Lps o55 b5

Immortalized BMDMs (seeded at 250 000 cells per ml in 96-well plates) were primed for 4 h with 100 ng ml⁻¹ LPS O55:B5 (Invivogen). The NLRP3 inflammasome was triggered by addition of 5 mM extracellular ATP (Sigma-Aldrich) for 60 min. The AIM2 inflammasome was triggered by transfection of 1 μg ml⁻¹ of the synthetic DNA analogue poly(dA:dT) (Invivogen) using Lipofectamine 2000 (Invitrogen), according to the manufacturer's protocol, in OptiMEM (Gibco) for 3 h. The NLRC4 inflammasome was triggered by infection of the cells with S. enterica serovar Typhimurium SL1344 at a multiplicity of infection of 10. The infection was synchronized by centrifugation and continued for 60 min. The pyrin inflammasome was triggered by addition of 1 μg ml⁻¹ (final concentration) of C. difficile toxin B (CdtB, Enzo Biotech) for 2.5 h.

For experiments, colonoids were passaged using TrypLE techniques as already described but with modifications^{3 (link), 27 (link), 29 (link)}. First, Matrigel domes were disrupted by adding ice-cold PBS containing 5 mM EDTA and incubating on ice for 15 min. Colonoids were washed twice with base media, resuspended in TrypLE express (Gibco) and incubated at 37 °C with 5% CO₂ for 2 × 5 min. The colonoids were then rapidly disrupted into single cell suspensions with repeated pipetting through a p1000 tip, and an equal volume of organoid media was added. Cells were centrifuged, then resuspended in base media. The cells were counted using an automated cell counter and the appropriate number of cells (15,000 cells/well for human; 10,000 cells/well for mouse) were seeded using Matrigel as described in the colonoid isolation section in a 24-well plate. Colonoids were stimulated (days 5–7 after seeding for mice, day 10–14 for human) with base media with FliC (100 ng/ml; InvivoGen) or human IL-1β (10 ng/mL; Sigma) or murine IL-1β (10 ng/mL; PeproTech) or LPS O55:B5 (1000 ng/ml; InvivoGen) with or without recombinant IL-37 (100 to 0,1 ng/ml; R&D) or corresponding volume of base media for 4 h.

Recombinant Ts-Hsp70 (rTs-Hsp70) was expressed in E. coli (BL21) and purified as previously described [14 (link)]. The contaminated endotoxin was effectively removed by ToxOut High Capacity Endotoxin Removal Kit (Biovision, USA). The residual endotoxin was 0.1984 EU/mg in the final purified rTs-Hsp70, approximately equivalent to 20 pg/mg endotoxin in rTs-Hsp70 [30 (link)], which is lower than the minimal amount that could stimulate TLR2/4 based on the instruction of standard LPS O55: B5 (Invivogen, USA). The rTs-Hsp70 was labeled with phycoerythrin (PE) by Beijing Biosynthesis Biotechnology company, LTD.