Thioglycolate medium

Peritoneal macrophages were isolated from Nur77^+/+ and Nur77^−/− mice. Mice were intraperitoneally (i.p) injected with 2 ml of 4 % thioglycolate medium (BD Biosciences) for 3d, then, macrophages in the peritoneal exudates were collected by peritoneal lavage with 10 ml ice-cold DMEM. Collected cells were incubated in DMEM supplemented with 10 % fetal bovine serum (FBS) at 37 °C for 4 h and washed with PBS to eliminate non-adherent cells. The adherent cells were taken as peritoneal macrophages.

Three days before the extraction of peritoneal macrophages, 8-week-old C57BL/6J mice were injected intraperitoneally with 2 mL of 3% thioglycolate medium (BD Biosciences, Sparks, MD). Peritoneal macrophages were isolated by washing the peritoneal cavity with endotoxin-free phosphate-buffered saline (PBS; HyClone, Logen, UT) and cultured at 37°C in 5% CO₂ in RPMI 1640 media supplemented with 10% fetal bovine serum (HyClone). The cells were seeded in 6-well culture plates (2 × 10⁶/well) in 1 mL of fresh culture medium and washed thrice with PBS before stimulation. Macrophages were stimulated with LPS (1 μg/mL) and BLP (1 μg/mL) alone or in combination or stimulated with E. coli (DH5α or JE5505, 1 × 10⁷ CFU/well, multiplicity of infection [MOI] of 5:1). After 1 h of infection, extracellular E. coli was eliminated via washing with fresh medium containing 10 mg/mL ovalbumin and 20 U/mL interferon gamma (58 (link)). In the groups treated with the ERK inhibitor FR180204 (Tocris, Bristol, UK) or p38 inhibitor SB203580 (Tocris), macrophages were incubated in culture media supplemented with 1 μM FR180204 for 2 h or 3 μM SB203580 for 30 min before stimulation. In the groups treated with the NF-κB inhibitor celastrol (InvivoGen, San Diego, CA), macrophages were incubated in culture media supplemented with 1 μM celastrol in combination with stimulation.

The extraction and culture of primary mouse macrophages were completed as previously described (14 (link)). In brief, WT, NLRP3^−/−, and NLRC4^−/− mice were injected with 2 ml of 3% thioglycolate medium (BD Biosciences, USA). Three days after the injection, peritoneal macrophages were isolated by washing the peritoneal cavity with PBS. The primary mouse macrophages and murine macrophage cell line J774A.1 were cultured in DMEM media (HyClone, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, USA), 100 U/ml penicillin and 100 µg/ml streptomycin in 5% CO₂ atmosphere at 37°C. Before infection, cells were washed three times by DMEM media to dislodge the penicillin and streptomycin.

The human acute monocytic leukemia cell line THP-1 obtained from the American Type Culture Collection (United States) was cultured in RPMI 1640. The mice monocytic macrophage cell line RAW264.7 was purchased from China Infrastructure of Cell Line Resource (China) and human aortic smooth muscle cells (HVASMCs, BeNa Culture Collection, China) were cultured in DMEM, supplemented with 10% of FBS, 1% L-glutamine, streptomycin (100 μg ml⁻¹), and penicillin (100 units ml⁻¹) and incubated at 37°C in a 5% CO2 atmosphere.
Three days before the extraction of peritoneal macrophage, 8-week-old C57BL/6 J mice were injected with 1 ml of 3% thioglycolate medium (BD Biosciences, United States). Peritoneal macrophage was isolated by washing the peritoneal cavity with endotoxin-free phosphate buffered saline (PBS, Hyclone, United States) and cultured at 37°C in 5% CO2 in RPMI 1640 medium supplemented by 10% fetal bovine serum (FBS, Hyclone). Peritoneal macrophage cells (2 × 10⁶·per well) were seeded into six-well plates with 1 ml fresh medium and washed three times with PBS before treatment.

Culture of primary hamster macrophages: Hamsters were injected with 2 ml of 3% thioglycolate medium (BD Biosciences, Sparks, MD). Three days after the injection, peritoneal macrophages were isolated by washing the peritoneal cavity with dulbecco's modified eagle medium (DMEM, HyClone^TM). Cells were cultured at 37°C in 5% CO₂ in DMEM supplemented with 10% fetal bovine serum. A total of 1X10⁶ cells in each well were seeded in 6-well culture plates in 2 ml of fresh culture medium. After incubation for 6 hours at 37°C and 5% CO₂, each experimental well was prestimulated for 24 hours with 0.5 ml of β-glucan (10 mg/ml) suspended in DMEM. After 5 days, each well was stimulated with Leptospira at an MOI of 100 per cell. Cells were then incubated for 24 h; thereafter, cells were harvested, and total RNA extraction was performed.

The strains used in this study were wild-type Staphylococcus aureus strain NCTC 8325-4 and the hla-deficient mutant DU1090, which were cultured in TSB (tryptic soy broth) broth or on TSB agar as previously described (Ragle and Juliane, 2009 (link)). Bacteria picked from a single colony were cultured overnight in 2 ml of TSB with shaking at 180 r/min and 37°C. The cultures were then diluted 1:100 in 20 ml of TSB for subculture under the same conditions until the OD600 reached 1.5 and 0.6 for cell infection and animal infection assays, respectively. The cultures were centrifuged (12,000 rpm, 1 min), and the bacterial pellet was collected for the infection assay. Human alveolar epithelial A549 cells were cultured in DMEM supplemented with 10% fetal serum at 37°C in 5% CO₂. Primary peritoneal macrophages were extracted from male C57BL/6 mice as previously described (Zhang et al., 2017 (link)). Briefly, mice were injected intraperitoneally with 2 ml of 3% thioglycolate medium (BD Bioscience, USA) 3 days before cells were isolated, and then the euthanized mice were injected intraperitoneally with 5 ml of RPMI medium to perform peritoneal lavage. The macrophages were collected by centrifugation (1,200 rpm, 5 min) and cultured in RPMI supplemented with 10% FBS at 37°C in 5% CO₂.