Hy 10197

Primary microglia were isolated from the brain of neonatal wild-type and TLR4 knockout mice at P1-P2 as described previously 6 (link), 26 (link) with minor modifications. Briefly, mixed glial cells were cultured in high glucose DMEM medium supplemented with 20% fetal bovine serum (FBS) at 37 °C in a 95% O₂ and 5% CO₂ incubator for 8-10 days, with the medium refreshed every 3 days. Microglia were dislodged by shaking at 200 rpm at 37°C for 2 h. The obtained microglia were seeded into 6-well plates in high glucose DMEM medium supplemented with 20% FBS at a density of 1×10⁶/well for 24 h before further treatment. The purity of cultured microglia was identified to be more than 98% by immunofluorescent staining for anti-Iba1 (Wako, 1:500).
LPS (100ng/ml) or IL-4 (20 ng/mL) was added to microglial cultures for 24 hours to induce different phenotypes. To determine the role of autophagy in microglial activation, cultured microglia stimulated by LPS were also incubated with the autophagy inhibitors bafilomycin (10nm, HY-100558, MedChem Express, MCE)27 (link), 28 (link) or wortmannin (100nm, HY-10197, MedChem Express, MCE)18 (link), or the autophagy agonist rapamycin (10nm, HY-10219, MedChem Express, MCE) 28 (link).

Cell viability was assessed using an MTT assay (M2128, Sigma-Aldrich, Germany). HaCaT cells were seeded in 96-well plates at a density of 5×10⁴ cell/well. After overnight growth, cells were pretreated with SAL for 2 h and exposed to UVB. In some experiments, cells were also co-incubated with Wortmannin (HY-10197, MedChemExpress) or EX-527 (HY-15452, MedChemExpress). Cells were washed with PBS and incubated with MTT solution (0.5 mg/mL, 20 µL/well) for 4 h at 37°C. Then, the supernatant was removed and DMSO (100 µL/well) was added to each well to dissolve the formazan crystals, and the absorbance was measured at 570 nm of optical density (OD) using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Cell viability was calculated as the percentage of viable cells of the treated cells versus untreated cells.19 (link) Experiment was performed in triplicates and repeated at least three times.