Surface staining and intracellular staining
A total of 1.5x106 and 2x106 fresh
PBMCs were used for Tfh and Tfreg analysis, respectively. PBMCs were
stained with a fixable viability dye-700-alexa fluorophore at
40C for 15 minutes in the dark (for dead cell
discrimination) prior to surface and intracellular staining. PBMCs were
subsequently stained with surface markers at 40C for
30 minutes in the dark with the following monoclonal antibodies:
anti-CD3-PE-cy7, anti-CD4-Buv496, anti-CD8-FITC, anti-CXCR5-Percp-cy5.5,
anti-PD-1-BV786, anti-CXCR3-PE, anti-CCR6-BV711, and anti-CD25-APC-cy-7.
All monoclonal antibodies were purchased from BD Bioscience. For
intracellular cytokine staining (ICS), PBMCs were stimulated for 5 hours
with phorbol 12-myristate 13-acetate (PMA) (50 ng/mL), ionomycin (500
ng/mL) (Sigma-Aldrich), and golgistop (monensin; 0.6 µL/mL) (BD
Bioscience). Following stimulation, PBMCs were fixed and permeabilized
with cytofix/cytoperm cell permeabilization buffer (BD Bioscience). ICS
was then performed using monoclonal antibodies against IFN-γ-APC,
IL-17-BV650, IL21-BV421, IL-10-PE-CF594 (BD Bioscience) and IL-4-BV605
(Bio-legend) following incubation under the same conditions described
above for surface staining. Simultaneously, FoxP3 staining was performed
for 30 minutes in dark at 40C using anti-FoxP3-PE
antibody according to the manufacturer’s instructions (eBioscience).