Blocking antibody for mouse il 1β

Ultrapure Escherichia coli O111:B4 LPS and CpG were from Invivogen. Recombinant mouse IL-23 and IL-1β, neutralizating antibody for IL-23p19 and isotype control (rat IgG) were purchased from eBioscience. Blocking antibody for mouse IL-1β was obtained from Leinco Technologies. Kanamycin (Km)-resistant wild-type C. rodentium strain DBS120 (pCRP1::Tn5) was a gift of Dr David Schauer (Massachusetts Institute of Technology, Massachusetts). The isogenic C. rodentium Δler, Δtir, Δeae, ΔescN, ΔescU mutants were generated from DBS120. Salmonella enterica serovar Typhimurium strain SL1344 was a gift from Dr D. Monack (Stanford University, California). For preparation of heat-killed C. rodentium, the pathogen was cultured overnight in Luria-Bertani (LB) medium, inoculated into DMEM at a 1:50 dilution and cultured under standard cell culture conditions (37 °C with 5% CO₂) for 8 h without shaking. Bacteria were then harvested and washed twice with ice-cold PBS, and heat inactivated at 60°C for 30 min. Complete killing of bacteria was confirmed by 72 h incubation at 37 °C on bacteria growth plates.