Elisa kits for mouse il 1β and tnfα

Immortalized macrophage cell lines were generated from wt, NLRP3^−/−, ASC^−/−, caspase1^−/−, TLR9^−/−, ctsb^−/−, ctsl^−/− and 3d mice (Hornung et al., 2008 (link)). Primary BMDMs were generated as described (Rathinam et al., 2010 (link)). Human PBMCs were isolated from whole blood of healthy volunteers by density gradient centrifugation. All cells, primary and cell lines were cultured in DMEM supplemented with Ciprofloxacin and 10% FBS. For all experiments for immunoblot analysis, serum-free DMEM medium was used. For stimulations, poly (dAdT) (1.5 µg/ml) or genomic DNA was transfected with lipofectamine 2000 (Invitrogen). Nigericin (10 µM) was added 1 h before supernatants were collected. 2×10⁵ macrophages were plated and stimulated the day after with indicated amounts of CpG, sHz, nHz, sHz/CpG and sHz/Pf gDNA, iRBCs and RBCs. Cytokine measurements were performed using ELISA kits for mouse IL-1β and TNFα (R&D systems). Immunoblot analysis was performed with anti– mouse IL-1β antibody (AF-401-NA; R&D Systems) and rabbit anti-goat IgG-HRP (Santa Cruz Biotechnology).