Il 6 fitc

For determination of ex vivo cytokine production, PBMCs were suspended in 1640 RPMI medium supplemented with 10% heat-inactivated FBS (fetal bovine serum), 1% L-glutamine (200 mM), 1% penicillin/streptomycin (100 ×), 1% Hepes (1 M), 1% 2-ME (14.3 M). For T cell stimulation, 1 × 10⁶ PBMCs were placed in a 96 well plate and stimulated for 4 hours at 37 degrees C with a mixture of PMA (Sigma) and ionomycin (Sigma) at a concentration of 1 µg/mL each. For monocyte stimulation, 1 × 10⁶ PBMCs were placed in a 96 well plate and stimulated for 4 hours at 37 degrees C with LPS at a concentration of 1 µg/mL. Brefeldin A (GolgiPlug, BD Biosciences) was added to all cells after 1 hour of stimulation at a concentration of 1 µg/mL. Intracellular staining was performed after fixation and permeabilization according to manufacturer’s instructions (BD Biosciences) utilizing fluorophore-labeled antibodies to IFNγ-APC, IL-6-FITC, IL-17-APC-Cy7, IL-10-APC (all antibodies were purchased from Biolegend). Cytokine production was determined via flow cytometry.