Wst 8 kit

To determine the characteristic absorption peak of the purified proteins Rhd1α and Rhd1β, a UV-2550 (SHIMADZU, Japan) was used for full-band scanning. For the enzyme activity, the enzyme reaction system contained 0.5 mmol/L benzo(a)pyrene, 0.1 μmol/L Rhd1α, 0.1 μmol/L Rhd1β, 0.5 μmol/L FMN, 0.1 mmol/L Fe (NH₄)₂·(SO₄)₂·6H₂O, and 10 mmol/L NADH [27 (link)]. For the determination of the optimum temperature, reaction systems were heated using a water bath at 25, 30, 35, 40, 45, 50, and 55 °C, respectively. For the pH values, the reaction solutions were pH 5.0–6.0 citric acid-Na₂HPO₄ (CPBS), pH 6.0–8.0 KH₂PO₄-NaOH, and pH 8.0–9.0 Na₂CO₃-NaHCO₃. The reaction lasted for 5 min, and the concentration of NADH was determined using a WST-8 kit (Beyotime Biotechnology, China). The activity of Rhd1 was determined by measuring the consumption of NADH. The condition with the highest enzyme activity was used as a reference point (100%) to calculate the relative enzyme activity under each experimental condition.

Breast cancer cell proliferation was determined with WST-8 kit (Beyotime Inst Biotech, China). Briefly, 2 000 cells/well were seeded into a 96-well flat-bottomed plate. The cells were incubated at 37 °C for 24 h, then were cultured in the presence of different concentrations of E2 or 4-OHT for 5 days. Fresh WST-8 dye was added and the cells were incubated at 37 °C for 2 h before the absorbance was determined by Multiskan Spectrum 1500 (Thermo Scientific, PA) at 450 nm.