (middle), and Foxp3 + cells (Treg, right) of all cells or gated cells in tumor on day 7 ( n = 6). b Flow-cytometry analysis of IA/IE + CD11c + cells (DC, top) and F4/80 + CD11b + cells (MΦ, bottom) of all cells in tumor on day 7 ( n = 6). c Flow-cytometry analysis of CD8 + cells (left), CD4 + cells (middle), and Foxp3 + cells (right) of all cells or gated cells in spleen on day 7 ( n = 6). d Flow-cytometry analysis of IA/IE + CD11c + cells (top) and F4/80 + CD11b + cells (bottom) of all cells in spleen on day 7 ( n = 6). Data represent mean ± SEM; * P

    Journal: EJNMMI Research

    Article Title: Anti PD-1 treatment increases [18F]FDG uptake by cancer cells in a mouse B16F10 melanoma model

    doi: 10.1186/s13550-018-0433-1

    Figure Lengend Snippet: Flow-cytometry analysis of immune cell populations. a Flow-cytometry analysis of CD8 + cells (left), CD4 + cells (middle), and Foxp3 + cells (Treg, right) of all cells or gated cells in tumor on day 7 ( n = 6). b Flow-cytometry analysis of IA/IE + CD11c + cells (DC, top) and F4/80 + CD11b + cells (MΦ, bottom) of all cells in tumor on day 7 ( n = 6). c Flow-cytometry analysis of CD8 + cells (left), CD4 + cells (middle), and Foxp3 + cells (right) of all cells or gated cells in spleen on day 7 ( n = 6). d Flow-cytometry analysis of IA/IE + CD11c + cells (top) and F4/80 + CD11b + cells (bottom) of all cells in spleen on day 7 ( n = 6). Data represent mean ± SEM; * P

    Article Snippet: For intracellular staining, cells were fixed and permeabilized using Foxp3/Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA, USA) after cell surface staining and were stained with labeled antibodies against the intracellular molecules foxp3 (eBioscience, clone FJK-16s), glucose transporter 1 (GLUT1, Abcam, Cambridge, UK, clone EPR3915), and hexokinase II (HX2, Abcam, clone EPR20839).

    Techniques: Flow Cytometry, Cytometry, IA

    M/D-driven tumors recapitulate key features of human HR + breast cancer. a Schedule of oncogenic challenges for the induction of M/D-driven tumors and representative images of M/D-driven tumors established in WT C57BL/6 mice. Scale bar = 1 cm. b Tumor-free survival (TFS) and time-to-death (TTD) in WT C57BL/6 mice subjected to M/D-driven oncogenesis. Number of mice is reported. c Representative histology of M/D-driven tumors as compared to human HR + breast cancers. Scale bar = 50 µm. d Probeset distribution comparing the transcriptomic profile of 6 M/D-driven mammary tumors established in C57BL/6 mice with that of human breast cancers from the TCGA public database. Probability of molecular subtyping is reported for each mouse tumors. e , f TFS and overall survival (OS) of WT C57BL/6 mice and C57BL/6 bearing ER mutations that cause nuclear exclusion ( e ) or nuclear accumulation ( f ) subjected to M/D-driven oncogenesis. Number of mice, hazard ratio (HR) and p values (two-sided log-rank) are reported. g TFS and OS of WT C57BL/6 mice subjected to M/D-driven oncogenesis in control conditions or along with tamoxifen administration in the drinking water. Number of mice, HR and p values (two-sided log-rank) are reported. h . Non-supervised hierarchical clustering of the transcriptomic profile of M/D-driven tumors established in WT C57BL/6 mice ( n = 6), mammary glands exposed to M/D but not developing tumors ( n = 6) and M/D-naïve mammary glands ( n = 6). Gene Ontology analysis, fold change (FC) and adjusted, two-sided p values are reported. Red, upregulation. Yellow, downregulation. i Relative amount of CD3 + , CD8 + , CD4 + and CD25 + FOXP3 + cells infiltrating M/D-driven tumors in C57BL/6 mice vs syngeneic M/D-naïve mammary glands. Results are means ± SEM plus individual data points. Number of mice and p values (unpaired, two-sided Student’s t , as compared to M/D-naïve mammary glands) are reported.

    Journal: Nature Communications

    Article Title: Immunoprophylactic and immunotherapeutic control of hormone receptor-positive breast cancer

    doi: 10.1038/s41467-020-17644-0

    Figure Lengend Snippet: M/D-driven tumors recapitulate key features of human HR + breast cancer. a Schedule of oncogenic challenges for the induction of M/D-driven tumors and representative images of M/D-driven tumors established in WT C57BL/6 mice. Scale bar = 1 cm. b Tumor-free survival (TFS) and time-to-death (TTD) in WT C57BL/6 mice subjected to M/D-driven oncogenesis. Number of mice is reported. c Representative histology of M/D-driven tumors as compared to human HR + breast cancers. Scale bar = 50 µm. d Probeset distribution comparing the transcriptomic profile of 6 M/D-driven mammary tumors established in C57BL/6 mice with that of human breast cancers from the TCGA public database. Probability of molecular subtyping is reported for each mouse tumors. e , f TFS and overall survival (OS) of WT C57BL/6 mice and C57BL/6 bearing ER mutations that cause nuclear exclusion ( e ) or nuclear accumulation ( f ) subjected to M/D-driven oncogenesis. Number of mice, hazard ratio (HR) and p values (two-sided log-rank) are reported. g TFS and OS of WT C57BL/6 mice subjected to M/D-driven oncogenesis in control conditions or along with tamoxifen administration in the drinking water. Number of mice, HR and p values (two-sided log-rank) are reported. h . Non-supervised hierarchical clustering of the transcriptomic profile of M/D-driven tumors established in WT C57BL/6 mice ( n = 6), mammary glands exposed to M/D but not developing tumors ( n = 6) and M/D-naïve mammary glands ( n = 6). Gene Ontology analysis, fold change (FC) and adjusted, two-sided p values are reported. Red, upregulation. Yellow, downregulation. i Relative amount of CD3 + , CD8 + , CD4 + and CD25 + FOXP3 + cells infiltrating M/D-driven tumors in C57BL/6 mice vs syngeneic M/D-naïve mammary glands. Results are means ± SEM plus individual data points. Number of mice and p values (unpaired, two-sided Student’s t , as compared to M/D-naïve mammary glands) are reported.

    Article Snippet: Cells were then fixed and permeabilized in 1X Foxp3 Transcription Factor Staining Buffer (part of #A25866A, from Thermo Fisher).

    Techniques: Mouse Assay

    Increased Proportion of IL-17-Producing T Cells in the SI LP and spleen of anti-NMDAR encephalitis FMT mice ( n = 8). a Representative staining of IL-17A + CD4 T cell subsets and statistical analysis of the percentages in spleen and SI LP, gated on TCRβ + CD4 + . b Representative staining of FOXP3 + CD4 T cell subsets and statistical analysis of the percentages in spleen and SI LP, gated on TCRβ + CD4 + . Each dot represents a mouse and this is one of three different experiments performed with similar results in each experiment. (* P

    Journal: Cell Death Discovery

    Article Title: Fecal microbiota transplantation from patients with autoimmune encephalitis modulates Th17 response and relevant behaviors in mice

    doi: 10.1038/s41420-020-00309-8

    Figure Lengend Snippet: Increased Proportion of IL-17-Producing T Cells in the SI LP and spleen of anti-NMDAR encephalitis FMT mice ( n = 8). a Representative staining of IL-17A + CD4 T cell subsets and statistical analysis of the percentages in spleen and SI LP, gated on TCRβ + CD4 + . b Representative staining of FOXP3 + CD4 T cell subsets and statistical analysis of the percentages in spleen and SI LP, gated on TCRβ + CD4 + . Each dot represents a mouse and this is one of three different experiments performed with similar results in each experiment. (* P

    Article Snippet: Intra-nuclear Foxp3 was stained using the Foxp3 Staining Buffer Set (eBioscience, San Diego, CA, USA).

