Enzyme-Linked Immunosorbent Assay (ELISA)

For the quantification of TNF-α in the conditioned medium at each time point, sandwich TNF-α ELISA (KHC3012, TermoFisher Scientific, Carlsbad, CA, USA) was performed according to the manufacturer. Absorbance was read at 450 nm on a microplate reader (Synergy HT, BioTek, Winooski, VT, USA). A standard curve was built with concentrations ranging from 0 to 1000 pg/ml. The final concentrations were calculated by subtracting the value of each sample (i.e., testing conditions 1–3) to the conditioned culture medium baseline (i.e., testing condition 4), and the average was plotted for each testing condition along time.
