2.6.4. Cytokine Measurements by ELISA

The supernatant of the pre-treated macrophages (4 and 24 h) was harvested and stored at −80 °C until analysis. Tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6) of the 4 and 24 h pre-treated samples were quantified via ELISA assay kit (eBioscience). Samples for TNF-α analysis were measured with the diluted supernatant (1/10) for all the samples, and for IL-6, supernatants were diluted by 1/10 for unstimulated and 1/50 for stimulated samples with a micro-plate reader (Molecular Devices) at 540 nm. With calibration curves (0 to 1000 pg/mL of TNFα or 0 to 500 pg/mL of IL-6), results are expressed as the % stimulation of TNF-α production and % inhibition of IL-6 production. All cytokine measurements were performed in triplicate.