    Techniques: Mouse Assay, Staining

    More-restricted expression and Treg activation phenotype of FoxP3-eGFP-Cre-ERT2 x FSF-Tim3 mice. (a) Crosses used to generate FoxP3-eGFP-Cre-ERT2 x FSF-Tim-3 mice. (b) Tim-3 transgene expression is restricted to CD4 + Treg when driven by the tamoxifen-inducible Cre. (c) Proportion of FoxP3 + CD25 + Treg in spleen and LN of Tim-3 Tg vs. WT (Cre only) animals. (d) Activation status of Treg from the Tim-3 Tg vs. WT animals. (e) Quantitation of Treg frequency (among CD4 + T cells). (f) Quantitation of activated (CD44 + CD62L − ) Treg. (g) Levels of expression of CD25 (based on MFI of flow cytometry data). Mice were analyzed at 6-8 weeks of age. Representative of three experiments with 3-4 mice per experiment.

    Journal: bioRxiv

    Article Title: Expression of Tim-3 drives naïve Treg to an effector-like state with enhanced suppressive activity

    doi: 10.1101/2020.07.31.230714

    Figure Lengend Snippet: More-restricted expression and Treg activation phenotype of FoxP3-eGFP-Cre-ERT2 x FSF-Tim3 mice. (a) Crosses used to generate FoxP3-eGFP-Cre-ERT2 x FSF-Tim-3 mice. (b) Tim-3 transgene expression is restricted to CD4 + Treg when driven by the tamoxifen-inducible Cre. (c) Proportion of FoxP3 + CD25 + Treg in spleen and LN of Tim-3 Tg vs. WT (Cre only) animals. (d) Activation status of Treg from the Tim-3 Tg vs. WT animals. (e) Quantitation of Treg frequency (among CD4 + T cells). (f) Quantitation of activated (CD44 + CD62L − ) Treg. (g) Levels of expression of CD25 (based on MFI of flow cytometry data). Mice were analyzed at 6-8 weeks of age. Representative of three experiments with 3-4 mice per experiment.

    Article Snippet: Samples analyzed for transcription factors were first stained for Live/Dead staining along with Fc Block (CD16/32) followed by cell surface marker staining, fixed/permeabilized with the eBioscience FoxP3 staining kit (#00-5523-00), followed by intracellular protein staining.

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

    Treg with enforced Tim-3 expression display enhanced suppressive activity and IL-10 production. (a-b) GFP + cells were sorted from Tim-3 Tg or WT (Cre-only) mice and mixed with sorted naïve conventional T cells from BL/6 mice for an in vitro suppression assay. Panel A shows representative CellTrace Violet (CTV) dilution, used to quantitate proliferation of the target conventional T cells. Panel B shows average suppression of triplicate samples over a range of ratios of Treg to conventional T cells. (c-d) Effects of Treg Tim-3 induction on transplanted tumor growth. The indicated mice were treated with tamoxifen or vehicle, followed by injection of B16 (c) or MC38 (d) tumors. Growth curves for individual animals are shown (left panels), as well as average tumor growth (right panels). (e-f) Effects of Treg Tim-3 expression on IL-10 production by exhausted CD8 + conventional T cells (e) or CD4 + Treg (f). Relative IL-10 expression heat-mapped onto Tim-3 x PD-1 expression for exhausted T cells or FoxP3 x CD25 expression for Treg. Representative of two experiments of four mice each, with six-week-old mice.

    Journal: bioRxiv

    Article Title: Expression of Tim-3 drives naïve Treg to an effector-like state with enhanced suppressive activity

    doi: 10.1101/2020.07.31.230714

    Figure Lengend Snippet: Treg with enforced Tim-3 expression display enhanced suppressive activity and IL-10 production. (a-b) GFP + cells were sorted from Tim-3 Tg or WT (Cre-only) mice and mixed with sorted naïve conventional T cells from BL/6 mice for an in vitro suppression assay. Panel A shows representative CellTrace Violet (CTV) dilution, used to quantitate proliferation of the target conventional T cells. Panel B shows average suppression of triplicate samples over a range of ratios of Treg to conventional T cells. (c-d) Effects of Treg Tim-3 induction on transplanted tumor growth. The indicated mice were treated with tamoxifen or vehicle, followed by injection of B16 (c) or MC38 (d) tumors. Growth curves for individual animals are shown (left panels), as well as average tumor growth (right panels). (e-f) Effects of Treg Tim-3 expression on IL-10 production by exhausted CD8 + conventional T cells (e) or CD4 + Treg (f). Relative IL-10 expression heat-mapped onto Tim-3 x PD-1 expression for exhausted T cells or FoxP3 x CD25 expression for Treg. Representative of two experiments of four mice each, with six-week-old mice.

    Article Snippet: Samples analyzed for transcription factors were first stained for Live/Dead staining along with Fc Block (CD16/32) followed by cell surface marker staining, fixed/permeabilized with the eBioscience FoxP3 staining kit (#00-5523-00), followed by intracellular protein staining.

    Techniques: Expressing, Activity Assay, Mouse Assay, In Vitro, Suppression Assay, Injection

    Evidence of partial Treg destabilization in the spleen after Tim-3 induction. (a-b) Expression of the Tim-3 transgene and GFP (as a surrogate for FoxP3) in unfixed Treg from FoxP3-eGFP-Cre-ERT2 heterozygous female mice. (c-d) Expression of the Tim-3 transgene and FoxP3 protein in fixed Treg. (e-f) Proportion of FoxP3 + CD25 + Treg among CD4 + T cells in spleen and lymph node, showing a slight but statistically significant decrease in the proportion of splenic, but not lymph node, Treg in the Tg animals, by six weeks of age after tamoxifen administration. (g) FoxP3 and Tim-3 expression among splenic CD4 + T cells at the indicated times after tamoxifen administration. Expression of Tim-3 is permanent once Cre-mediated recombination occurs at the modified Rosa26 locus, making Tim-3 expression a faithful fate-tracking reporter. (h) FoxP3-eGFP-Cre and Tim-3 expression in WT (Cre-only) vs. Tim-3 Tg Treg, indicating the three cell populations sorted for RNA sequencing.

    Journal: bioRxiv

    Article Title: Expression of Tim-3 drives naïve Treg to an effector-like state with enhanced suppressive activity

    doi: 10.1101/2020.07.31.230714

    Figure Lengend Snippet: Evidence of partial Treg destabilization in the spleen after Tim-3 induction. (a-b) Expression of the Tim-3 transgene and GFP (as a surrogate for FoxP3) in unfixed Treg from FoxP3-eGFP-Cre-ERT2 heterozygous female mice. (c-d) Expression of the Tim-3 transgene and FoxP3 protein in fixed Treg. (e-f) Proportion of FoxP3 + CD25 + Treg among CD4 + T cells in spleen and lymph node, showing a slight but statistically significant decrease in the proportion of splenic, but not lymph node, Treg in the Tg animals, by six weeks of age after tamoxifen administration. (g) FoxP3 and Tim-3 expression among splenic CD4 + T cells at the indicated times after tamoxifen administration. Expression of Tim-3 is permanent once Cre-mediated recombination occurs at the modified Rosa26 locus, making Tim-3 expression a faithful fate-tracking reporter. (h) FoxP3-eGFP-Cre and Tim-3 expression in WT (Cre-only) vs. Tim-3 Tg Treg, indicating the three cell populations sorted for RNA sequencing.

    Article Snippet: Samples analyzed for transcription factors were first stained for Live/Dead staining along with Fc Block (CD16/32) followed by cell surface marker staining, fixed/permeabilized with the eBioscience FoxP3 staining kit (#00-5523-00), followed by intracellular protein staining.

