CCK-8 assay

The cytotoxicity of BBR against cancer cells was assessed by using Cell Counting Kit-8. The assay was performed in triplicate. All cells were seeded in 96-well plate at a density of 5 × 10³ cells/well at 37 °C. After cell attachment overnight, each well was treated with different concentrations of BBR or 0.1% DMSO for 24 h, respectively. Then, 10 μL of WST-8 solution was added to each well and the cultures were incubated for another 2 h. The absorbance was read at 450 nm by a SYNERGY microplate reader (Bio Tek, Winooski, VT) (Tian et al. 2016).