Sterile pbs

Bone marrow-derived neutrophils (BMDNs) were isolated by using discontinuous Percoll gradients, as previously described.^{18 (link)} Briefly, following the surgical dissection of the bones, the femur and tibia were flushed with sterile PBS (Sangon, Shanghai, China) and passed through a 70 μm sterile filter (Fisher Scientific, Waltham, USA) to obtain the crude bone marrow cells. After centrifugation at 600 g for 5 min at 4°C with the brake off, cells were resuspended in 4 mL PBS and loaded on top of a discontinuous density gradient (62% and 81% Percoll) and centrifuged at 1500 g for 20 min at 4°C with the brake off. BMDNs were collected between 62% and 81% Percoll layers and suspended in 4 mL Red Cell Lysis Buffer (eBiosciences, multispecies) for 5 min on a rocker, washed with PBS, and centrifuged at 600 g for 5 min before resuspending in RPMI1640 (Hyclone, Logan, USA). Trypan blue dye exclusion indicated that cell viability was 95%–97% after the isolation. The purity of the isolated neutrophils was routinely 90%, as confirmed by flow cytometric analysis.

Cells were seeded on glass coverslips and incubated with S. Typhimurium described in the figure legends. After being washed 3 times with sterile PBS (Sangon Biotech), the samples were fixed with 4% paraformaldehyde (Servicebio) at room temperature for 20 min and incubated with 0.3% (vol/vol) Triton X-100 (MilliporeSigma) for 10 min. After being blocked with 3% bovine serum albumin (BSA; Beyotime Biotechnology), the samples were incubated with primary antibodies at 4 °C overnight, developed with the appropriate Alexa Fluor® 555 secondary antibody (Thermo Fisher Scientific) at room temperature for 1 h, and then incubated with DAPI (Sigma-Aldrich) for 10 min. Photomicrographs were taken with a Nikon Eclipse Ni–U fluorescence microscope (Nikon Corporation, Tokyo, Japan) with NIS-Elements F (Nikon Corporation). Primary antibody to NRF2 (ab31163) was purchased from Abcam (Cambridge, MA, USA). Primary antibody to FOXO3A (10849-1-AP) was purchased from Proteintech (Rosemont, IL, USA).

Wild-type S. Typhimurium strain SL1344 was a gift from Professor Qian Yang (Nanjing Agricultural University, Nanjing, China) and used for all experiments in this study. SL1344 was grown under sterile conditions on Luria-Bertani (LB) agar (Hangwei, Hangzhou, China) plates or in LB medium (Hangwei). On the day of experiments, overnight bacteria cultures were diluted in a ratio of 1:100 and agitated for a further another 3 h until reaching the late-logarithmic phase. Upon washing 3 times with sterile PBS (Sangon Biotech, Shanghai, China), numbers of S. Typhimurium in fresh bacterial suspension were estimated and adjusted based on the optical density at 600 nm absorbance and ready for the infection experiment.

Hepatocytes, non-parenchymal liver cells and liver macrophages were isolated as previously described with minor modifications [9 ,41 (link)]. In brief, livers were harvested, perfused with calcium and magnesium-free Hank's Balanced Salt Solution (HBSS; Beyotime Biotechnology, Shanghai, China) and digested with collagenase type I (Worthington, Lakewood, NJ, USA). The dissociated cells were filtered through a 70 μm cell strainer (Sorfa, Zhejiang, China) and then centrifuged at 50 g for 5 min at 4 °C. The hepatocyte pellets were collected and processed to eliminate erythrocyte using the Red Blood Cell Lysis Buffer (Beyotime Biotechnology). The supernatant fluids were transferred to a clean collection tube and centrifuged at 500 g for 10 min at 4 °C. The non-parenchymal liver cell pellets were collected and processed to eliminate erythrocyte using the Red Blood Cell Lysis Buffer (Beyotime Biotechnology). Liver macrophages were isolated from the non-parenchymal liver cell fraction as previously described with minor modifications [42 (link)]. Briefly, the non-parenchymal liver cell pellets were resuspended in sterile PBS (Sangon Biotech), gently layered on a double Percoll gradient (25% and 50%; GE Healthcare, Buckinghamshire, UK), and centrifuged at 900 g for 20 min at 4 °C. The layer between the 25%–50% gradient interface was collected.