Flow Cytometric Analysis of Th17 and Treg Cells

PBMCs will be cultured in 48-well culture plates at 0.5 to 1 × 10⁶ cells/mL per well and will be stimulated with 5 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 μg/ml ionomycin (both from Sigma Aldrich, Oakville, Ontario, Canada) for two hours at 37°C. A total of 2 μg/ml brefeldin A (Sigma Aldrich, Oakville, Ontario, Canada) will then be added to block cytokine secretion and cells will be cultured for 18 hours at 37°C. PBMCs will be surface stained followed by fixation and permeabilization using the Cytofix/Cytoperm Permeabilization kit (BD Bioscience, Mississauga, Ontario, Canada) for intracellular staining for IL-17A and IFN-ɣ (positive control). Treg cells will be defined by flow cytometry as being CD3+ CD4+ CD25^high CD127^low FoxP3^high and Th17 cells as being CD3+ CD4+ IL-17A+ upon PMA and ionomycin stimulation [27 (link)].