Anti mouse il 1β ab

Tibiae and femora were obtained and BM cells were flushed out using a‐MEM/2% FBS. After red blood cells were lysed, BM cells were cultured in α‐MEM supplemented with 10% foetal Bovine serum (FBS), 1% penicillin/streptomycin (P/S) and 25 ng/mL M‐CSF for 3 days to generate osteoclast precursors (OCPs). OCPs were primed with 100 ng/mL LPS for 3 hours in serum free medium, and then treated with MCC950 (0.1 µmol/L) for 30 minutes before being stimulated with Nigericin (10 μmol/L) for 30 minutes. Cell lysates and supernatants were collected to detect NLRP3 inflammasome activation via Western blot and ELISA. For osteoclastogenic assays, BM cells were harvested and cultured in 96‐well plates (5 × 10⁴ cells/well) in α‐MEM with 10% FBS, 1% penicillin/streptomycin (P/S) containing 25 ng/mL M‐CSF and 10 ng/mL RANKL for 4 days. On day 5, stimulus were used to activate inflammasome as above. Then cells were fixed by 4% paraformaldehyde and stained for TRAP activity. TRAP‐positive cells with more than 3 nuclei were counted for TRAP⁺ osteoclast number by using inverted microscopy. For blocking experiments with neutralizing Ab, anti‐mouse IL‐1β Ab (R&D System, Cat#AF‐401‐NA) was purchased from R & D systems and used according to the manufacturer's instructions.

Immunoblot analysis of inflammasome activation was performed as described previously [18] (link). Briefly, BMDMs were seeded in six-well plates and cultured overnight. Cells were primed with LPS (10 ng/mL) for 6 hours at 37°C. After replacing the media with serum-free media containing recombinant human M-CSF (50 ng/mL; Peprotech Inc., Rocky Hill, NJ), cells were stimulated with 0.3 mg/mL of silica particles for two hours at 37°C. Culture supernatants and total cell lysates were pooled and then clarified by centrifugation. Proteins were precipitated with Strataclean Resin (Stratagene, La Jolla, CA) and detected with anti-mouse IL-1β Ab (R&D systems, Minneapolis, MN), anti-mouse caspase-1 Ab (Santa Cruz, CA), anti-mouse caspase-11 mAb (Santa Cruz), anti-mouse β-actin mAb (Bio Legend).