Il 17a bv711

Cryopreserved PBMCs were available for a subset of all participants (n=17). PBMCs were thawed, washed with complete media (90% RPMI, 10% FBS, 1.0% penicillin-streptomysin; (Sigma Aldrich, St. Louis, MO, USA)), and rested overnight at 37°C and 5% CO₂. The next morning, cell viability (trypan blue) was determined using an automated cell counter (TC-20, BioRad, Hercules, CA, USA) with viability >90%. PBMCs were then stimulated for 4 hours with 2 ng/μL of phorbol 12-myristate 13-acetate (PMA) and 1 ug/μL ionomycin (Sigma Aldrich, St. Louis, MO, USA) at 37°C and 5% CO₂. Following the stimulation, cells were washed and surface markers were labelled as described above, except that CCR4, CCR5 and CCR6 were omitted. Cells were washed in 1x phosphate buffered saline and were treated with a fixation and permeabilization kit (BD Biosciences Cytofix/Cytoperm, San Jose, CA, USA) following manufacturer instructions. Intracellular staining was performed using mouse anti-human monoclonal antibodies in 100 μL of permeabilization buffer to quantify cytokine production [IFNγ (APC); TNFα (BV650); IL-17A (BV711), Biolegend, San Diego, CA, USA)] for 30 minutes at 4°C in the dark. Cells were washed to remove excess antibody prior to being suspended in 200 μL of cell staining buffer for flow cytometry analysis.