Generation and Culture of Murine Bone Marrow-Derived Macrophages

Cryopyrin^−/−, caspase-1^−/−, Abca1^−/−, Abcg1^−/−, Abca1^−/−Abcg1^−/−, Kir6.1^−/−, Kir6.2^−/−, and SUR2^−/− mice have been described previously (Miki et al., 1998 (link), 2002 (link); Schott et al., 2004 (link); Mariathasan et al., 2006 (link); Stoller et al., 2007 (link); Yvan-Charvet et al., 2008 (link)). P2X₇^−/− mice were obtained from Lexicon Pharmaceuticals. Mice were housed in a pathogen-free facility. All experiments were conducted in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Genentech. BMDMs were prepared as described previously (Lamkanfi et al., 2007 (link)). In brief, BMDMs were isolated from femurs of 6–12-wk-old mice and were cultured in Iscove's modified Dulbecco's medium containing 10% heat-inactivated FBS, 20% L cell–conditioned medium, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. After 5–7 d of incubation, cells were collected and plated in 6- or 24-well plates in Iscove's modified Dulbecco's medium containing 10% heat-inactivated FBS and 100 mg/ml thymidine and antibiotics. Macrophages were cultured for an additional 24 h before use.