BD PharmingenTM Transcription Factor Phospho Buffer Set

Description

Multicolor flow cytometric analysis Stat5 pY694 expression by human T cell subsets in response to different IL-2 doses. Human peripheral blood mononuclear cells were not cultured No Stim or were cultured IL-2; 37oC for 15 min with 1, 10, or 100 ng/mL doses of Recombinant Human IL-2 protein Cat. No. 554603 as indicated. The cells were then fixed, permeabilized, and stained with fluorescent antibodies specific for surface CD3, CD4, CD8, CD25, CD45RA and intracellular FoxP3, T-bet, pStat5 pY694 markers using the BD Pharmingen TFP Buffer Set and protocol. The fluorescence histograms showing Stat5 pY694 expression were derived from CD3-negative or CD3-positive gated events with CD45RA+ T-bet-, CD45RA- Tbet+, or CD45RA- T-bet- phenotypes and with the forward and side light-scatter characteristics of intact lymphocytes as indicated. Multicolor flow cytometric analysis of cytokine signaling responses in human T-cell and other leucocyte subsets defined by the correlated expression of FOXP3+ and T-bet+ or other markers. Human peripheral blood mononuclear cells were treated with 100 ng/mL of recombinant human IFN- alpha; 100 ng/mL IL-6; or 0.1, 1, 10, or 100 ng/mL of IL-2 for 15 minutes at 37oC and then fixed, permeabilized, and stained using the BD Pharmingen TFP Buffer Set as described in the protocol. Leucocyte subsets were identified by the correlated expression of FoxP3 and T-bet, and additional phenotypic markers specific for Th, Treg, NK, CD8 T cells, B cells, and monocytes using surface antibodies Anti-Human CD45RA, CD3, CD4, CD20, and/or CD25 . Patterns of Stat1, Stat 3, Stat 4, Stat 5 phosphorylation in response to cytokine stimulation are shown across leucocyte subsets in five donors. Heat maps display fold-change in phosphorylated STAT staining in response to stimulation. Not detected, 50 events acquired, Multicolor flow cytometric analysis of surface and intracellular markers coexpressed by previously-frozen and thawed human CD4+ T cells. Top Panel - IL-2-induced pSTAT5 pY694 signaling responses by human CD4 T cell subsets defined by FoxP3. Frozen human peripheral blood mononuclear cells were thawed and cultured 37C, 15 min with 0.2, 0.4, 1 and 10 U/mL or 12, 24, 61, 611 pg/mL, respectively of recombinant human IL-2 protein. The cells were then fixed, permeabilized, and stained with fluorescent antibodies specific for CD3, CD4, FoxP3, and phosphorylated Stat5 pY694 using the BD Pharmingen TFP Buffer Set and protocol. Two-color flow cytometric contour plots showing the correlated expression of FoxP3 versus phosphorylated Stat5 pY694 were derived from CD3+ CD4+ gated events with the forward and side light-scatter characteristics of intact lymphocytes. Bottom Panel - CD4 T cell subsets defined by surface and intracellular markers. Frozen human peripheral blood mononuclear cells were thawed and then fixed and permeabilized using the BD Pharmingen TFP Buffer Set and protocol. Cells were labeled with surface Anti-human CD4, CD45RA, and CD25 and intracellular Anti-FoxP3 antibodies after treatment with Perm III. Two-color flow cytometric contour plots showing the correlated expression of FoxP3 versus CD25 Left Plot or CD45RA Right Plot were derived from CD3+ CD4+ gated events with the forward and side light-scatter characteristics of intact lymphocytes. Data courtesy of Yang, Hsiu-Mien, King College London, UK. Multicolor flow cytometric analysis Stat5 pY694 expression by human T cell subsets in response to different IL-2 doses. Human peripheral blood mononuclear cells were not cultured No Stim or were cultured IL-2; 37oC for 15 min with 1, 10, or 100 ng/mL doses of Recombinant Human IL-2 protein Cat. No. 554603 as indicated. The cells were then fixed, permeabilized, and stained with fluorescent antibodies specific for surface CD3, CD4, CD8, CD25, CD45RA and intracellular FoxP3, T-bet, pStat5 pY694 markers using the BD Pharmingen TFP Buffer Set and protocol. The fluorescence histograms showing Stat5 pY694 expression were derived from CD3-negative or CD3-positive gated events with CD45RA+ T-bet-, CD45RA- Tbet+, or CD45RA- T-bet- phenotypes and with the forward and side light-scatter characteristics of intact lymphocytes as indicated. Show More Multicolor flow cytometric analysis of cytokine signaling responses in human T-cell and other leucocyte subsets defined by the correlated expression of FOXP3+ and T-bet+ or other markers. Human peripheral blood mononuclear cells were treated with 100 ng/mL of recombinant human IFN- alpha; 100 ng/mL IL-6; or 0.1, 1, 10, or 100 ng/mL of IL-2 for 15 minutes at 37oC and then fixed, permeabilized, and stained using the BD Pharmingen TFP Buffer Set as described in the protocol. Leucocyte subsets were identified by the correlated expression of FoxP3 and T-bet, and additional phenotypic markers specific for Th, Treg, NK, CD8 T cells, B cells, and monocytes using surface antibodies Anti-Human CD45RA, CD3, CD4, CD20, and/or CD25 . Patterns of Stat1, Stat 3, Stat 4, Stat 5 phosphorylation in response to cytokine