White walled 96 well plates

Activity of caspases-3/7 and -8 were assessed using the corresponding Caspase-Glo Assays in white-walled 96-well plates (Promega).

Cells were seeded in cRPMI in white-walled 96-well plates (Promega, Madison, WI, USA) at a density of 15,000 cells/well. At 24 h post seeding, medium was removed to waste, and cells were treated with 100 µL of metformin (10 mM) or vehicle control (H₂O) diluted in cRPMI and incubated at 37°C in 5% CO₂/95% humidified air. After 24 h treatment, media was removed to waste and GSH/GSSG levels measured using the GSH/GSSG-Glo™ luminescent assay (Promega), according to the manufacturer’s instructions. Luminescence was measured at 1 s integration time using the Explorer Luminometer (Promega). An identical plate of cells was set up and treated as with the experimental plate and was used for normalisation of assay results by crystal violet assay.