The cells were seeded into 6-well plates at a density of 2 × 105 cells/mL, and incubated in the medium of folate-free RPMI-1640 for 24 h. Then, the culture medium was replaced by fresh medium containing DOX and its derivatives (the equivalent DOX concentration was 20 μg/mL). After being cultured for 12 h or 24 h, they were analyzed by a Becton Dickinson FACScan (excitation at 488 nm) equipped with Cell Quest software (BD Biosciences, Franklin Lakes, Franklin Lakes, NJ, USA).
In addition, the effects of DOX and its derivatives on caspase-3 level in MDA-MB-231 cancer cells were measured by using the caspase-3 detection kit (Beyotime Institute of Biotechnology, Nanjing, China). The MDA-MB-231 cancer cells were cultured in 96-well plates at a density of 2 × 104 cells/mL and incubated for 24 h. Then, the culture medium was replaced by fresh medium containing DOX and its derivatives (the equivalent DOX concentration was 20 μg/mL). After being cultured for 12 h or 24 h, the caspase-3 level was determined at 405 nm on a microplate reader according to the manufacturer’s instructions.
In addition, the effects of DOX and its derivatives on caspase-3 level in MDA-MB-231 cancer cells were measured by using the caspase-3 detection kit (Beyotime Institute of Biotechnology, Nanjing, China). The MDA-MB-231 cancer cells were cultured in 96-well plates at a density of 2 × 104 cells/mL and incubated for 24 h. Then, the culture medium was replaced by fresh medium containing DOX and its derivatives (the equivalent DOX concentration was 20 μg/mL). After being cultured for 12 h or 24 h, the caspase-3 level was determined at 405 nm on a microplate reader according to the manufacturer’s instructions.