Determination of Cell viability
The cell viability effect of TC on AMDC was tested by Cell Counting Kit-8 assay (CCK-8 Sparkjade, CT001, Shandong, China) assay. Briefly, AMDC was dispensed into 96-well plates (3.5 × 103cells/well, 50% cells/well for 24h) in 100 μL of complete DMEM and various concentrations of TC were added (0, 8, 16, 32, 64μg/mL). After incubated at 37 ° C for 48h, 10 μL of CCK-8 solution in 90 μL complete DMEM was added to each well and incubation continued for 4 h. The absorbance of each well was determined at 450 nm by Microplate Reader (Thermo, Shanghai, China). The cell viability was calculated using the following equation:
Cellviability (%)
= (Absorbance individual test group – Absorbance blank group)/ (Absorbance control group – Absorbance blank group)
The IC50 values (concentration that inhibits cell growth by 50%) were determined using regression analysis. We reset new concentration to analyze the maximum drug concentration that does not affect cell viability (Compared with 0 drug concentration, cell viability P>0.05), 0, 6, 12, 18, 24 μg/mL, according to the maximum drug concentration measured and decline turning concentration in this study, using immunofluorescence for cytoskeleton, scratching, transwell and Western Blot were used on AMDC in subsequent experiments respectively.