Flow cytometry determination of CD4+ T helper T cell subpopulation ratios in peripheral blood

Detection of the proportion of Th1, Th2, and Th17 cells in CD4+ T cells in human peripheral blood using the Human Th1/Th2/Th17 Staining Kit (MultiSciences Biotech Co., Ltd., China). First, 125 μl of fresh heparin anticoagulated blood was mixed with an equal amount of RPMI1640 medium (GIBCO, USA), 1 μl each of the stimulant and blocker supplied with the kit was added, shaken and mixed, and incubated at a constant temperature incubator at 37 °C for 5 h. The 100 μl cell suspension was taken into another clean tube, and CD3-APC-Cy7 antibody with CD8α-PerCP-Cy5.5 antibody was added to each tube and incubated at room temperature and in the dark. Next, FIX & PERM medium A was added and washed after incubation. FIX & PERM Medium B, IFN-γ-FITC, IL-4-APC and IL-17A-PE monoclonal antibodies, were added sequentially, mixed, incubated, and washed. The cells were blown and mixed by adding 350 μl FBS and detected by FACS Canto II flow cytometry (BD, USA). Data were analyzed using Flow Jo v10 software.