Measurement of cytokines release using ELISA

The immunomodulatory effect of respective C5aR1 ligands on LPS-induced cytokine release was assessed in primary human macrophages as previously described (34). HMDMs were seeded in 96-well TC plates (100,000/well) for 24 h before treatment. All ligands were prepared in serum-free IMDM containing 0.1% BSA. For stimulation, the cells were co-treated with LPS and respective C5aR1 ligands for 24 h (37 °C, 5% C0₂). The supernatant was collected and stored at −20 °C till use. IL-6 levels in the supernatant were quantified using respective human ELISA kits (BD OptEIA) as per the manufacturer's protocol.