Cell cycle analysis
HaCaT cells were seeded in six-well plates and allowed to adhere. Then
the cells were serum-starved for 24 h, followed by treatment with
10%-FBS/DMEM containing various concentrations of allicin (6.4, 9.6,
12.8μg/mL) for 48 h. Later, the cells were collected by trypsinization
and fixed in 70% ethanol overnight at
−20°C. After being washed with
PBS, HaCaT cells were resuspended and incubated with PI/RNase staining
buffer according to the manufacturer’s instructions (C1052, Beyotime)
for 30 min at 37°C in the dark and then subjected to flow cytometric
analysis. The samples were acquired with the Attune NxT (Thermo Fisher
Science), and the cell cycle distribution was further analyzed using
ModFit software (Verity Software House).