Fluorescence plate reader

HEK293 cells (1 × 10⁴ cells per well) were incubated in 96-well black plates in 100 μL of DMEM medium containing 10% FBS, Streptomycin (100 μg/mL) and Penicillin (100 unit/mL) at 37 °C for 24 h. The medium was exchanged to fresh medium with different concentrations of compound and cells were incubated for 72 h. Fluorometric microculture assay (FMCA) was used for determination of cell proliferation using a fluorescence plate reader (Thermo Fisher Scientific Inc., Waltham, USA).
HPB-ALL cells (1 × 10⁴ cells per well) were incubated in 96-well black plates in 100 μL of RPMI-1640 medium with 10% FBS, Streptomycin (100 μg/mL) and Penicillin (100 unit/mL). Media also contained different concentrations of compound. Cells were incubated at 37 °C for 72 h. After incubation, AlamarBlue (00–025, DAL1025; Thermo Fisher Scientific Inc., Waltham, USA) was added 10 μL per well. After incubation for 4 h, using a fluorescence plate reader (Thermo Fisher Scientific Inc., Waltham, USA), fluorescence was measured and cell viability was determined.

The antiproliferative activities of GUR extracts and flavonoids were investigated using the MTT assay [14 (link)]. Firstly, B16-F10 cells were cultured in 96-well plates at approximately 7.5 × 10³ cells per well and incubated for 12 h. Then cells were treated with different concentrations of GUR extracts (5, 10, 25, 50, and 100 μg/mL) or GUR flavonoids (10, 20, 40, 60, and 80 μM). After incubation of 48 h, MTT solution (5 mg/mL in PBS) was added to each well, and cells were incubated at 37 °C for 4 h. Subsequently, 150 μL DMSO was added to each well, and the plate was put on a shaker for 5 min, the absorbance were measured at 570 nm using a fluorescence plate reader (Thermo scientific, Waltham, MA, USA). Inhibition percentage (%) was calculated according to the following formula: Inhibition percentage of cell viability (%) = [1−(OD treated well/OD control well)] × 100%. The IC₅₀ value was calculated using the SPSS v. 22.0 Statistics Software (IBM Corp., Armonk, New York, NY, USA).

Cells (3 × 10³/96-well plates) were seeded in medium supplemented with CS-FBS for 24 h in the presence of DEX and/or RU486. Cells were stained with 10 μM DCFH-DA (Beyotime, SH, CHN) in phenol-red free DMEM without serum at 37°C for 20 min, washed with medium, and subjected to fluorescence plate reader (Thermo, NY, USA) (details in Supplementary Table 1).