For primary cultures, bone marrow–derived macrophages and microglia were isolated as described42 (link),43 (link) and were cultured in DMEM supplemented with l -glutamine, ciprofloxacin (Cellgro) and 10% (vol/vol) FCS (Hyclone). Immortalized macrophage and microglial cell lines were generated with J2 recombinant retrovirus (carrying the v-myc and v-raf oncogenes)44 (link),45 (link). For the induction of macrophage differentiation, primary bone marrow cells were incubated for 3–4 d in medium conditioned by L929 mouse fibroblasts. Cells were then infected with J2 recombinant retrovirus and were maintained in culture until cells were growing in the absence of conditioned medium. Similarly, primary mixed glial cultures were cultured until fully confluent. After two consecutive infections with J2 retrovirus, cells were maintained in normal medium until there was growth of microglial cells in colonies. Semiadherent colonies of microglial cells were washed off and were cultured in new flasks. Microglial and macrophage cell lines were tested extensively. Immortalized microglial cells showed 100% purity and morphology and surface marker expression highly similar to primary microglia (Supplementary Fig. 1a,b ). Microglial cell lines from wild-type (C57BL/6) mice and mice in deficient caspase-1 or IL-1R as well as macrophage cell lines from wild-type (C57BL/6) mice and mice in deficient NALP3, IPAF or ASC were also generated and used for experiments.
Cells were stimulated in serum-free DMEM. Microglial cells and macrophages were primed with interferon-γ (100 U/ml; Griess assay, enzyme-linked immunosorbent assay (ELISA) of TNF and real-time quantitative PCR) or ultrapure LPS (100 ng/ml; ELISA of IL-1β, assays with FAM-YVAD-fmk (fluorescence-labeled inhibitor of caspases; 5-carboxyfluorescein–Tyr-Val-Ala-Asp–fluoromethylketone), assay of the microglia cell line expressing CFP-ASC and immunoblot analysis) 1–3 h before stimulation with Aβ, revAβ, zymosan or ATP or transfection with poly(dA:dT).
Cells were stimulated in serum-free DMEM. Microglial cells and macrophages were primed with interferon-γ (100 U/ml; Griess assay, enzyme-linked immunosorbent assay (ELISA) of TNF and real-time quantitative PCR) or ultrapure LPS (100 ng/ml; ELISA of IL-1β, assays with FAM-YVAD-fmk (fluorescence-labeled inhibitor of caspases; 5-carboxyfluorescein–Tyr-Val-Ala-Asp–fluoromethylketone), assay of the microglia cell line expressing CFP-ASC and immunoblot analysis) 1–3 h before stimulation with Aβ, revAβ, zymosan or ATP or transfection with poly(dA:dT).
