Foxp3 buffer system

For cellular phenotyping, cells were washed, incubated with anti-CD16 to block Fc receptors and stained with VIVID (Invitrogen, Carlsbad, CA) to exclude dead cells. Cells were surface stained in cocktails with different combinations of fluorescent dye-conjugated monoclonal antibodies (mAbs) depending on the panel that included antibodies raised against CD3ε, CD4, CD8, CD19, CD11b, Ly6C, Ly6G, F4/80, NKp46, CD127 (IL-7Rα), CCR6 and γδTCR (BD Biosciences, San Jose, CA). For intracellular cytokine staining (ICS) experiments, single cell suspensions of spleen, MLN, small intestinal IELs (sIEL) and sLPLs, colonic IELs (cIEL) and cLPLs were re-stimulated for 4 hours with 40ng/mL phorbol-12-myristate-13-acetate (PMA) and 2μg/mL ionomycin (Millipore, Billerica, MA, USA) in the presence of brefeldin A (BD Biosciences, San Jose, CA). Cells were surface stained as above then were fixed and permeabilized using the eBioscience Foxp3 buffer system and stained with antibodies against mouse IL-10, IFN-γ, IL-17, IL-22, RORγt and/or Foxp3 (BD Biosciences, San Jose, CA). Cells were acquired on a BD LSRII. Data were analyzed with FlowJo software (Tree Star, Ashland, OR).