Microglial BV2 Cell-Based Experiments

BV2 cells were cultured following previous published methods⁵³ and grew in DMEM media containing 10% heat-inactivated fetal bovine serum, 2 mM l-Glutamine, and 1% penicillin/streptomycin (Life Technologies). For treatment, BV2 cells were first incubated with DMSO (vehicle) or lipopolysaccharide (LPS) (1 μg/mL) for 4 h, then incubated with YM-I-26 in the presence of inflammasome activators, either nigericin (20 μM) or ATP (5 mM) for 30 min. Subsequently, media was collected and assayed for LDH to assess cell viability; cells were extensively washed with PBS and were lysed in MPER lysis buffer (MPER++) supplemented with EDTA-free protease inhibitors (Roche), Halt phosphatase inhibitor cocktail (Thermo Fisher Scientific). Lysates were centrifuged⁵³ (12,000 rpm at 4 °C for 15 min) and supernatants were collected for further analysis.