Alamarblue high sensitivity cell viability reagent

Cell viability was assessed using the sulforhodamine B (SRB) assay. A total of 3 × 10³ wild type (WT) or SHBG-overexpressing (SHBG_OE) prostate cancer cells were seeded per well in 96-well microtiter plates containing supplemented growth media, and incubated at 37 °C in humidified 5% CO₂. After 48 h, cell viability was measured following the manufacturer’s instructions. After fixing the WT or SHBG_OE prostate cancer cells with 10% trichloroacetic acid (TCA) and carefully washing with ddH₂O, the cells were stained with 0.4:1 (w/v) SRB–acetic acid solution. We carefully washed off unbound SRB dye from the cells using 1% acetic acid thrice, followed by air-drying the plates, and solubilization of bound SRB dye in 10 mM Tris base. For cell proliferation, invitrogen alamarBlue™ high sensitivity cell viability reagent (#A50100, Thermo Fisher Scientific Inc., Bartlesville, OK, USA) was used, strictly following the manufacturer’s instructions. Briefly, after seeding cells in triplicates with three biological replicas for each assay at each time point (day 1–8), the cells were incubated with alamarBlue™ for 2 h at 37 °C. The number of dye-stained viable/proliferating cells was read at 570 nm absorbance wavelength in the Molecular Devices Spectramax M3 multimode microplate reader (Molecular Devices LLC., San Jose, CA, USA).

For cell proliferation, Invitrogen alamarBlue™ high-sensitivity cell viability reagent (#A50100, Thermo Fisher Scientific Inc., Bartlesville, OK, USA) was used strictly following the manufacturer’s instruction. Briefly, after 1 × 10³ wild-type (WT) or MED10-silenced (shMED10) SW1738 or JMSU1 cell lines were seeded per well in triplicates with three biological replica for each assay in 96-well microtiter plates containing supplemented growth media and incubated at 37°C in humidified 5% CO₂. After 24 h, the cells were incubated with alamarBlue™ for 2 h at 37°C. The number of dye-stained viable proliferating cells was read at 570-nm absorbance wavelength in the Molecular Devices SpectraMax M3 multimode microplate reader (Molecular Devices LLC., San Jose, CA, USA).