2.3. Cytotoxicity and MDR Reversal Experiments

The cytotoxicity of rutaecarpine and MDR reversal experiments were performed by CCK8 assay. Briefly, for rutaecarpine cytotoxicity study, each type of cell was harvested and resuspended before being seeded in a 96-well plate at a final quantity of 5 × 10³ cells per well in 100 μL of medium, and was then incubated overnight. Different concentrations of rutaecarpine were added. For reversal study, rutaecarpine and positive control drug were added 2 h prior to incubation with or without anticancer drugs. After 48 h of further incubation, CCK8 was added to each well and the cells were incubated for 2 h at 37 °C. Subsequently, the absorbance value was read at 450 nm. Graphpad Prism software was used to calculate the concentration for 50% inhibition of cell viability (IC₅₀) of the anticancer drug. For positive control drug, tariquidar (20 μM) was used as reference inhibitor to reverse ABCB1-mediated MDR. Cisplatin, a non-substrate drug of ABCB1, was used as a negative control anticancer drug for reversal study.