Percp cy5.5 conjugated anti human cd4

For Th1 and Th17 analysis, cells were stimulated for 4-5 h with 50 ng/mL PMA (Sigma-Aldrich, St. Louis, MO, USA), 750 ng/mL ionomycin (Calbiochem, La Jolla, CA, USA), and Golgiplug at the recommended concentrations (BD Pharmingen, San Diego, CA, USA) at 37°C under a 5% CO₂ environment. Then cells were stained with Percp cy5.5-conjugated anti-human CD4 (BD Pharmingen, San Diego, CA, USA) at room temperature for 15 min, fixed and permeabilized with Cytofix/Cytoperm solution (BD Pharmingen, San Diego, CA, USA), and then labeled with APC-conjugated anti-human IFN-γ (Biolegend, San Diego, CA, USA) and PE-conjugated anti-human IL-17 (Santa Cruz Biotechnology, CA, USA). For Treg analysis, cells were labeled with Percp cy5.5-conjugated anti-human CD4 and PE-conjugated anti-human CD25 (BD Pharmingen, San Diego, CA, USA). For intracellular Foxp3 staining, cells were fixed and permeabilized according the manufacturer's protocol for the staining set (eBioscience, San Diego, CA, USA) and labeled with FITC-conjugated anti-human Foxp3 (eBioscience, San Diego, CA, USA). For Tfh analysis, cells were incubated with Percp cy5.5-conjugated anti-human CD4, FITC-conjugated anti-CCR7 (BD Pharmingen, San Diego, CA, USA) and APC-conjugated anti-PD-1 (BD Pharmingen, San Diego, CA, USA). The percentage of positive stained cells was analyzed using a FACS instrument (BD Biosciences, San Jose, CA, USA).