    Techniques: Expressing, Mouse Assay, Modification, RNA Sequencing Assay

    Detailed cell-surface phenotype of Treg from FoxP3-eGFP-Cre-ERT2 x FSF-Tim3 mice. (a-e) Statistically significant increases in ICOS, CD103, Nrp1, CD39 and CD73 were all observed on Tim-3 Tg Treg, in comparison to WT Treg (Cre-only). (f) By contrast, there was a corresponding decrease in expression of CTLA-4 on the Tim-3 Tg Treg. Mice were analyzed at 6-8 weeks of age. Representative of three experiments with 3-4 mice per experiment.

    Journal: bioRxiv

    Article Title: Expression of Tim-3 drives naïve Treg to an effector-like state with enhanced suppressive activity

    doi: 10.1101/2020.07.31.230714

    Figure Lengend Snippet: Detailed cell-surface phenotype of Treg from FoxP3-eGFP-Cre-ERT2 x FSF-Tim3 mice. (a-e) Statistically significant increases in ICOS, CD103, Nrp1, CD39 and CD73 were all observed on Tim-3 Tg Treg, in comparison to WT Treg (Cre-only). (f) By contrast, there was a corresponding decrease in expression of CTLA-4 on the Tim-3 Tg Treg. Mice were analyzed at 6-8 weeks of age. Representative of three experiments with 3-4 mice per experiment.

    Article Snippet: Samples analyzed for transcription factors were first stained for Live/Dead staining along with Fc Block (CD16/32) followed by cell surface marker staining, fixed/permeabilized with the eBioscience FoxP3 staining kit (#00-5523-00), followed by intracellular protein staining.

    Techniques: Mouse Assay, Expressing

    Phenotype of conventional T cells in peripheral lymphoid organs of FoxP3-eGFP-CRE-ERT2 x FSF-Tim-3 mice. (a) Proportion of CD4 + and CD8 + T cells in the spleen and lymph nodes of WT (FoxP3-eGFP-Cre-ERT2 only) and Tim-3 Tg animals, showing normal proportions of both cell types. (b-c) Proportions of antigen-experienced conventional T cells, based on CD44 and CD62L expression, again showing no evidence of basal lymphocyte activation in the Cre-ERT2 model. (d) Gross phenotypes of spleen and lymph nodes were also indistinguishable between WT and Tim-3 Tg animals. Quantitation of conventional T cell frequencies (e) and the proportions of antigen-experienced (CD44 + ) cells in spleen (f) and lymph node (g) from three animals per genotype. Representative of three experiments with 3-4 mice per experiment.

    Journal: bioRxiv

    Article Title: Expression of Tim-3 drives naïve Treg to an effector-like state with enhanced suppressive activity

    doi: 10.1101/2020.07.31.230714

    Figure Lengend Snippet: Phenotype of conventional T cells in peripheral lymphoid organs of FoxP3-eGFP-CRE-ERT2 x FSF-Tim-3 mice. (a) Proportion of CD4 + and CD8 + T cells in the spleen and lymph nodes of WT (FoxP3-eGFP-Cre-ERT2 only) and Tim-3 Tg animals, showing normal proportions of both cell types. (b-c) Proportions of antigen-experienced conventional T cells, based on CD44 and CD62L expression, again showing no evidence of basal lymphocyte activation in the Cre-ERT2 model. (d) Gross phenotypes of spleen and lymph nodes were also indistinguishable between WT and Tim-3 Tg animals. Quantitation of conventional T cell frequencies (e) and the proportions of antigen-experienced (CD44 + ) cells in spleen (f) and lymph node (g) from three animals per genotype. Representative of three experiments with 3-4 mice per experiment.

    Article Snippet: Samples analyzed for transcription factors were first stained for Live/Dead staining along with Fc Block (CD16/32) followed by cell surface marker staining, fixed/permeabilized with the eBioscience FoxP3 staining kit (#00-5523-00), followed by intracellular protein staining.

    Techniques: Mouse Assay, Expressing, Activation Assay, Quantitation Assay

    Phenotypic and functional effects of FoxP3-YFP-Cre-driven Tim-3 expression on Treg. (a) Crosses used to generate FoxP3-YFP-Cre x FSF-Tim-3 mice. (b) Efficiency of Tim-3 expression on Treg. Shown is a representative male mouse. Similar results were observed with Cre-homozygous female mice. (c) Ectopic Tim-3 expression via FoxP3-YFP-Cre resulted in an increase in the proportion of FoxP3 + cells among CD4 + T cells in spleen and lymph nodes. (d) Increase in activated Treg in the context of ectopic Tim-3 expression. (e-f) Expression of selected markers of Treg effector status and function in WT vs. Tim-3 Tg Treg. (g) In vitro suppression assay performed with FoxP3 − YFP-Cre + Treg sorted from Cre-only (WT) or Tim-3 Tg mice. (h) Growth of B16 tumors in WT vs. Tim-3 Treg Tg animals. Left – tumor growth in individual mice; right – average tumor growth. (i-j) Organ size and lymphocyte cell number of WT and Tim-3 Tg animals at eight weeks of age. (k) left – similar proportions of previously activated CD4 + T cells in spleen of WT vs. Tim-3 Tg animals. (l) Leaky expression of the Tim-3 transgene in multiple cell types from the spleen. Data are from a single experiment, representative of three experiments, with 3-4 mice per experiment, except for tumor experiments, which are representative of two experiments of 6-8 mice per experiment.

    Journal: bioRxiv

    Article Title: Expression of Tim-3 drives naïve Treg to an effector-like state with enhanced suppressive activity

    doi: 10.1101/2020.07.31.230714

    Figure Lengend Snippet: Phenotypic and functional effects of FoxP3-YFP-Cre-driven Tim-3 expression on Treg. (a) Crosses used to generate FoxP3-YFP-Cre x FSF-Tim-3 mice. (b) Efficiency of Tim-3 expression on Treg. Shown is a representative male mouse. Similar results were observed with Cre-homozygous female mice. (c) Ectopic Tim-3 expression via FoxP3-YFP-Cre resulted in an increase in the proportion of FoxP3 + cells among CD4 + T cells in spleen and lymph nodes. (d) Increase in activated Treg in the context of ectopic Tim-3 expression. (e-f) Expression of selected markers of Treg effector status and function in WT vs. Tim-3 Tg Treg. (g) In vitro suppression assay performed with FoxP3 − YFP-Cre + Treg sorted from Cre-only (WT) or Tim-3 Tg mice. (h) Growth of B16 tumors in WT vs. Tim-3 Treg Tg animals. Left – tumor growth in individual mice; right – average tumor growth. (i-j) Organ size and lymphocyte cell number of WT and Tim-3 Tg animals at eight weeks of age. (k) left – similar proportions of previously activated CD4 + T cells in spleen of WT vs. Tim-3 Tg animals. (l) Leaky expression of the Tim-3 transgene in multiple cell types from the spleen. Data are from a single experiment, representative of three experiments, with 3-4 mice per experiment, except for tumor experiments, which are representative of two experiments of 6-8 mice per experiment.

    Article Snippet: Samples analyzed for transcription factors were first stained for Live/Dead staining along with Fc Block (CD16/32) followed by cell surface marker staining, fixed/permeabilized with the eBioscience FoxP3 staining kit (#00-5523-00), followed by intracellular protein staining.

    Techniques: Functional Assay, Expressing, Mouse Assay, In Vitro, Suppression Assay

    Effects of enforced Tim-3 expression on Treg gene expression. (a) Volcano plot of RNA sequencing data comparing Tim-3 + FoxP3 hi cells from Tg animals to FoxP3 + cells from WT (Cre-only) animals. Only comparisons with a p value

    Journal: bioRxiv

    Article Title: Expression of Tim-3 drives naïve Treg to an effector-like state with enhanced suppressive activity

    doi: 10.1101/2020.07.31.230714

    Figure Lengend Snippet: Effects of enforced Tim-3 expression on Treg gene expression. (a) Volcano plot of RNA sequencing data comparing Tim-3 + FoxP3 hi cells from Tg animals to FoxP3 + cells from WT (Cre-only) animals. Only comparisons with a p value

    Article Snippet: Samples analyzed for transcription factors were first stained for Live/Dead staining along with Fc Block (CD16/32) followed by cell surface marker staining, fixed/permeabilized with the eBioscience FoxP3 staining kit (#00-5523-00), followed by intracellular protein staining.

