BMDM and iMAC culture and stimulation

Bone marrow cells were differentiated into macrophages in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) low-endotoxin fetal bovine serum (FBS; Omega Scientific) and 20% (v/v) L929-conditioned medium at 37°C with 5% CO₂. Unless otherwise specified, BMDMs were harvested on day 5. iMACs were maintained in RPMI 1640 medium supplemented with 10% (v/v) low-endotoxin FBS, murine granulocyte-macrophage colony-stimulating factor (GM-CSF) (20 ng/ml) (eBioscience), and 1 μM β-estradiol (Sigma) and then were differentiated with L929-conditioned medium for 5 days at 37°C with 5% CO₂. For stimulation, cells were plated overnight at ~1.0 × 10⁶ cells/ml in 100 μl on 96-well plates or in 500 μl for 24-well plates. For 96-well–based inflammasome stimulations, cells were primed with Pam3CSK4 (1 μg/ml) for 5 hours, which was followed by stimulation with 5 mM ATP, or else the cells were transfected with LPS (5 μg/ml) and 0.25% (v/v) FuGENE HD (Promega) in Opti-MEM I media (Gibco) (24). For FasL stimulations, cells were left unprimed and were stimulated with FasL (1 μg/ml) in Opti-MEM I. For electroporation, cells were collected and resuspended at 0.3 × 10⁶ to 0.5 × 10⁶ cells/10 μl of R buffer from the Neon Transfection System (Thermo Fisher Scientific). Stimuli were mixed with cells at the following ratios: LPS 2 μg/1 × 10⁶ cells; Cyto-c 50 μg/1 × 10⁶ cells. Cells were electroporated using the 10-μl Neon tip with 1720 Voltage, 10 Width, 2 Pulse settings according to the manufacturer’s instructions, added to medium to yield ~0.5 × 10⁶ to 1.0 × 10⁶ cells/ml, and then plated in 100 μl for analysis on 96-well plates. Where indicated in the figure legends, cells were electroporated with the Amaxa 4D-Nucleofector Y Unit (Lonza) in Opti-MEM I medium with LPS (5 μg/ml) or Cyto-c (100 μg/ml). For IFN treatments, cells were cultured with IFN-α (50 ng/ml), IFN-β (50 ng/ml), or IFN-γ (100 ng/ml).