Multitox glo multiplex cytotoxicity assay

Mouse 3T3-L1 (CL-173, ATCC) pre-adipocyte cells were cultured in DMEM supplemented with 10% newborn calf serum (Gibco), penicillin G (100 IU/mL) and streptomycin (100 µg/mL). Differentiation of the cell was induced by treating confluent cells with an adipogenic mixture consisting of 10 µg/mL insulin (Sigma), 500 µM isobutylmethylxanthine (Sigma) and 1 µM dexamethasone (Sigma) in the presence of 10% fetal bovine serum (FBS), at 37°C in a 5% CO 2 atmosphere (Duteil et al. 2014) . The medium containing the adipogenic mixture was replaced 3 days later with medium supplemented with 10 µg/mL insulin and incubated for 2 days. The medium was exchanged every 2 days until 9 days after the start of differentiation. 3T3-L1 cells were treated with ginsenoside Rg3 (0, 5, 25 and 50 µM) combined with the adipogenic mixture given at the start of differentiation. For determining the cytotoxicity of ginsenoside Rg3, cells were cultured in 96-well plates for 24 h. The MultiTox-Glo Multiplex Cytotoxicity Assay (G9270, Promega) was used to assess the viability of the cells after treatment; all steps were performed according to the manufacturer's instructions.