Anti mouse foxp3 fjk 16s

Naïve CD4⁺ T cells were incubated in T_reg cell differentiation condition in the presence of the following: plate-bound anti-CD3 (2 µg/ml, eBioscience), anti-CD28 (3 µg/ml, eBioscience), anti-IL-4 (10 µg/ml, BioXCell), anti-IFN-γ (10 µg/ml, BioXCell), anti-IL-12 (10 µg/ml, BioXcell), IL-2 100 U/ml, Peprotech) and TGF-β (5 ng/ml, R&D Systems). Cells were stained for surface markers with anti-mouse CD4 (RM4–5, eBioscience) and anti-mouse CD8 (53-6.7 eBiosciences). Intracellular staining was performed using Cytofix/Cytoperm buffer (eBioscience) to fix and permeabilize cells. Cells were then washed and stained with following antibodies: anti-mouse Helios (22F6, BioLegend), anti-mouse Ki67 (B56, BD Biosciences) and anti-mouse Foxp3 (FJK-16s, eBioscience). Dead cells were excluded from analysis using LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies). Sample were run on FACSCanto II cell analyzer (BD Biosciences) and analyzed with FlowJo software.

Various fluorochrome labeled antibodies specific for CD8 (53-6.72), CD4 (RM4-5), CD3 (145-2C11), TCRVα8.3 (B21.14), TCRVβ8.1.2 (KJ16), TCRVβ4 (KT4), CD44 (IM7.8.1), CD62L (MEL-14), CD25 (7D4), CD19 (ID3), CD11c (N418), CD11b (M1/70), PDCA-1 (927), CD80 (16-10A1), CD86 (GL-1), CTLA-4 (IB8), GITR (YGITR765), GITRL (YGL386), and CD70 (FR70), were purchased from BD Bioscience (San Jose, CA, USA) or BioLegend (San Diego, CA). Anti-mouse Foxp3 (FJK-16s) was purchased from eBioscience (San Diego, CA). NRP-V7-H-2K^d Tetramer-KYNKANVFL-PE was acquired from the NIH tetramer core facility (Atlanta, GA). Anti-mouse CD25 (PC61.5) and CD4 (GK1.5) were purchased from Bioxcell (West Lebanon, NH). Dead cells were excluded by DAPI staining. Stained cells were acquired using a FACS LSRII instrument (BD Bioscience). All flow cytometric data were analyzed with FlowJo (Tree Star, Ashland, OR). In one experiment the previously described flow cytometry based assay (13 (link)) was used to compare the ability of residual myeloid APC from NOD-scid and NSG mice to support regulatory T-cell (Treg) activity. In studies assessing their proliferative capacity by flow cytometry, Tregs (Foxp3-eGFP⁺) or conventional T-cells (Tconv; Foxp3-eGFP⁻) cells were stained following the manufacturers recommendations with the Dye eFluor^®670 (eBioscience, San Diego, CA) that dilutes with each division cycle.

IL-17a and IFN-γ cytokine expression were assessed by flow cytometry as described previously^{45 (link)}. Briefly, the cells were stimulated for 4 h in phorbol 1,2-myristate 1,3-acetate (5 ng/ml) and ionomycin (1 µg/ml) in the presence of protein transport inhibitor GolgiPlug (BD Pharmingen). Cells were stained for surface markers with following antibodies: anti-mouse TCRβ (H57–597; eBioscience), anti-mouse CD4 (RM4–5; eBioscience), anti-mouse CD45 (30-F11, eBioscience). Cells were washed and fixed using Cytofix/Cytoperm buffer (BD Pharmingen or eBioscience). Intracellular staining was performed with following antibodies: anti-mouse IL-17a (TC11-18H10.1; BioLegend), anti-mouse IFN-γ (XMG1.2, eBiosciences), anti-mouse Foxp3 (FJK-16s, eBioscience) and anti-human/mouse T-bet (eBio4B10, eBiosciences). Dead cells were excluded from analysis using Zombie Yellow Fixable Viability Kit (Biolegend) or LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies).