Nycodenz solution

Primary mouse HSC and HC were isolated by pronase/collagenase perfusion digestion followed by density gradient centrifugation, as previously described [17 (link)]. In brief, primary LSEC were isolated from the 8-week-old Balb/c mice by in situ perfusion with 30 ml SC1 solution and 30 ml 0.05% Collagenase IV solution sequentially. The cell suspension was centrifuged at 50g for 4 min and then the supernatant was centrifuged at 500g for 8 min at 4 °C. Pelleted cells were resuspended in 10 ml of 18% Nycodenz solution (Sigma-Aldrich, St. Louis, MO, USA); 6 ml of 12% Nycodenz solution, 6 ml of 8% Nycodenz, and 4 ml of DMEM were orderly loaded on the top of the cell suspension. The added gradient centrifugal liquid was centrifuged at 1450g and 4 °C for 22 min without brake. LSECs were recovered from the interface between the 8 and 12% Nycodenz solutions, mixed with an equal volume of DMEM and centrifuged at 600g for 6 min at 4 °C. Cells were resuspended and incubated with anti-CD146 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Finally, LSEC were cultured in collagen IV-coated plates with LSEC medium, and cell viability was determined by the trypan blue exclusion method.

Primary mouse HSCs were isolated by pronase/collagenase perfusion digestion, followed by density gradient centrifugation²³ Briefly, liver tissues were initially digested in situ with 0.05% pronase E (Roche, Shanghai, China) and 0.03% collagenase type IV (Sigma–Aldrich, Shanghai, China) and then further digested with collagenase type IV, pronase E, and DNase I (Roche) at 37 °C in a shaking bath for 20 min. HSCs were isolated from nonparenchymal cells using Nycodenz solution (Sigma–Aldrich) at 4 °C due to the presence of massive amounts of vitamin A-storing lipid droplets. Primary HSCs were cultured in high-glucose Dulbecco’s modified Eagle’s medium containing 10% FBS and 1% penicillin–streptomycin and were maintained in a humidified incubator with 5% CO₂ at 37 °C.
The isolation and culture of primary hepatocytes was performed according to the technical patent of this research team (2017.03.08, National Invention Patent, CN201510418641.3).

HSCs were isolated from 32-week old male Balb/c mice or mice treated with control or fish oil diet by in situ pronase, collagenase perfusion and Nycodenz gradient as previously reported50 (link) . Liver was initially in situ digested with 0.05% pronase E (Roche, Mannheim, Germany), 0.03% collagenase type IV (Sigma-Aldrich, St. Louis, MO, USA) and then further digested with collagenase type IV, pronase E and DNase I (Roche, Mannheim, Germany) solution at 37 °C bath shaking for 20 minutes. After hepatocytes were pelleted by centrifugation 50 g for 4 minutes, the supernatant containing nonparenchymal cells was further centrifuged at 500 g for 5 minutes. HSCs were isolated from nonparenchymal cells using 8.2%, 12% and 18% Nycodenz solution (Sigma-Aldrich, St. Louis, MO, USA) at 1450 g and 4 °C without brake for 22 minutes. The purity of HSCs was higher than 95% evaluated by retinoid autofluorescence. Cell viability was determined by the trypan blue exclusion method. HSCs counting were performed with a hemocytometer.