    Techniques: Expressing, RNA Sequencing Assay

    Treg phenotype and IL-10 production in orally infected Ahr -/- mice. Ahr -/- mice or wild type controls were infected orally with 20 Me49 cysts for 9 days. Results are pooled from 2–3 separate experiments. (A) Frequency of Tregs in the indicated tissues of wild type or Ahr -/- mice. In the lamina propria, the plots shown are gated on live CD45 + CD3 + CD4 + cells, and Tregs were gated as live CD45 + CD3 + CD4 + FoxP3 + cells. For the mesenteric lymph node and spleen, the plots shown are gated on CD3 + CD4 + cells, and Tregs were gated as CD3 + CD4 + FoxP3 + cells. (B) Expression of T-bet, CXCR3, and Ki67 on Tregs in the spleens of wild type or Ahr -/- mice. The plots are gated on CD3 + CD4 + FoxP3 + cells. (C) IL-10 secretion by cells isolated from the lamina propria or the spleen following restimulation with soluble Toxoplasma antigen. Results are pooled from 2 separate experiments with a total of 5–7 mice per group.

    Journal: PLoS ONE

    Article Title: The Group 3 Innate Lymphoid Cell Defect in Aryl Hydrocarbon Receptor Deficient Mice Is Associated with T Cell Hyperactivation during Intestinal Infection

    doi: 10.1371/journal.pone.0128335

    Figure Lengend Snippet: Treg phenotype and IL-10 production in orally infected Ahr -/- mice. Ahr -/- mice or wild type controls were infected orally with 20 Me49 cysts for 9 days. Results are pooled from 2–3 separate experiments. (A) Frequency of Tregs in the indicated tissues of wild type or Ahr -/- mice. In the lamina propria, the plots shown are gated on live CD45 + CD3 + CD4 + cells, and Tregs were gated as live CD45 + CD3 + CD4 + FoxP3 + cells. For the mesenteric lymph node and spleen, the plots shown are gated on CD3 + CD4 + cells, and Tregs were gated as CD3 + CD4 + FoxP3 + cells. (B) Expression of T-bet, CXCR3, and Ki67 on Tregs in the spleens of wild type or Ahr -/- mice. The plots are gated on CD3 + CD4 + FoxP3 + cells. (C) IL-10 secretion by cells isolated from the lamina propria or the spleen following restimulation with soluble Toxoplasma antigen. Results are pooled from 2 separate experiments with a total of 5–7 mice per group.

    Article Snippet: For the detection of T-bet, RORγt, FoxP3, and Ki67, cells were surface stained and then stained intracellularly for these proteins using the FoxP3/transcription factor staining buffer set (eBioscience).

    Techniques: Infection, Mouse Assay, Expressing, Isolation

    Ahr -/- mice exhibit increased T cell activation following infection. Wild type or Ahr -/- mice were orally infected with T . gondii for 9 days. (A) Group 3 ILC frequency in the lamina propria of wild type or Ahr -/- mice following infection. The plots on the left are gated on live CD90.2 + CD11c - B220 - cells. ( B) Weight loss was monitored at various days post-infection. Data are pooled from 2 experiments with 6–8 mice per group. (C) H E staining of small intestinal tissue sections. (D) H E staining of small intestinal tissue from an infected Ahr -/- mouse. The Peyer’s patch exhibits severe lymphocytolysis (*) and the lamina propria of adjacent villi is expanded by primarily lymphocytes and plasma cells (➜). (E) A higher magnification image of the section in Fig 2C shows that the Peyer’s patch exhibits severe lymphocytolysis characterized by pyknosis and other cellular debris. (F) H E staining of small intestinal tissue from an infected Ahr -/- mouse shows crypt loss (*) and multifocal necrotic enterocytes (➜). The lamina propria of the villi is expanded by lymphocytes and plasma cells (❋). (G) Expression of T-bet and Ki67 by FoxP3 - CD4 + T cells in the mesenteric lymph nodes of infected mice. Results are pooled from 3 separate experiments. (H) Cytokine production by CD4 + T cells following stimulation with PMA/ionomycin. Data are pooled from 2 experiments.

    Journal: PLoS ONE

    Article Title: The Group 3 Innate Lymphoid Cell Defect in Aryl Hydrocarbon Receptor Deficient Mice Is Associated with T Cell Hyperactivation during Intestinal Infection

    doi: 10.1371/journal.pone.0128335

    Figure Lengend Snippet: Ahr -/- mice exhibit increased T cell activation following infection. Wild type or Ahr -/- mice were orally infected with T . gondii for 9 days. (A) Group 3 ILC frequency in the lamina propria of wild type or Ahr -/- mice following infection. The plots on the left are gated on live CD90.2 + CD11c - B220 - cells. ( B) Weight loss was monitored at various days post-infection. Data are pooled from 2 experiments with 6–8 mice per group. (C) H E staining of small intestinal tissue sections. (D) H E staining of small intestinal tissue from an infected Ahr -/- mouse. The Peyer’s patch exhibits severe lymphocytolysis (*) and the lamina propria of adjacent villi is expanded by primarily lymphocytes and plasma cells (➜). (E) A higher magnification image of the section in Fig 2C shows that the Peyer’s patch exhibits severe lymphocytolysis characterized by pyknosis and other cellular debris. (F) H E staining of small intestinal tissue from an infected Ahr -/- mouse shows crypt loss (*) and multifocal necrotic enterocytes (➜). The lamina propria of the villi is expanded by lymphocytes and plasma cells (❋). (G) Expression of T-bet and Ki67 by FoxP3 - CD4 + T cells in the mesenteric lymph nodes of infected mice. Results are pooled from 3 separate experiments. (H) Cytokine production by CD4 + T cells following stimulation with PMA/ionomycin. Data are pooled from 2 experiments.

    Article Snippet: For the detection of T-bet, RORγt, FoxP3, and Ki67, cells were surface stained and then stained intracellularly for these proteins using the FoxP3/transcription factor staining buffer set (eBioscience).

    Techniques: Mouse Assay, Activation Assay, Infection, Staining, Expressing

    Regulatory T (Treg) cells repopulating B-cell-deficient (B –/– ) mice after depletion by anti-CD25 have reduced suppressive function and altered phenotype compared with unmanipulated Treg cells. (a). Naive B –/– Foxp3-GFP

    Journal: Immunology

    Article Title: Regulatory T cells in B-cell-deficient and wild-type mice differ functionally and in expression of cell surface markers

    doi: 10.1111/imm.12410

    Figure Lengend Snippet: Regulatory T (Treg) cells repopulating B-cell-deficient (B –/– ) mice after depletion by anti-CD25 have reduced suppressive function and altered phenotype compared with unmanipulated Treg cells. (a). Naive B –/– Foxp3-GFP

    Article Snippet: For detection of intracellular CTLA4, cells were stained for surface markers, then with eBioscience FoxP3 Staining Buffer Set and incubated (1 × 106 cells/100 μl) with anti-CTLA4 (UC10-2B9-PE) Cells were washed and the data were collected and analysed as above.

    Techniques: Mouse Assay

    Regulatory T (Treg) cells from B-cell-deficient (B –/– ) and wild-type (WT) mice can be distinguished phenotypically. (a, b). Splenocytes from naive WT or B –/– NOD.H-2h4 mice were stained for expression of CD4, Foxp3 and

    Journal: Immunology

    Article Title: Regulatory T cells in B-cell-deficient and wild-type mice differ functionally and in expression of cell surface markers

    doi: 10.1111/imm.12410

    Figure Lengend Snippet: Regulatory T (Treg) cells from B-cell-deficient (B –/– ) and wild-type (WT) mice can be distinguished phenotypically. (a, b). Splenocytes from naive WT or B –/– NOD.H-2h4 mice were stained for expression of CD4, Foxp3 and

    Article Snippet: For detection of intracellular CTLA4, cells were stained for surface markers, then with eBioscience FoxP3 Staining Buffer Set and incubated (1 × 106 cells/100 μl) with anti-CTLA4 (UC10-2B9-PE) Cells were washed and the data were collected and analysed as above.

