2.8. TNF-α and IL-6 ELISA

BV2 cells were exposed to different treatments as indicated. After 24 h treatment, culture supernatants were collected for TNF-α and IL-6 ELISAs, which were performed according to the manufacturer's instructions (Abcam, Cambridge, MA, USA). Briefly, the capture antibody was added in each well of a 96-well ELISA plate and incubated overnight at 4°C. The plate was washed with phosphate-buffered saline (PBS) with 0.05% Tween 20 solution (PBST) five times and then incubated with 100 μL supernatant/each well for 2 h at room temperature. Following five PBST washings, biotin-conjugated antibody was added to each well and incubated for 1 h at room temperature. After five PBST washings, diluted avidin-HRP was added and the plate was incubated at room temperature for 30 min. After washing the plate five times, tetramethylbenzidine (TMB) substrate was added to each well, and the color was developed in the dark for 10-30 min at room temperature. The color reaction was stopped by adding 1 M H₃PO₄. The absorbance at 450 nm was measured on a plate reader, and 620 nm was chosen as the reference wavelength. The concentration of TNF-α and IL-6 was calculated using a mouse TNF-α and IL-6 standard working curve, respectively.