Cytokine Production Assay in Leukocytes

As we described previously in our study^{20 (link)}, isolated leukocytes (2 × 10⁶ cells/mL) were resuspended in complete medium [RPMI 1640 medium-10% fetal bovine serum containing penicillin (200 μg/mL), streptomycin (200 U/mL), 4 mmol/L L-glutamine, and 5 × 10⁻⁵ mol/L 2-mercaptoethanol; all from Gibco, Carlsbad, CA, USA] and stimulated with lipopolysaccharide (LPS; 10 μg/mL; Escherichia coli serotype 0111: B4; Sigma-Aldrich, St louis, Mo, USA), phorbol 12 myristate 13-acetate (PMA; 50 ng/mL, Sigma-Aldrich), ionomycin (500 ng/mL, Sigma-Aldrich), and brefeldin A (3 μM, BioLegend) for IL-10 and PMA, ionomaycin, and brefeldin A for IL-17 for 5 h at 37℃. Dead cells were detected using a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen) before cell surface staining. Stained cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s instructions and stained with PE or APC -conjugated mouse anti-IL-10 mAb and APC-conjugated mouse anti-IL-17A mAb.