Mtt kit

The Pg extract was evaluated for possible in vitro anticancer activity against the MCF-7 human breast cancer cell line. The effect of black poplar bud extracts on MCF-7 breast cancer cells viability was evaluated by means of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The method was conducted as previously described [25 (link)]. Briefly, 1 × 10⁴ cells/well were seeded in 96-well culture plates and allowed to adhere overnight. On the second day, the cells were stimulated with different concentrations of poplar bud extracts (10, 25, 50, 75, 100 and 150 μg/mL) and were incubated for 72 h. The Control group is represented by cells treated with the solvent DMSO (Sigma-Aldrich). After the 72 h incubation period, the cells were treated with 10 μL of 5 mg/mL MTT solution from the MTT kit (Sigma-Aldrich) and incubated for an additional 3 h. The obtained formazan crystals were dissolved in 100 μL of lysis solution provided in the MTT kit. The absorbance was determined at 570 nm with a microplate reader (BioRad, xMark Microplate Spectrophotometer).

The effect of API (purchased from Sigma-Aldrich) on SK-MEL-24 human melanoma cells viability was evaluated by using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The method was performed as previously described [37 (link)]. In brief, a number of 1 × 10⁴ cells/well were seeded onto 96-well culture plates and allowed to adhere overnight. The next day, the cells were stimulated with different concentrations of API (0.3, 1, 3, 10, 30, and 60 μM) and further incubated for 72 h. After 72 h of incubation, a volume of 10 μL of 5 mg/mL MTT solution from the MTT kit (Sigma-Aldrich) was added in each well and incubated for an additional 3 h. The formed formazan crystals were dissolved by adding 100 μL/well of lysis solution provided in the MTT kit. The cells treated with the solvent dimethyl sulfoxide (DMSO, Sigma-Aldrich) were used as the control group. The absorbance was spectrophotometrically analyzed at 570 nm with a microplate reader (BioRad, xMark Microplate Spectrophotometer).

The viability of the cells after treatment was measured using a colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide) test (MTT kit, Millipore, Burlington, MA, USA), according to the manufacturer’s instructions. In brief, CFBE41o-cells were cultured in a 96-well plate and treated with PF. After the treatment, WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) was added to the wells and incubated for 4 h in a humidified incubator (37 °C, 5% CO₂). WST-8 was reduced to an orange formazan product that was directly proportional to the number of living cells, which were detected by measuring the absorbance at 450 nm. The percentage of viability of the cells exposed to the MBTP1 inhibitor was determined through comparing their absorbance with that of the untreated cells (100% of viability).