Foxp3 mouse regulatory t cell staining kit

Fluorescent dye–conjugated antibodies were purchased from BD Biosciences, Biolegend or Ebioscience (Thermofisher). The following clones were used: anti-CD45.1, A20; anti-Foxp3, FJK-16s; anti-CD4, RM4–5; anti-CD45.2, 104; anti-CD8α, 53–6.7; anti-CD8β, YTS 156.7.7; anti-CD44, IM7; anti-CD45, 30-F11; anti-CD62L, MEL-14; G8.8; anti-TCRβ, H57–597; anti-TCRγδ, eBioG23; anti-CD25, PC61.5;. Live/dead fixable dye Aqua (ThermoFisher Scientific) was used according to manufacturer’s instructions. Intracellular staining of Foxp3 was conducted using Foxp3 Mouse Regulatory T Cell Staining Kit (eBioscience, USA). Flow cytometry data was acquired on a LSR-II flow cytometer (Becton Dickinson, USA) and analyzed using FlowJo software package (Tri-Star, USA). Anti-ThPOK ChIP antibody was kindly provided by T. Egawa (Wash. U.).

Fluorescent-dye-conjugated antibodies were purchased from BD-Pharmingen (anti-CD4, 550954; anti-CD25, 553866; anti-CD103, 557495; anti-IL-17a, 559502; anti-T-bet, 561312) or eBioscience (anti-CD8α, 56-0081; anti-CD44, 56-0441; anti-CD45.1, 25-0453; anti-CD45.2, 47-0454; anti-CD62L, 48-0621; anti-TCR-β, 47-5961; anti-IFN-γ, 25-7311; anti-IL-22, 24-7221; anti-RORγt, 12-6981; anti-Foxp3, 17-5773). Flow cytometry data was acquired on a LSR-II flow cytometer (Becton Dickinson) and analyzed using FlowJo software package (Tree Star). Intracellular staining of Foxp3 was conducted using Foxp3 Mouse Regulatory T cell Staining Kit (eBioscience).
For flow cytometric analysis of cytokine-secreting cells, cells were incubated in the presence of 100ng/ml PMA (Sigma), 500ng/ml Ionomycin (Sigma) for 4.5h and 10μg/ml brefeldin A (BFA) (Sigma) for the last 2.5h prior staning. Cell populations were first stained with antibodies against the indicated cell surface markers, followed by permeabilization in Fix/Perm buffer, and intracellular staining in Perm/Wash buffer (BD Pharmingen).