The THP1 (no. TIB-202) cell line was obtained from the American Type Culture Collection (ATCC). Immortalized BMDMs from wild-type, Nlrp3−/−, Nlrc4−/−, and Nlrp1−/− mice were a gift from K. Fitzgerald (University of Massachusetts Medical School). BMDMs from Casp11−/− mice were obtained using 30% L929 cell–conditioned medium as a source of granulocyte/macrophage colony stimulating factor. CRISPR-Cas9–mediated Gsdmd−/− BMDMs were a gift from D. Abbott (Case Western Reserve University). Human peripheral blood monocytes were obtained from STEMCELL Technologies (no. 70034). These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; no. 11995073, Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (no. TMS-013-B, Millipore) and 1% penicillin and streptomycin (no. 15070-063, Thermo Fisher Scientific) at 37°C, 95% humidity, and 5% CO2. Cells were primed with LPS (200 ng/ml, 6 hours), poly(I:C) (5 μg/ml, 6 hours), or Pam3CSK4 (1 μg/ml, 6 hours) and then stimulated with LPS electroporation (1 μg, 16 hours) or E. coli [multiplicity of infection (MOI), 25; 16 hours] infection (5, 37, 38). All cells used were authenticated using short tandem repeat (STR) profiling, and mycoplasma testing was negative.

E. coli (no. 11775) was obtained from the ATCC and then added to cells at an MOI of 25 in medium without antibiotics. After 30 min, cells were washed and incubated for 1.5 hours at 37°C in fresh medium supplemented with gentamicin (100 μg/ml; no. G1397, Sigma-Aldrich) to kill extracellular bacteria.

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