ELISA

IL-1β, TNF-α, IL-6, CCL5 (RANTES), CXCL2 (MIP-2), and CXCL10 (IP-10) concentrations in the cellular supernatants were quantitated using the murine ELISA development kits (PeproTech, NJ, USA) [43]. Briefly, BV-2 and PMM were seeded in triplicate onto 12-well and PDL-coated 24-well plates at a density of 5 × 10⁴ and 2.5 × 10⁵ cells/well, respectively. After overnight serum starvation, cells were incubated in serum-free medium containing LPA (1 μM) in the absence or presence of the antagonists for the indicated time periods. For each time point, the supernatants were collected and kept at − 70 °C until further use. The assays were performed according to the manufacturer’s instructions. Standard curves for each ELISA were done in triplicates. The concentrations of the cytokines and chemokines were determined using the external standard curve.