stimulation are shown across leucocyte subsets in five donors. Heat maps display fold-change in phosphorylated STAT staining in response to stimulation. Not detected, 50 events acquired, Show More Multicolor flow cytometric analysis of surface and intracellular markers coexpressed by previously-frozen and thawed human CD4+ T cells. Top Panel - IL-2-induced pSTAT5 pY694 signaling responses by human CD4 T cell subsets defined by FoxP3. Frozen human peripheral blood mononuclear cells were thawed and cultured 37C, 15 min with 0.2, 0.4, 1 and 10 U/mL or 12, 24, 61, 611 pg/mL, respectively of recombinant human IL-2 protein. The cells were then fixed, permeabilized, and stained with fluorescent antibodies specific for CD3, CD4, FoxP3, and phosphorylated Stat5 pY694 using the BD Pharmingen TFP Buffer Set and protocol. Two-color flow cytometric contour plots showing the correlated expression of FoxP3 versus phosphorylated Stat5 pY694 were derived from CD3+ CD4+ gated events with the forward and side light-scatter characteristics of intact lymphocytes. Bottom Panel - CD4 T cell subsets defined by surface and intracellular markers. Frozen human peripheral blood mononuclear cells were thawed and then fixed and permeabilized using the BD Pharmingen TFP Buffer Set and protocol. Cells were labeled with surface Anti-human CD4, CD45RA, and CD25 and intracellular Anti-FoxP3 antibodies after treatment with Perm III. Two-color flow cytometric contour plots showing the correlated expression of FoxP3 versus CD25 Left Plot or CD45RA Right Plot were derived from CD3+ CD4+ gated events with the forward and side light-scatter characteristics of intact lymphocytes. Data courtesy of Yang, Hsiu-Mien, King College London, UK. Show More Multicolor flow cytometric analysis Stat5 pY694 expression by human T cell subsets in response to different IL-2 doses. Human peripheral blood mononuclear cells were not cultured No Stim or were cultured IL-2; 37oC for 15 min with 1, 10, or 100 ng/mL doses of Recombinant Human IL-2 protein Cat. No. 554603 as indicated. The cells were then fixed, permeabilized, and stained with fluorescent antibodies specific for surface CD3, CD4, CD8, CD25, CD45RA and intracellular FoxP3, T-bet, pStat5 pY694 markers using the BD Pharmingen TFP Buffer Set and protocol. The fluorescence histograms showing Stat5 pY694 expression were derived from CD3-negative or CD3-positive gated events with CD45RA+ T-bet-, CD45RA- Tbet+, or CD45RA- T-bet- phenotypes and with the forward and side light-scatter characteristics of intact lymphocytes as indicated. Multicolor flow cytometric analysis of cytokine signaling responses in human T-cell and other leucocyte subsets defined by the correlated expression of FOXP3+ and T-bet+ or other markers. Human peripheral blood mononuclear cells were treated with 100 ng/mL of recombinant human IFN- alpha; 100 ng/mL IL-6; or 0.1, 1, 10, or 100 ng/mL of IL-2 for 15 minutes at 37oC and then fixed, permeabilized, and stained using the BD Pharmingen TFP Buffer Set as described in the protocol. Leucocyte subsets were identified by the correlated expression of FoxP3 and T-bet, and additional phenotypic markers specific for Th, Treg, NK, CD8 T cells, B cells, and monocytes using surface antibodies Anti-Human CD45RA, CD3, CD4, CD20, and/or CD25 . Patterns of Stat1, Stat 3, Stat 4, Stat 5 phosphorylation in response to cytokine stimulation are shown across leucocyte subsets in five donors. Heat maps display fold-change in phosphorylated STAT staining in response to stimulation. Not detected, 50 events acquired, Multicolor flow cytometric analysis of surface and intracellular markers coexpressed by previously-frozen and thawed human CD4+ T cells. Top Panel - IL-2-induced pSTAT5 pY694 signaling responses by human CD4 T cell subsets defined by FoxP3. Frozen human peripheral blood mononuclear cells were thawed and cultured 37C, 15 min with 0.2, 0.4, 1 and 10 U/mL or 12, 24, 61, 611 pg/mL, respectively of recombinant human IL-2 protein. The cells were then fixed, permeabilized, and stained with fluorescent antibodies specific for CD3, CD4, FoxP3, and phosphorylated Stat5 pY694 using the BD Pharmingen TFP Buffer Set and protocol. Two-color flow cytometric contour plots showing the correlated expression of FoxP3 versus phosphorylated Stat5 pY694 were derived from CD3+ CD4+ gated events with the forward and side light-scatter characteristics of intact lymphocytes. Bottom Panel - CD4 T cell subsets defined by surface and intracellular markers. Frozen human peripheral blood mononuclear cells were thawed and then fixed and permeabilized using the BD Pharmingen TFP Buffer Set and protocol. Cells were labeled with surface Anti-human CD4, CD45RA, and CD25 and intracellular Anti-FoxP3 antibodies after treatment with Perm III. Two-color flow cytometric contour plots showing the correlated expression of FoxP3 versus CD25 Left Plot or CD45RA Right Plot were derived from CD3+ CD4+ gated events with the forward and side light-scatter characteristics of intact lymphocytes. Data courtesy of Yang, Hsiu-Mien, King College London, UK.