Isolation and Activation of Neonatal Microglia

Neonatal microglia were isolated from C57BL/6 P1-4 mouse pups (Charles River, UK) as previously described (Carrillo-Jimenez et al., 2018 (link)). In brief, brains were dissected by removing meninges, cerebellum, and olfactory bulbs before tissue was finely minced with a scalpel then digested with papain solution for 20 min at 37 °C. Tissue was dissociated and the resulting cell suspension was passed through a 40 μm cell strainer (Greiner Bio-One). Cells were centrifuged at 300 g for 5 min then resuspended in culture medium (DMEM, 10% FBS, 1% PenStrep) and plated in T75 collagen-coated flasks (Greiner Bio-One). A full medium change was caried out on day 2 while a half medium was changed on day 5 in the presence of 5 ng/ml of mouse GM-CSF (R&D Systems). Microglia were shaken off between day 9 and 11 and plated onto poly-d-lysine coated 96-well plates (μclear, Greiner Bio-One) at a density of 20,000 cells/well. The following day cells were primed with LPS (100 ng/ml) for 3.5 h prior to treatment with C101248, MCC950 (Tocris), or vehicle (0.1% DMSO) for 30 min. NLRP3 was then activated by a complete medium change with an isotonic K⁺-free buffer (148 mM NaCl, 10 mM HEPES, 10 mM glucose, 2 mM CaCl₂, 1 mM MgCl₂ at pH 7.4) in the presence of test compounds or DMSO for 1 h after which supernatants were collected and stored at −20 °C until the measurement of IL-1β.