    Techniques: Mouse Assay, Staining, Expressing

    Regulatory T (Treg) cells that repopulate B-cell deficient (B –/– ) mice after depletion by diphtheria toxin (DT) have reduced suppressor function and differ phenotypically from Treg cells in unmanipulated B –/– Foxp3-DTR

    Journal: Immunology

    Article Title: Regulatory T cells in B-cell-deficient and wild-type mice differ functionally and in expression of cell surface markers

    doi: 10.1111/imm.12410

    Figure Lengend Snippet: Regulatory T (Treg) cells that repopulate B-cell deficient (B –/– ) mice after depletion by diphtheria toxin (DT) have reduced suppressor function and differ phenotypically from Treg cells in unmanipulated B –/– Foxp3-DTR

    Article Snippet: For detection of intracellular CTLA4, cells were stained for surface markers, then with eBioscience FoxP3 Staining Buffer Set and incubated (1 × 106 cells/100 μl) with anti-CTLA4 (UC10-2B9-PE) Cells were washed and the data were collected and analysed as above.

    Techniques: Mouse Assay

    Comparison of different commercial intracellular staining kits on digested lamina propria cells. Dots plots of Collagenase A digested lamina propria cells from naïve C57BL/6 mice stained with Zombie NIR, CD45, CD4, ki67, FoxP3 and RORγt using four different commercial intracellular staining kits following the respective manufacturers’ instructions (representative of two independent experiments). The eBioscience FoxP3/Transcription Factor Staining Buffer showed the best separation of our cell populations of interest and was used henceforth.

    Journal: eLife

    Article Title: High-dimensional analysis of intestinal immune cells during helminth infection

    doi: 10.7554/eLife.51678

    Figure Lengend Snippet: Comparison of different commercial intracellular staining kits on digested lamina propria cells. Dots plots of Collagenase A digested lamina propria cells from naïve C57BL/6 mice stained with Zombie NIR, CD45, CD4, ki67, FoxP3 and RORγt using four different commercial intracellular staining kits following the respective manufacturers’ instructions (representative of two independent experiments). The eBioscience FoxP3/Transcription Factor Staining Buffer showed the best separation of our cell populations of interest and was used henceforth.

    Article Snippet: In our hands, the eBioscience FoxP3/Transcription Factor Staining Buffer Set yielded the best results and was used henceforth.

    Techniques: Staining, Mouse Assay

    CD28+/+ Treg suppress ISAT development in CD28−/− mice. (A) CD28−/− mice were given three i.v. injections of 10 6 sorted CD4+FoxP3+ Treg or CD4+FoxP3− T cells from CD28+/+ FoxP3−GFP NOD.H-2h4 mice as described

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

    Article Title: Reduced effectiveness of CD4+FoxP3+ regulatory T cells in CD28 deficient NOD.H-2h4 mice leads to increased severity of spontaneous autoimmune thyroiditis

    doi: 10.4049/jimmunol.1301253

    Figure Lengend Snippet: CD28+/+ Treg suppress ISAT development in CD28−/− mice. (A) CD28−/− mice were given three i.v. injections of 10 6 sorted CD4+FoxP3+ Treg or CD4+FoxP3− T cells from CD28+/+ FoxP3−GFP NOD.H-2h4 mice as described

    Article Snippet: Cells were washed and then fixed and permeabilized using the eBioscience FoxP3 buffer set (eBioscience, San Diego, CA) and stained for IFNγ (XMG1.2, PE).

    Techniques: Mouse Assay

    CD28−/− NOD.H-2h4 mice have fewer Treg than WT NOD.H-2h4 mice. CD4+FoxP3+ cells in spleens and lymph nodes of WT and CD28−/− NOD.H-2h4 mice were determined by flow cytometry. Dot plots of representative mice from each group

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

    Article Title: Reduced effectiveness of CD4+FoxP3+ regulatory T cells in CD28 deficient NOD.H-2h4 mice leads to increased severity of spontaneous autoimmune thyroiditis

    doi: 10.4049/jimmunol.1301253

    Figure Lengend Snippet: CD28−/− NOD.H-2h4 mice have fewer Treg than WT NOD.H-2h4 mice. CD4+FoxP3+ cells in spleens and lymph nodes of WT and CD28−/− NOD.H-2h4 mice were determined by flow cytometry. Dot plots of representative mice from each group

    Article Snippet: Cells were washed and then fixed and permeabilized using the eBioscience FoxP3 buffer set (eBioscience, San Diego, CA) and stained for IFNγ (XMG1.2, PE).

    Techniques: Mouse Assay, Flow Cytometry, Cytometry

    Conditional elimination of NR4A function in B cells. A-C . Lymphocytes from Nr4a1 +/+ and Nr4a1 -/- mice harboring NUR77-EGFP BAC Tg were stimulated with 10 μg/mL anti-IgM for the indicated times. qPCR was performed to determine relative expression of Nr4a1, Nr4a2 and Nr4a3 transcripts. Samples were also subjected to surface staining in parallel to detect % B cells via flow cytometry. Mean % B cells for Nr4a1+/+ samples was 37.6 ± 0.15 (SEM), and for Nr4a1 -/- samples was 33.5 ± 1.02 (SEM). N=3 biological replicates for all conditions. Nr4a1 +/+ samples correspond to data in Figs 1A-C . D . Purified B cells from Nr4a1 +/+ and Nr4a1 -/- mice were stimulated for 2 h with 10 μg/mL anti-IgM, and analyzed by RNAseq as described in Fig 1D and methods. Displayed is the FPKM of Nr4a2 and Nr4a3 . N=1 for the unstimulated condition, and N=4 for the stimulated condition. E . Left: Schematic showing the extent of nucleotide deletion in exon 3 of the Nr4a3 gene harboring ATG translation initiation site, resulting in the introduction of a premature stop codon. Right: Representative PCR showing absence of the WT Nr4a3 gene and the presence of a truncated product in Nr4a3 -/- mice. F . Purified B cells from WT, Nr4a1 -/- and Nr4a3 -/- mice were incubated +/- PMA and ionomycin for 2 h. Subsequently whole cell lysates were blotted with Ab to detect NOR1, c-MYC, pERK, and GAPDH. Red arrow indicates presence of low abundance truncated NOR1 protein. G . Representative flow plots showing the gated splenic Treg cell population in WT, Nr4a1-/-, Nr4a3-/- and Nr4a1-/- x Nr4a3-/- mice, as determined by CD25 expression and intra-cellular staining for Foxp3 in splenic CD4+ T cells. Plots are representative of at least 3 WT, Nr4a1-/- and Nr4a3-/- mice, and 2 DKO mice ( Nr4a1-/- Nr4a3-/- ). H . Chimeras were generated by reconstituting lethally irradiated CD45.1+ WT mice with bone marrow from either CD45.2+ mb1-cre mice (cre chimera), or CD45.2+ mb1-cre x Nr4a1 fl/fl x Nr4a3-/- mice (cDKO chimera). 8-10 weeks after reconstitution, mice were sacrificed for analysis by flow cytometry. Shown are the relative sizes of developmental B cell subsets in the spleen as determined via expression of CD21, CD23 and CD93 on gated B cells. N= 5 chimeras / genotype. I . Different genotypes are defined as the following: mb1-cre: mb1-cre mice; Nr4a1 cKO: mb1-cre x Nr4a1 fl/fl; cDKO: mb1-cre x Nr4a1 fl/fl x Nr4a3-/- . Purified B cells were isolated from whole lymph node via MACs purification, and stimulated for the indicated times with 10 μg/mL anti-IgM. Relative Nr4a2 transcript was determined via qPCR. Data in this figure depict N=3 biological replicates for all panels except (D) and (H) as noted above. Mean +/- SEM displayed for all graphs. Statistical significance was assessed with student’s t-test with Holm-Sidak (A, B, C); student’s t-test without correction (D, H); two-way ANOVA with Tukey’s (I). *p

