3.7. Enzyme-Linked Immunosorbent Assay (ELISA) for TNF-α and IL-6

The amount of TNF-α and IL-6 was measured using an ELISA kit according to the manufacturer’s instructions. RAW264.7 cells were plated at a density of 5 × 10⁴ cells/well in 24-well cell culture plates and incubated for 24 h. After removing the medium, HM at concentrations of 0.1, 0.5, and 1 μg/mL were added to each well and incubated for 1 h. The cells were further co-incubated with 1 μg/mL of LPS for 24 h. Post-incubation, the culture medium was collected from each well and stored at −70 °C for the cytokine analysis. Sixty-fold diluted samples were used for detecting TNF-α and IL-6 to not exceed the standard range.