Methyl thiazolyl tetrazolium (mtt)

The concentration and treatment time of TM (# 5045700001; Sigma Corporation, Missouri, United States) were screened by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (# D0801; Tokyo Chemical Industry, Tokyo, Japan) method. The cells were divided into four groups after synchronization and treated with 0, 1, 2, and 4 μg/ml TM for 24 h, respectively. Twenty microliters of MTT was added to the cells in the dark, and they were incubated at 37 C with 5% CO₂ for 4 h. One hundred fifty microliters of dimethyl sulfoxide (DMSO) solution per well was added to the cells after discarding the culture medium. After the HSCs were oscillated at low speed for 10 min, the absorbance (OD) value was read and the proliferation rate (PR) (PR = T/C × 100%, T was the OD value of the treatment group, and C was the OD value of the 0 μg/ml TM group) was calculated after adjusting the wavelength of the plate analyzer to 490 nm. The cells were divided into three groups after synchronization and treated with 2 μg/ml TM for 12, 24, and 36 h, respectively. The OD value was detected by the same method as above.

HaCaT cells were seeded into 96-well plate at a density 1 × 10⁵ cells/mL and were incubated overnight at 37 °C in humidified atmosphere of 5% CO₂. Then, the cells were treated with isolated compounds at various concentrations for 24 h. Subsequently, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, purchased from Tokyo Chemical Industry, Tokyo, Japan) solution (5 mg/mL in phosphate buffered saline, PBS) was added to each well and incubated for additional 4 h. Then, supernatant was discarded, and 40 mM HCl-isopropanol (100 μL) was added to dissolve formazan crystals. The absorbance was measured at 570 nm using microplate reader. The percentage of cell viability measured in control cells treated with DMSO without samples was used to calculate cell viability.

MSCs exposed to Atrazine or vehicle were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (TCI America, OR, USA) (0.5mg/ml final concentration) for five hours. Supernatants were removed and formazan crystals were dissolved with organic solvent. Optical densities were measured at 560 nm, and background at 670nm was subtracted.

Cells cultured on a 96-well plate were incubated in medium containing 0.4 mg/mL 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl-2H-tetrazolium bromide (MTT; TCI America) at 37°C for 1 h. Reduced MTT was solubilized in DMSO (Sigma-Aldrich) for determination of absorbance at 570 nm.