70 mm nylon mesh cell strainers

KCs were isolated as previously described^{45 (link)}. Briefly, livers were perfused in situ via the portal vein with calcium- and magnesium-free HBSS supplemented with 2% heat-inactivated FBS, followed by 0.27% collagenase IV (Sigma, St. Louis, MO). Perfused livers were dissected and teased through 70 mm nylon mesh cell strainers (BD Biosciences, San Diego, CA), followed by suspension in 40 ml of DMEM supplemented with 10% FBS, 10 mM HEPES, 2 mM GlutaMax, 100 U/ml penicillin, and 100 mg/ml streptomycin for 15 min at 37 °C. Nonadherent cells were removed by replacing the culture medium. The adherent cells were used for further ex vivo experiments. KCs were cultured in vitro for 6 h, and then cells or supernatants were collected for further analysis. For M2 polarization, KCs isolated from mice were treated with 10 ng/ml IL-4 (Sigma, Saint Louis, MO, USA).

KCs were isolated as previously described.59 In brief, livers were perfused in situ via the portal vein with warmed (37°C) Hanks’ balanced salt solution, followed by collagenase IV (Sigma). Perfused livers were dissected and teased through 70‐mm nylon mesh cell strainers (BD Biosciences, San Diego, CA, USA). Nonparenchymal cells were separated from hepatocytes by three 2‐min centrifugations at 50 g. Nonparenchymal cells were suspended in Hanks’ balanced salt solution and layered onto a 50%/25% two‐step Percoll gradient (Sigma) and centrifuged at 1800g at 4°C for 15 min. KCs in the middle layer were collected and allowed to attach to cell culture plates in Dulbecco's Modified Eagle Medium with 10% FBS, 10 mm (4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid) (HEPES), 100 U mL⁻¹ penicillin, 100 μg mL⁻¹ streptomycin and 2 mm glutamine at 37°C for 15 min. Nonadherent cells were removed by replacing the culture medium. KCs were cultured for 6 h in vitro. Cells and supernatants were collected for further experiments.

Primary hepatic macrophages were isolated from Cav1 KO and WT mice as previously described (27 (link)). In brief, mice were anesthetized with an open midline incision and portal vein intubation. The liver was sequentially perfused with 0.04% collagenase IV (Sigma-Aldrich, Shanghai, China) in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, Shanghai, China) at a rate of 10 mL/min through the portal vein. Perfused livers were dissected and teased through 70-mm nylon mesh cell strainers (BD Biosciences, Breda, The Netherlands) and suspended in RPMI 1640 medium (Sigma-Aldrich, Shanghai, China). Then, a 25%/50% two-step percoll (Sigma-Aldrich, Shanghai, China) gradient was made and cell suspension was added to the top of 25% percoll. After centrifugation at 1500 g for 15 min, primary hepatic macrophages were collected from the top of 50% percoll and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and antibiotics. Then cells were collected for RT-PCR and Western blot analysis.