Crystal violet assay

Crystal violet is a basic dye, which stains cell nuclei, and spectrophotometric reading of color intensity is an indicator of DNA content, and cell number (33). For determining cell viability in relation to the colony formation, proliferation and death of HepG2 cells, an adaptation of the crystal violet staining procedure was applied, as follows: In the experiment for cell proliferation, the cells (1×10⁵/ml per well) were seeded into 24-well plates and cultured in α-MEM (containing 10% FBS, 1% P/S and 1% fungizone) in the presence of either the vehicle (1% DMSO) or TCDD (1 or 10 nM) for 3 days. In the experiment for cell death, the cells (1×10⁵/ml per well) were seeded into 24-well plates and cultured in α-MEM (containing 10% FBS, 1% P/S and 1% fungizone) for 3 days to reach subconfluency. They were then cultured for 24 h in the presence of either the vehicle (1% DMSO) or TCDD (1 or 10 nM). The cells were washed with PBS and fixed with methanol for 20 min at room temperature, and then washed 3 times with PBS. Crystal violet solution (0.5%, in 20% methanol) was added to the fixed cells for 30 min. Thereafter, the plates were immersed in running tap water for 15 min. After the plates had dried, 300 µl 0.2% Triton X-100 (in distilled water) was added to each well followed by incubation at room temperature for 90 min, and 100 µl of the liquid content subsequently transferred to 96-well microtiter plates. The absorbance (OD) was read on an ELX800 Universal Microplate Reader (Bio-Tek Instruments Inc.) at a wavelength of 570 nm. Triton X-100 (0.2% in distilled water) was used as a blank. The results are presented as absorbance.