Dnase

LPS was extracted by the hot-phenol method as follows. The culture was collected, washed with PBS, and then suspended in double-distilled water (ddH₂O). After freezing and thawing three times, an equal volume of 90% phenol was added and followed by vigorous shaking at 68 °C for 15 min. After centrifugation, the water layer was collected in a new tube. The phenol layer was added to ddH₂O, and this process was repeated again. After that, the collected water layer was dialyzed into ddH₂O for at least three days, then DNase (5 µg/mL; Solarbio, Beijing, China), RNase (5 µg/mL; Solarbio), and proteinase K (20 µg/mL; Solarbio) were sequentially added and incubated for 2 h at the optimal temperature of each enzyme. After boiling in a water bath for 10 min, the supernatant was collected as the LPS solution. To prepare OPS, glacial acetic acid was added to the extracted LPS solution with a final concentration of 1% (v/v). After boiling in a water bath for 90 min, the mixture was adjusted to a pH of 7.0 with 1 mol/L NaOH and centrifuged at 40,000 g for 5 h. The supernatant was collected as the OPS solution.

LPS extraction was performed as described previously [35 (link)]. Briefly, the culture was collected by centrifugation and then resuspended in ddH₂O. After freezing and thawing, an equal volume of 90% phenol was added to each sample followed by vigorous shaking at 68 °C and then centrifugation to collect the uppermost part of the supernatant (the water layer). The phenol layer was re-extracted with ddH₂O and the previous step was repeated. The extracts were dialyzed into ddH₂O, then DNase (5 µg/mL; Solarbio, Beijing, China), RNase (1 μg/mL; Solarbio), and proteinase K (20 μg/mL; Merck) were sequentially added to the dialyzed samples and incubated at the optimal temperature for each enzyme. After a boiling water bath for 10 min, samples were centrifuged to obtain the LPS.
Glacial acetic acid was added to the extracted LPS to achieve a final concentration of 1% (v/v). After boiling for 90 min, the pH of the LPS extract was adjusted to 7.0 with NaOH. Finally, the mixture was centrifuged at 40,000 ×g for 5 h, and the supernatant was collected as the OPS fraction.

Primary cortical neurons were harvested from newborn mice (C57BL/6, postnatal 24 h) as described in previous studies [11 (link)]. Briefly, the newborn mice were disinfected with 75% ethanol and euthanized by decapitation, and the meninges were removed out and the cerebral cortex was made into single suspended cells with digesting buffer (papain (2 mg/ml)) (Worthington, America)/DNase I (Solarbio life science, Beijing, China) (10 mg/ml) = 1/100). The cells were seeded into the plates coated with polylysine (Solarbio Life Science, Beijing, China). Culture media (Neurobasal-A (Gibco, Carlsbad, USA) containing 2% B27 (Invitrogen, America) and 2 Mm glutamine (Gibco, Carlsbad, USA)) were regularly replaced every 3 days, and adenovirus were used to infect neurons at a MOI = 50 for 8 h.

Tumor tissues were harvested and subsequently cut into small pieces and then placed in RPMI 1640 media containing 0.4 mg/mL collagenase D (Roche, Switzerland) and 0.2 mg/ mL DNase (Solarbio, China) for 30 min at 37 ℃ with gentle shaking for CD3⁺CD4⁺ and CD3⁺CD8⁺ T cell isolation. The cell suspension was passed through a 70 μm strainer and then leukocytes were obtained with lymphocyte separation medium by density gradient centrifugation (DAKEWE, China). The isolated cells were further stained with anti-CD3, anti-CD8a and anti-CD4a antibodies (Biolegend, USA) for flow cytometric analysis.

We first anesthetized the mice by intraperitoneally injecting 7% chloral hydrate (25 mg/g). Five minutes later, the peritoneal cavity was opened. The inferior vena cava was perfused with EGTA solution (Sigma Aldrich). When the liver color became lighter, we cut the portal vein and perfused the liver using the solution of protease and collagenase (Sigma Aldrich). At the end of the perfusion, a white texture was visible on the liver. After perfusion, the mice were sacrificed, and the organ was cut and transferred to a 6 cm culture dish. After the addition of pre-warmed protease and collagenase in vitro hydrolyzate, we disrupted the liver tissue by forceps. Then, the broken liver tissue was transferred into a 50 ml centrifuge tube and incubated with the 20 ml pre-warmed protease and collagenase in vitro hydrolyzate and 1% DNase (Solarbio) in a 37°C hybridization chamber for 20 min. The liver tissue was then filtered by the 70 μm pore size Falcon filter (BD Biosciences Discovery Labware, Bedford, MA) and centrifuged 20 ~ 30 × g for 4 ~ 5 min at 4°C. The bottom primary hepatocytes were finally obtained.

After the excess connective tissue and blood stains removed from the theca lays, the theca layers were digested in DPBS containing 1 mg/ mL type I collagenase (Solarbio), 0.4mg/ mL DNase (Solarbio) and 0.1%BSA for 60 min in a constant temperature water bath at 37°C. DMEM (HyClone) (containing 10% FBS) was added, filtered by nylon mesh (70 μm) and centrifuged at 250 g for 5 min. Meanwhile, pre-cooled 35% Percoll (Solarbio) was centrifuged at 30,000 g at 4°C for 15 min. The cell pellets were resuspended in DMEM and tiled on the centrifuged Percoll. After centrifuging for 1 min at 400 g in a vertical centrifuge, the solution was divided into 3 layers. Cells in middle layer (a density of 1.033–1.047g/ Ml; Hou et al., 2020 (link)) were carefully absorbed into a 15 mL centrifuge tube, and centrifuged for 5 min at 250 g. The supernatant was discarded. The cell pellets were resuspended by ECM medium containing 10%FBS, 50 μg/mL ECGs (Sigma-Aldrich, St. Louis, MO), and 1% penicillin-streptomycin double antibody. The cells were cultured at 37°C under 5% CO₂ for 24 h, changing the ECM medium every 2 to 3 d. Only 3 to 6th generations of cells were used for follow-up tests (Sakurai et al., 2011 (link)).

LPS extracted by hot‐phenol method, details as follow. The culture was collected, washed, with PBS and then suspended in ddH₂O. After freezing and thawing 3 times, an equal volume of 90% phenol was added and followed by vigorous shaking at 68 ℃ for 15 min. Then centrifugation was performed. The water layer was collected in a new tube and the phenol layer was re‐extracted with ddH₂O, repeated twice. The collected water layer was dialyzed into ddH₂O for at least 3 days, then DNase (5 µg mL⁻¹; Solarbio, Beijing, China), RNase (5 µg mL⁻¹; Solarbio), and proteinase K (20 µg mL⁻¹; Solarbio) were sequentially added and incubated at the optimal temperature of each enzyme. After a boiling water bath for 10 min, the supernatant was collected as the LPS solution. Glacial acetic acid was added to the extracted LPS solution with a final concentration of 1% (v v⁻¹). After boiling water bath for 90 min, the mixture was adjusted pH to 7.0 with 1 × 10⁻³ m NaOH and centrifuged at 40 000 × g for 5 h, the supernatant was collected as the OPS solution.