    Journal: bioRxiv

    Article Title: “A negative feedback loop mediated by the NR4A family of nuclear hormone receptors restrains expansion of B cells that receive signal one in the absence of signal two”

    doi: 10.1101/2020.03.31.017434

    Figure Lengend Snippet: Conditional elimination of NR4A function in B cells. A-C . Lymphocytes from Nr4a1 +/+ and Nr4a1 -/- mice harboring NUR77-EGFP BAC Tg were stimulated with 10 μg/mL anti-IgM for the indicated times. qPCR was performed to determine relative expression of Nr4a1, Nr4a2 and Nr4a3 transcripts. Samples were also subjected to surface staining in parallel to detect % B cells via flow cytometry. Mean % B cells for Nr4a1+/+ samples was 37.6 ± 0.15 (SEM), and for Nr4a1 -/- samples was 33.5 ± 1.02 (SEM). N=3 biological replicates for all conditions. Nr4a1 +/+ samples correspond to data in Figs 1A-C . D . Purified B cells from Nr4a1 +/+ and Nr4a1 -/- mice were stimulated for 2 h with 10 μg/mL anti-IgM, and analyzed by RNAseq as described in Fig 1D and methods. Displayed is the FPKM of Nr4a2 and Nr4a3 . N=1 for the unstimulated condition, and N=4 for the stimulated condition. E . Left: Schematic showing the extent of nucleotide deletion in exon 3 of the Nr4a3 gene harboring ATG translation initiation site, resulting in the introduction of a premature stop codon. Right: Representative PCR showing absence of the WT Nr4a3 gene and the presence of a truncated product in Nr4a3 -/- mice. F . Purified B cells from WT, Nr4a1 -/- and Nr4a3 -/- mice were incubated +/- PMA and ionomycin for 2 h. Subsequently whole cell lysates were blotted with Ab to detect NOR1, c-MYC, pERK, and GAPDH. Red arrow indicates presence of low abundance truncated NOR1 protein. G . Representative flow plots showing the gated splenic Treg cell population in WT, Nr4a1-/-, Nr4a3-/- and Nr4a1-/- x Nr4a3-/- mice, as determined by CD25 expression and intra-cellular staining for Foxp3 in splenic CD4+ T cells. Plots are representative of at least 3 WT, Nr4a1-/- and Nr4a3-/- mice, and 2 DKO mice ( Nr4a1-/- Nr4a3-/- ). H . Chimeras were generated by reconstituting lethally irradiated CD45.1+ WT mice with bone marrow from either CD45.2+ mb1-cre mice (cre chimera), or CD45.2+ mb1-cre x Nr4a1 fl/fl x Nr4a3-/- mice (cDKO chimera). 8-10 weeks after reconstitution, mice were sacrificed for analysis by flow cytometry. Shown are the relative sizes of developmental B cell subsets in the spleen as determined via expression of CD21, CD23 and CD93 on gated B cells. N= 5 chimeras / genotype. I . Different genotypes are defined as the following: mb1-cre: mb1-cre mice; Nr4a1 cKO: mb1-cre x Nr4a1 fl/fl; cDKO: mb1-cre x Nr4a1 fl/fl x Nr4a3-/- . Purified B cells were isolated from whole lymph node via MACs purification, and stimulated for the indicated times with 10 μg/mL anti-IgM. Relative Nr4a2 transcript was determined via qPCR. Data in this figure depict N=3 biological replicates for all panels except (D) and (H) as noted above. Mean +/- SEM displayed for all graphs. Statistical significance was assessed with student’s t-test with Holm-Sidak (A, B, C); student’s t-test without correction (D, H); two-way ANOVA with Tukey’s (I). *p

    Article Snippet: FOXP3 staining FOXP3 staining was performed utilizing a FOXP3/transcription factor buffer set (eBioscience) in conjunction with an APC anti-FOXP3 Ab (eBioscience), as per manufacturer’s instructions.

    Techniques: Mouse Assay, BAC Assay, Real-time Polymerase Chain Reaction, Expressing, Staining, Flow Cytometry, Purification, Polymerase Chain Reaction, Incubation, Generated, Irradiation, Isolation, Magnetic Cell Separation

    High dietary salt intake enhances bone loss by modulating Th17-Treg cell balance. Cells from bone marrow (BM) of normal, low salt diet (LSD) and high salt diet mice groups (HSD) were isolated at the end of experiment, labelled and analysed by flow cytometry for percentage of Foxp3 + and Rorγt + , IL-10 + Foxp3 + and Rorγt + IL-17 + cells. Gating was first performed for CD4 + and later analysed for the expression of CD4 + Foxp3 + (Tregs), CD4 + Rorγt + (Th17), CD4 + IL-10 + (Tregs) and CD4 + IL-17 + (Th17). ( A ) Average percentage of CD4 + Foxp3 + T cells ( B ) Average percentage of CD8 + Foxp3 + T cells ( C ) Average percentage of CD4 + Rorγt + T cells. ( D ) Average percentage of Foxp3 + IL-10 + (Treg). ( E ) Average percentage of Rorγt + IL-17 + (Th17). The results were evaluated by using ANOVA with subsequent comparisons by Student t test for paired or nonpaired data, as appropriate. Analysis was performed using Sigma plot software (Systat Software, Inc., Germany). Values are reported as mean ± SEM (n = 10) and similar results were obtained in three independent experiments. Statistical significance was defined as p ≤ 0.05 (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001) with respect to low salt diet group.

    Journal: Scientific Reports

    Article Title: High dietary salt intake correlates with modulated Th17-Treg cell balance resulting in enhanced bone loss and impaired bone-microarchitecture in male mice

    doi: 10.1038/s41598-018-20896-y

    Figure Lengend Snippet: High dietary salt intake enhances bone loss by modulating Th17-Treg cell balance. Cells from bone marrow (BM) of normal, low salt diet (LSD) and high salt diet mice groups (HSD) were isolated at the end of experiment, labelled and analysed by flow cytometry for percentage of Foxp3 + and Rorγt + , IL-10 + Foxp3 + and Rorγt + IL-17 + cells. Gating was first performed for CD4 + and later analysed for the expression of CD4 + Foxp3 + (Tregs), CD4 + Rorγt + (Th17), CD4 + IL-10 + (Tregs) and CD4 + IL-17 + (Th17). ( A ) Average percentage of CD4 + Foxp3 + T cells ( B ) Average percentage of CD8 + Foxp3 + T cells ( C ) Average percentage of CD4 + Rorγt + T cells. ( D ) Average percentage of Foxp3 + IL-10 + (Treg). ( E ) Average percentage of Rorγt + IL-17 + (Th17). The results were evaluated by using ANOVA with subsequent comparisons by Student t test for paired or nonpaired data, as appropriate. Analysis was performed using Sigma plot software (Systat Software, Inc., Germany). Values are reported as mean ± SEM (n = 10) and similar results were obtained in three independent experiments. Statistical significance was defined as p ≤ 0.05 (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001) with respect to low salt diet group.

    Article Snippet: The reagents Anti-Human/Mouse-Rorγt-PE-(AFKJS-9) (12-6988), Anti-Mouse/Rat-Foxp3-APC-(FJK-16s) (17-5773), Foxp3/Transcription factor staining buffer (0-5523-00), RBC lysis buffer (00-4300-54) were obtained from eBioscience (USA).

