Cytokine analysis in cell culture supernatants

ELISA MAX sandwich enzyme-linked immunosorbent assay (ELISA) kits (BioLegend) were used to assess the level of IL-17a/f, IFN-γ, and TNF-α cytokines. Cells were isolated from spleen of DTH mice, plated at 1×10⁶ cells/well, cultured overnight (18–20 hours) for spontaneous cytokine secretion in complete RPMI media (1 %v/v penicillin/streptomycin, 1 %v/v HEPES buffer, 10 %v/v heat inactivated FBS, and 0.0002 %v/v 2-mercaptoethanol). Cytokine production was quantified from cell supernatants (stored at −20 °C). Absorbance was measured at 450 nm using a Victor2 1420 Multilable counter (Wallac).