Human-specific antibodies used for flow cytometry included: CD4(RPA-T4), CD8((RPA-T8), CD25(M-A251), CD45RA(HI100), Annexin V(PE), 7-AAD(FITC) were purchased from BD Pharmingen, while FoxP3 (clone 249D) is from BioLegend and Ki67 is from eBioscience. The annexin V (PE)/7-AAD(FITC) were applied to assess the apoptosis of tTreg. Acquisition was performed using a CATON (BD Bioscience) and data were analyzed using FlowJo software (TreeStar).
Foxp3 clone 249d
Manufactured by BioLegend
The FoxP3 (clone 249D) is a mouse monoclonal antibody that recognizes the transcription factor FoxP3, a master regulator of regulatory T cells (Tregs). This antibody can be used for the identification and characterization of FoxP3-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd25 m a251, by BD (1 mentions)
Fixation permeabilization buffer set, by Thermo Fisher Scientific (1 mentions)
Cd4 clone rpa t4, by Thermo Fisher Scientific (1 mentions)
Foxp3 intracellular staining kit, by BioLegend (1 mentions)
Fitc conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Facscalibur machine, by BD (1 mentions)
Ifn γ, by BD (1 mentions)
Il 17a, by BD (1 mentions)
Annexin 5 pe 7 aad, by BD (1 mentions)
3 protocols using foxp3 clone 249d
1
Analyzing Apoptosis in Treg Cells
2
T Cell Intracellular Staining Protocol
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For intracellular staining, indicated populations of T cells were fixed and permeabilizied with Fixation/Permeabilization buffer set (00–5523; eBioscience), washed, and stained (30 min, 4 °C) with primary antibodies (PKC-θ or Dlgh1). Then, the cells were incubated (30 min, 4 °C) with a FITC-conjugated secondary antibodies (Jackson ImmunoResearch Lab. Inc., West Grove, PA). Human-specific antibodies used for flow cytometry CD4 (clone RPA-T4) and CD25 (M-A251), were purchased from eBioscience, while FOXP3 (clone 249D) is from BioLegend. Cells were stained for CD4, CD25, FOXP3 using the BioLegend FOXP3 intracellular staining kit. We analyzed samples in a FACSCalibur machine (BD, Franklin Lakes, NJ).
3
Flow Cytometry Analysis of Regulatory T Cells
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Example 10
Human-specific antibodies used for flow cytometry included: CD4 (RPA-T4), CD8 (RPA-T8), CD25 (M-A251), CD45RA (HI100), IFN-gamma, IL-17a, annexin V (PE), 7-AAD (FITC) were purchased from BD Pharmingen, while Foxp3 (clone 249D) was obtained from BioLegend and Ki67 from eBioscience. For cytokine analysis, cells were pre-treated by PMA, ionomycin and brefeldin A for 6 hours and then stained by different cytokine antibodies. The annexin V (PE)/7-AAD (FITC) were applied to assess the apoptosis of tTreg. Acquisition was performed using a LSRII (BD Bioscience) and data were analyzed using FlowJo software (TreeStar) or IDEA (Admin). Nuclear localization of NF-kB was quantitated using an imaging flow cytometer (Imagestream, Amnis Corp; Seattle, Wash.) by intracellularly staining tTreg with anti-NF-kB (vendor) and, after washing, incubating with DRAQS for 15 minutes at 10 μM final concentration.
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