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.