Intracellular Cytokine Staining

0.5 × 10⁶ PBMCs were stimulated with RPMI, PMA/ionomycin, PPD, a peptide pool of the TB-specific antigens ESAT-6/CFP-10 (2.5 µg/mL, ProImmune, UK), heat-killed SP (10⁵ cells/mL), C. albicans (10⁵ cells/mL), or E. coli (10⁶ cells/mL) per test. Cells were incubated at 37°C 5% CO₂ for 18 h with Brefeldin A (10 µg/mL) added after the first 2 h of incubation. Cells were stained with a live/dead aqua yellow stain (Invitrogen, USA), incubated for 10 min at room temperature before staining with a surface marker cocktail consisting of either CD3-PerCP Cy5.5 or CD3 APC efluor 750 and CD8 APC efluor 780. Cells were incubated for 30 min at room temperature and washed with 1 mL of PBS/1% FCS/0.2% Sodium Azide (FACS buffer). Supernatant was removed and cells washed in perm/wash solution (Ebioscience, UK). Permeabilization was then performed using fix/perm solution (Ebioscience) and cells incubated for 20 min at 4°C before centrifugation at 1,800 rpm for 5 min. A cocktail of intracellular cytokines IL-2 FITC, IL-17 PE, TNF-α PE-Cy7, IL-10 efluor-450, and IFN-γ APC (all from ebioscience) was added, and cells incubated for 30 min at room temperature. Cells were washed with perm/wash solution and 300 µL FACS buffer was added before acquisition. At least 100,000 lymphocytes were acquired for each sample on a CyanADP (Beckman Coulter, USA) flow cytometer using Summit v4 software and analyzed using FlowJo v10.0.2 (Tree Star, USA) according to the gating strategy shown (Figures S1 and S2 in Supplementary Material).