Briefly, protein concentration of each sample was determined using bicinchoninic acid (BCA; G-Biosciences, St. Louis, MO, USA) and equal concentration of protein was used for the assay. Then, 25 µL of each sample and cytokine standards of different concentration were added to a 96-well plate. Further, samples were checked for the concentrations of the cytokines (IL-6, IL-8, IL-10, IL-17, IL-1β, and TNF-α) by MILLIPLEX enzyme-linked immunosorbent assay (ELISA) using MILLIPLEX Human Cytokine/Chemokine/Growth Factor Panel A (HCYTA-60K-08; Millipore Sigma, Burlington, MA, USA). The experiment was conducted according to manufacturer's instructions. After incubation with primary and secondary antibodies, the plate was washed rigorously with wash buffer and the plate was resuspended in drive fluid. The plate was read and analyzed in the Luminex MAGPIX Multiplex System (Merck Millipore). Standard curves of known concentrations of recombinant human cytokines (R&D Systems, Minneapolis, MN, USA) were used to convert fluorescence units to cytokine concentration (pg/mL).34 (link)