In vitro cytotoxicity

The in vitro cytotoxicity of Bufalin was measured by Cell Counting Kit-8(CCK-8) (Dojindo Laboratories, Kumamoto, Japan), as described in the literature. Briefly, 5×10³ cells per well were plated in 96-well plates and treated with Bufalin, paclitaxel, Bufalin plus paclitaxel or DMSO (diluent) at various concentrations for 48 h. Then, the medium with compounds or DMSO was replaced with 180 μL of fresh medium along with 20 μL CCK-8 solution in each well and incubated at 37°C for 2 h. Last, the CCK-8-containing medium was discarded and 150 μL of DMSO per well was added to dissolve the newly formed formazan crystals. Absorbance of each well was determined by a microplate reader (Synergy H4, Bio-Tek) at a 450 nm wavelength. Growth inhibition rates were calculated with the following equation, Inhibition ratio = (OD_DMSO-OD_drug)/(OD_DMSO-OD_blank)×100%.