MTT assay

An MTT assay was performed to determine the cytotoxicity of AA. ESCs were seeded into 96-well plates at a density of 1.5x10⁵/ml (0.1 ml) per well. Different concentrations of AA (50.0, 25.0, 12.5, 10.0, 6.25, 5.0, 3.13 and 2.5 µg/ml; Tianjin Yifang Science & Technology Co., Ltd.) were added to each well, followed by incubation at 37˚C in an atmosphere with 5% CO₂ for 48 h. The supernatant was discarded and 20 µl of MTT (0.5 mg/ml; Sigma-Aldrich Merck KGaA) was added to each well, followed by incubation for 4 h at 37˚C in an atmosphere with 5% CO₂. The supernatant was discarded, the purple formazan crystals that had formed were dissolved using DMSO (150 µl) and cytotoxicity was subsequently measured at a wavelength of 570 nm. The concentration that reduced the number of viable cells by 10% (IC₁₀) of AA was calculated using SPSS software (version 11.5; SPSS, Inc.) and then AA was used at this concentration in the following experiments. There were 3 independent replicates and 6 multiple wells per experiment.