    Techniques: Mouse Assay, Isolation, Flow Cytometry, Cytometry, Expressing, Software

    The percentage of CD4 + FOXP3 + T cells in Pg-AS patients, Pg patients, and HCs. A. Representative profiles of Pg-AS, Pg and HC for detecting CD4 + FOXP3 + T cells by flow cytometry within the CD4 + gate. Cells were stained with anti-FOXP3 monoclonal antibody (clone 236A/E7) and Fixation/Permeabilization buffer (no. 00-5521) from eBioscience. B. Representative profiles of Pg-AS, Pg and HC for detecting CD4 + FOXP3 + T cells by flow cytometry within the CD4 + gate. Cells were stained with anti-FOXP3 monoclonal antibody (clone 259D/C7) and fixation permeabilization reagents (no. 560098) from BD Bioscience. C. The percentage of CD4 + FOXP3 + T cells stained with eBioscience reagents in Pg-AS (n = 4), Pg (n = 4) and HC (n = 4) groups. D. The percentage of CD4 + FOXP3 + T cells stained with BD reagents in Pg-AS (n = 4), Pg (n = 4) and HC (n = 4) groups. Boxes with the 25th to 75th percentiles and the lines with the 5th to 95th percentiles are presented.* P

    Journal: PLoS ONE

    Article Title: Porphyromonas gingivalis Infection Reduces Regulatory T Cells in Infected Atherosclerosis Patients

    doi: 10.1371/journal.pone.0086599

    Figure Lengend Snippet: The percentage of CD4 + FOXP3 + T cells in Pg-AS patients, Pg patients, and HCs. A. Representative profiles of Pg-AS, Pg and HC for detecting CD4 + FOXP3 + T cells by flow cytometry within the CD4 + gate. Cells were stained with anti-FOXP3 monoclonal antibody (clone 236A/E7) and Fixation/Permeabilization buffer (no. 00-5521) from eBioscience. B. Representative profiles of Pg-AS, Pg and HC for detecting CD4 + FOXP3 + T cells by flow cytometry within the CD4 + gate. Cells were stained with anti-FOXP3 monoclonal antibody (clone 259D/C7) and fixation permeabilization reagents (no. 560098) from BD Bioscience. C. The percentage of CD4 + FOXP3 + T cells stained with eBioscience reagents in Pg-AS (n = 4), Pg (n = 4) and HC (n = 4) groups. D. The percentage of CD4 + FOXP3 + T cells stained with BD reagents in Pg-AS (n = 4), Pg (n = 4) and HC (n = 4) groups. Boxes with the 25th to 75th percentiles and the lines with the 5th to 95th percentiles are presented.* P

    Article Snippet: After washing with PBS, cells were treated with fixation permeabilization reagents (no. 560098, BD Biosciences) or the FOXP3/Transcription Factor Fixation/Permeabilization buffer (no. 00-5521, eBioscience), then incubated with anti-human FOXP3 antibody clone 259D/C7 (BD Biosciences, San Jose, CA), or clone 236A/E7 (eBioscience, San Jose, CA) for 30 minutes at 4°C to stain intracellular marker.

    Techniques: Flow Cytometry, Cytometry, Staining

    PI3Kδ is the major isoform involved in AKT phosphorylation in Treg cells following in vitro stimulation. ( A ) Lymph node single cell suspensions from naive wild-type mice were incubated with biotinylated anti-CD3 and anti-ICOS, and cells were stimulated for 5 min at 37°C by cross-linking with streptavidin in the presence of selective inhibitors for p110α (alpelisib, 500 nM), p110δ (idelalisib, 100 nM), or both combined (A+I). Unstimulated samples (US) were treated with DMSO and incubated for 5 min without the addition of streptavidin. Selective inhibition of p110δ and combined inhibition resulted in significantly lower AKT phosphorylation in Treg cells as measured by flow cytometry. ( B ) Lymph node single cell suspensions from naive FYC-WT, FYC-p110α fl , FYC-p110δ fl , or FYC-p110α fl δ fl mice were stimulated with pervanadate for 1 min at 37°C. Treg cell–specific deletion of p110δ and combined p110α/δ deletion resulted in significantly lower AKT phosphorylation in Treg cells as measured by flow cytometry. Treg cells were identified by flow cytometry by excluding debris based on forward and side scatter and then gating on single cells, followed by CD4 + cells, and then gating on CD25 + Foxp3 + Treg cells. n = 5; results from five independent experiments combined. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Loss of PI3K activity in regulatory T cells leads to neuronal inflammation

    doi: 10.4049/jimmunol.2000043

    Figure Lengend Snippet: PI3Kδ is the major isoform involved in AKT phosphorylation in Treg cells following in vitro stimulation. ( A ) Lymph node single cell suspensions from naive wild-type mice were incubated with biotinylated anti-CD3 and anti-ICOS, and cells were stimulated for 5 min at 37°C by cross-linking with streptavidin in the presence of selective inhibitors for p110α (alpelisib, 500 nM), p110δ (idelalisib, 100 nM), or both combined (A+I). Unstimulated samples (US) were treated with DMSO and incubated for 5 min without the addition of streptavidin. Selective inhibition of p110δ and combined inhibition resulted in significantly lower AKT phosphorylation in Treg cells as measured by flow cytometry. ( B ) Lymph node single cell suspensions from naive FYC-WT, FYC-p110α fl , FYC-p110δ fl , or FYC-p110α fl δ fl mice were stimulated with pervanadate for 1 min at 37°C. Treg cell–specific deletion of p110δ and combined p110α/δ deletion resulted in significantly lower AKT phosphorylation in Treg cells as measured by flow cytometry. Treg cells were identified by flow cytometry by excluding debris based on forward and side scatter and then gating on single cells, followed by CD4 + cells, and then gating on CD25 + Foxp3 + Treg cells. n = 5; results from five independent experiments combined. * p

    Article Snippet: Cells were incubated 15 min on ice, washed in Foxp3 Transcription Factor Wash Buffer (Invitrogen), and then stained with Abs against CD4, CD8, CD25, Foxp3 and pAKT for 1 h at room temperature in Foxp3 Transcription Factor Wash Buffer (Invitrogen).

    Techniques: In Vitro, Mouse Assay, Incubation, Inhibition, Flow Cytometry

    PI3Kδ inhibition does not influence EAE disease progression because of attenuation of both proinflammatory and Treg cell responses. EAE was induced in mice by s.c. injection of MOG 35–55 peptide in CFA, followed by two doses of pertussis toxin at 0 and 48 h postimmunization. ( A ) No difference in clinical score was observed when comparing wild-type and p110δ D910A/D910A littermate controls or when comparing C57BL/6 mice receiving twice daily doses of IC87114 (30 mg/kg) to mice receiving vehicle control by oral gavage. Mean ± SEM, n = 12; results representative of at least two independent experiments. ( B ) We found reduced proportions of IFN-γ–producing CD4 + T cells in the draining lymph nodes and spinal cords of p110δ D910A/D910A mice compared with wild-type controls at 14 d post–EAE induction. We also found a significant decrease in IL-17–producing CD4 + T cells and Foxp3 + Treg cells in the draining lymph nodes, but not spinal cord at 14 d post–EAE induction. CD4 + T cells were identified by flow cytometry by excluding debris based on forward and side scatter and then gating on single cells, followed by live CD45 + cells and then CD3 + T cells and then gating on CD4 + T cells. ( C ) Naive T cells were isolated from p110δ D910A/D910A and C57BL/6 mice and cultured for 5 d in the presence of anti-CD3/anti-CD28 stimulation and polarizing cytokines: Th1, 10 μg/ml anti–IL-4, 4 ng/ml IL-12; Th17, 10 μg/ml anti–IL-4 and anti–IFN-γ, 1 ng/ml TGF-β, 20 ng/ml IL-6, and 10 ng/ml IL-23 and IL-1β; Treg cells, 10 μg/ml anti-CD4 and anti–IFN-γ, 10 ng/ml TGF-β, and 20 ng/ml IL-2. Cells isolated from p110δ D910A/D910A mice maintained the potential for differentiation into Th1, Th17, and Treg cells, but differentiation was less efficient than wild-type cells. Live cells were identified by flow cytometry by excluding debris based on forward and side scatter and then gating on single cells, followed by live cells. Mean + Scatter, n = 5–6; results representative of at least two independent experiments. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Loss of PI3K activity in regulatory T cells leads to neuronal inflammation

