Nycodenz

HN-EVs were isolated from spider venom using density gradient centrifugation as previously described (Figure 2A), with minor modifications [19 (link),48 (link)]. Briefly, the venom was centrifuged at 10,000× g for 5 min to remove glandular endothelial cells and other cell debris. The supernatant was layered on preformed 10–50% (w/v) Nycodenz (Sigma Aldrich, St. Louis, MO, USA) gradients in PBS in 12.5 mL Ultra-Clear tubes. First, the tubes were centrifuged at 100,000× g for 120 min at 4 °C in an SW41Ti rotor in a Beckman L90K centrifuge (Beckman Coulter, Woerden, Netherlands). Then, the light-scattering band was washed with PBS and centrifuged at 100,000× g for 70 min. Finally, the pellet (which contained EVs) was resuspended in 100 μL PBS. The protein concentration was determined by the BCA protein assay kit (Thermo Fisher, Waltham, MA, USA). The EVs were either used right away or stored at −80 °C for later use.

Anti-β-actin antibodies, 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), dexamethasone (DEX), formaldehyde, insulin, HEPES, Nycodenz, propidium iodide (PI), and Triton X-100 were purchased from Sigma-Aldrich (Missouri, USA). Fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific Inc. (NY, USA). Percoll and pronase were purchased from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). Trypan blue solution (0.5%) was purchased from BioWest (Nuaille, France). All other reagents were purchased from Invitrogen (CA, USA).

Primary HSCs were isolated from fresh livers in mice. In brief, hepatic specimens were digested by pronase and collagenase. The digest mixtures were subjected to density gradient centrifugation in 8.5% Nycodenz (Sigma-Aldrich, St. Louis, MO, USA) as previously described [25 (link),26 (link)]. HSCs expressed autofluorescence of retinoids in lipid droplets of cell cultures. HSC lipid droplets were verified under a fluorescence microscope. Trypan blue exclusion assays demonstrated that the viability of cell culture was more than 95%. 95% to 99% of cells were positive for Oil red O staining [26 (link)]. Cells were incubated in Dulbecco’s Modified Eagle’s Medium supplemented with 5% newborn calf serum. After one day in culture, the HSCs exhibited a dormant phenotype, followed by an activated phenotype at 7–14 days after incubating. Cell cultures were incubated till confluence; and those within 2–6 passages were used for the study. Each cell culture experiment was carried out at least 6 times.