Sa211

BV2 (YBC061, ybio Biotechnology, Shanghai, China), a mouse-derived microglial cell line, was cultured with high-glucose Dulbecco’s modified Eagle’s medium (DMEM, C11995500BT, Gibco, Grand Island, NY, USA), containing 10% fetal bovine serum (SA211.02, CellMax, Beijing, China), 100 U/ml penicillin, and 100 g/ml Streptomycin in a 5% CO₂ incubator (37 °C).

In order to obtain isolated SirNeoblasts, the tails of the planarians (> 8 mm in length) were amputated, then pooled and rinsed in calcium and magnesium free buffer with 1% bovine serum albumin (CMFB). Cells were macerated by rocking in the tube on a rotating platform for 20 min with agitation every 3 min. After filtering the macerated cells through a 70 μm cell-strainer cap, the dispersed cells were centrifuged at 290 x g for 10 min. Cells were then resuspended in isotonic planarian medium (IPM) with 10% Fetal Bovine Serum (FBS, CellMax SA211.02) for SiR-DNA staining by incubation in SiR-DNA (1 μM, Cytoskeleton Inc., CY SC007) for 1 h and Cell Tracker green CMFDA stains (2.5 μg/ml, Thermo Fisher Technologies, C7025) for 10 min. Target cells were sorted using a BD Influx cell sorter equipped with a 100 tip and purity sort mode.

HepG2 hepatocyte cells were cultured at 37°C, 5% CO₂, and constant pH (7.2-7.4) in Dulbecco’s Modified Eagle Medium (DMEM; C11995500BT, Gibco, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; SA211.02, Cellmax, Australia), 100 U/mL penicillin, and 100 μg/mL streptomycin (C0222, Beyotime, China). OA (O7501, Sigma, Germany) was conjugated to 10% (v/v) bovine serum albumin (BSA) (ST025, Beyotime, China). To induce hepatic steatosis, HepG2 cells were exposed to 0.5 mM OA for 24 h to induce lipid accumulation (62 (link)–64 (link)). Cells were then washed, cultured in fresh complete medium, and treated with different drugs for further analysis.