    doi: 10.4049/jimmunol.2000043

    Figure Lengend Snippet: PI3Kδ inhibition does not influence EAE disease progression because of attenuation of both proinflammatory and Treg cell responses. EAE was induced in mice by s.c. injection of MOG 35–55 peptide in CFA, followed by two doses of pertussis toxin at 0 and 48 h postimmunization. ( A ) No difference in clinical score was observed when comparing wild-type and p110δ D910A/D910A littermate controls or when comparing C57BL/6 mice receiving twice daily doses of IC87114 (30 mg/kg) to mice receiving vehicle control by oral gavage. Mean ± SEM, n = 12; results representative of at least two independent experiments. ( B ) We found reduced proportions of IFN-γ–producing CD4 + T cells in the draining lymph nodes and spinal cords of p110δ D910A/D910A mice compared with wild-type controls at 14 d post–EAE induction. We also found a significant decrease in IL-17–producing CD4 + T cells and Foxp3 + Treg cells in the draining lymph nodes, but not spinal cord at 14 d post–EAE induction. CD4 + T cells were identified by flow cytometry by excluding debris based on forward and side scatter and then gating on single cells, followed by live CD45 + cells and then CD3 + T cells and then gating on CD4 + T cells. ( C ) Naive T cells were isolated from p110δ D910A/D910A and C57BL/6 mice and cultured for 5 d in the presence of anti-CD3/anti-CD28 stimulation and polarizing cytokines: Th1, 10 μg/ml anti–IL-4, 4 ng/ml IL-12; Th17, 10 μg/ml anti–IL-4 and anti–IFN-γ, 1 ng/ml TGF-β, 20 ng/ml IL-6, and 10 ng/ml IL-23 and IL-1β; Treg cells, 10 μg/ml anti-CD4 and anti–IFN-γ, 10 ng/ml TGF-β, and 20 ng/ml IL-2. Cells isolated from p110δ D910A/D910A mice maintained the potential for differentiation into Th1, Th17, and Treg cells, but differentiation was less efficient than wild-type cells. Live cells were identified by flow cytometry by excluding debris based on forward and side scatter and then gating on single cells, followed by live cells. Mean + Scatter, n = 5–6; results representative of at least two independent experiments. * p

    Article Snippet: Cells were incubated 15 min on ice, washed in Foxp3 Transcription Factor Wash Buffer (Invitrogen), and then stained with Abs against CD4, CD8, CD25, Foxp3 and pAKT for 1 h at room temperature in Foxp3 Transcription Factor Wash Buffer (Invitrogen).

    Techniques: Inhibition, Mouse Assay, Injection, Flow Cytometry, Isolation, Cell Culture

    Gamma-secretase inhibitors alter Th1 and Th17 responses but do not inhibit EAE. B6 mice were immunized with MOG35-55 to induce active EAE. Beginning on the day after immunization, the mice were randomized and treated with DMSO or GSI every other day for 18 days. A. Clinical course of EAE. B. Peak clinical score of DMSO and GSI-treated mice. C. Cumulative EAE scores for DMSO and GSI-treated animals. T cell cytokine expression in CNS-infiltrating CD4+ T cells from DMSO or GSI-treated mice following the Peak of EAE. On day 17 post-immunization, CNS cells were isolated and intracellular cytokine staining performed. D. Expression of IL-17 and IFNγ. E. Expression of GM-CSF and IFNγ. Distribution of the percentages of cells expressing IFNγ (F), IL-17 (G), GM-CSF (H) or FoxP3 (I) among CNS-infiltrating CD4+ T cells. J-L, Presence of cytokine expression in the spleens of EAE mice on day 17 post-immunization. Distribution of the percentages of cells expressing IFNγ (J), IL-17 (K), GM-CSF (L). Symbols indicate the percentage of cells from individual mice. Also plotted are the mean and SEM for each treatment group. Open circles indicate DMSO treatment, filled squares indicate treatment with GSI. Error bars represent SEM. Asterisks indicate significant differences (* p

    Journal: PLoS ONE

    Article Title: Presenilin1 regulates Th1 and Th17 effector responses but is not required for experimental autoimmune encephalomyelitis

    doi: 10.1371/journal.pone.0200752

    Figure Lengend Snippet: Gamma-secretase inhibitors alter Th1 and Th17 responses but do not inhibit EAE. B6 mice were immunized with MOG35-55 to induce active EAE. Beginning on the day after immunization, the mice were randomized and treated with DMSO or GSI every other day for 18 days. A. Clinical course of EAE. B. Peak clinical score of DMSO and GSI-treated mice. C. Cumulative EAE scores for DMSO and GSI-treated animals. T cell cytokine expression in CNS-infiltrating CD4+ T cells from DMSO or GSI-treated mice following the Peak of EAE. On day 17 post-immunization, CNS cells were isolated and intracellular cytokine staining performed. D. Expression of IL-17 and IFNγ. E. Expression of GM-CSF and IFNγ. Distribution of the percentages of cells expressing IFNγ (F), IL-17 (G), GM-CSF (H) or FoxP3 (I) among CNS-infiltrating CD4+ T cells. J-L, Presence of cytokine expression in the spleens of EAE mice on day 17 post-immunization. Distribution of the percentages of cells expressing IFNγ (J), IL-17 (K), GM-CSF (L). Symbols indicate the percentage of cells from individual mice. Also plotted are the mean and SEM for each treatment group. Open circles indicate DMSO treatment, filled squares indicate treatment with GSI. Error bars represent SEM. Asterisks indicate significant differences (* p

    Article Snippet: The cells were fixed and then permeabilized using the FoxP3 transcription factor buffer kit (eBioscience).

    Techniques: Mouse Assay, Expressing, Isolation, Staining

    PSEN1 cKO mice are susceptible to EAE. WT and PSEN1 cKO mice were immunized with MOG35-55 to induce active EAE. A. Clinical course of EAE. B. Peak clinical score of EAE mice. C. Cumulative EAE scores for DMSO and GSI-treated animals. T cell cytokine expression in CNS-infiltrating CD4+ T cells from DMSO or GSI-treated mice following the Peak of EAE. On day 17 post-immunization, CNS cells were isolated and intracellular cytokine staining performed. D. Expression of IL-17 and IFNγ. E. Expression of GM-CSF and IFNγ. Distribution of the percentages of cells expressing IFNγ (F), IL-17 (G), GM-CSF (H) or FoxP3 (I) among CNS-infiltrating CD4+ T cells. Symbols indicate the percentage of cells from individual mice. Open circles indicate results from WT and filled squares indicate PSEN1 cKO mice. Error bars represent SEM. Asterisks indicate significant differences (* p

    Journal: PLoS ONE

    Article Title: Presenilin1 regulates Th1 and Th17 effector responses but is not required for experimental autoimmune encephalomyelitis

    doi: 10.1371/journal.pone.0200752

    Figure Lengend Snippet: PSEN1 cKO mice are susceptible to EAE. WT and PSEN1 cKO mice were immunized with MOG35-55 to induce active EAE. A. Clinical course of EAE. B. Peak clinical score of EAE mice. C. Cumulative EAE scores for DMSO and GSI-treated animals. T cell cytokine expression in CNS-infiltrating CD4+ T cells from DMSO or GSI-treated mice following the Peak of EAE. On day 17 post-immunization, CNS cells were isolated and intracellular cytokine staining performed. D. Expression of IL-17 and IFNγ. E. Expression of GM-CSF and IFNγ. Distribution of the percentages of cells expressing IFNγ (F), IL-17 (G), GM-CSF (H) or FoxP3 (I) among CNS-infiltrating CD4+ T cells. Symbols indicate the percentage of cells from individual mice. Open circles indicate results from WT and filled squares indicate PSEN1 cKO mice. Error bars represent SEM. Asterisks indicate significant differences (* p

    Article Snippet: The cells were fixed and then permeabilized using the FoxP3 transcription factor buffer kit (eBioscience).

    Techniques: Mouse Assay, Expressing, Isolation, Staining