2.3. Cell viability assay

The viability of HEK293/pcDNA3.1 and HEK293/ABCB1 cells to anticancer drugs was measured using the MTT assay. Cells (5000 cells/well) were seeded in 96-well plates (160 μL/well) and cultured for 24 h. For the cytotoxicity experiment of Y₆, 40 μL of varying concentrations of Y₆ were added into each well to the final concentrations of: 100,30,10,3,1,0.3,0.1,0.03 μmol/L. They were incubated for 48 h at 37 °C. For the reversal experiment, Y₆, EGCG, and the control modulator (verapamil) (20 μL/well) were added 1 h prior. Then, twenty microliters of different concentrations of chemotherapeutic drugs (doxorubicin or cisplatin) were added into the designated wells and incubated for 48 h at 37 °C. Subsequently, 20 μL of the MTT solution (4 mg/mL) was added to each well and incubated for an additional 4 h. The solution was discarded and 100 μL of DMSO were added to dissolve the formazan crystals. Finally, light absorbance was determined at 490 nm using the OPSYS microplate reader (Dynex Technology, Chantilly, VA, USA). Verapamil (1 μmol/L) was used as positive control inhibitors of ABCB1.