Quantifying Murine Immune Cell Subsets

To quantify the populations of Th1, Th2, Th17 and Foxp3-positive Treg cells, murine splenocytes and stromal vascular cells were stimulated with 25ng/mL phorbol myristate acetate (PMA) and 250ng/mL ionomycin (both from Sigma-Aldrich, St. Louis, MO, USA) and Golgi Stop (BD Biosciences, San Diego, CA, USA) in a 24-well plate and incubated for 4 hours. The stimulated splenocytes were stained with PerCP-conjugated anti-CD4 antibody (eBiosciences), then fixed and permeabilized using the Cytofix/Cytoperm Plus Kit (BD Biosciences) following the manufacturer’s protocol. Splenocytes were then reacted with fluorescein isothiocyanate (FITC)-conjugated anti-IL-17 antibody (eBiosciences). For analysis of the number of Treg cells, splenocytes were labeled with anti-CD4 and anti-CD25 antibodies, followed by fixation, permeabilization, and intracellular staining with anti-Foxp3 antibody as per the manufacturer’s instruction. To quantify the population of F4/80+CD11c+CD206- (M1) and F4/80+CD11c-CD206+ (M2) cells, mouse splenocytes were stained using allophycocyanin (APC)-conjugated anti-F4/80 antibody, FITC-conjugated anti-CD11c antibody and Phycoerythrin (PE)-conjugated anti-CD206 antibody (eBiosciences). All samples were analyzed by a FACS Calibur device (BD Pharmingen) [48 (link)].