Crystal violet assay for in vitro cell growth

For determining the effect of TSTs on cell growth, 4,000 cells were seeded per well in 96-well plates. After 24 hours, cells were treated with different concentrations of TST-A or TST-D. At the indicated time, cells were washed in PBS once, fixed in 100 µl/well 1% glutaraldehyde (Sigma-Aldrich) for 15 minutes, after the removal, then stained with 100 µl/well 0.5% crystal violet (Sigma-Aldrich) for 15 minutes. After washing out excess dye, 100 µl/well Sorenson’s solution was added to elute dye from stained cells and subjected to densitometry measurement at OD_{560 nm}. The relative cell number was determined using control =1.