BV2 microglial cell culture and treatment

The murine BV2 cell line was obtained from the China Center for Type Culture Collection (Wuhan, China) and cultured as previously described 11. In summary, cells were cultivated in Dulbecco’s Modified Eagle’s Medium (Cat. No. 11965092; Gibco, Shanghai, China) supplemented with 10% fetal bovine serum (Cat. No. 11011-8615; Every Green, Hangzhou, China), 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C in a humidified incubator with 5% CO₂. For the experiments, confluent cultures were passaged by trypsinization and treated in culture medium overnight before treatments.

In the first set of experiments, propofol (Cat. No. 56931; Sigma, Shanghai, China) at final concentration of 50 µM was applied 1 h before or 0, 1, 2, 4, 6 h after the onset of LPS (10 ng/ml, Cat. No. L-4391; Sigma) stimulation to determine the treatment window for propofol. Then, different concentrations (0, 12.5, 25, 50 and 100 µM) of propofol were incubated with the cells 1 h after the addition of LPS stimulation to identify the optimal dosage of propofol. In the second set of experiments, 50 µM of propofol was incubated with the cells 1 h after the addition of LPS stimulation. In the third set of experiments, the specific PI3K inhibitor wortmannin (1 µM, Cat. No. W-1628; Sigma) was added to the incubation medium during propofol application to determine if the PI3K/PKB signaling pathway might be involved in propofol’s action on BV2 cells. In the fourth set of experiments, wortmannin (1 µM) and propofol were added to the incubation medium 1 h after LPS exposure.