4.3. Cell Viability Assay

The colourimetric MTT assay is routinely used to measure cell viability in the context of ferroptosis [10,11,13]. The assay principle is that nicotinamide adenine dinucleotide phosphate (NADPH)-dependent cellular oxidoreductase enzymes and/or succinate dehydrogenase in the mitochondria of metabolically active cells reduce the yellow MTT to purple formazan crystals. As such, metabolic changes may impact the MTT signal in the absence of cell death; however, in the context of acute ferroptosis, we have regularly observed the MTT results to parallel other viability assays, such that any metabolic contribution would be marginal. To conduct this experiment, we seeded immortalised cells in 96-well plates at a density of 2 × 10⁴ cells/well, while primary cortical neurons were 1 × 10⁵ cells/well. Cells were then co-treated with tested compounds and ferroptosis inducers for 24 h (but tBH was for 4 h), and 20  μL of MTT (5  mg/mL) was added to each well and incubated for 2–4 h. The overlying media were aspirated from each well, and DMSO was added to the wells to dissolve formed formazan crystals in viable cells. The absorbance at 570 nm of the dissolved formazan demonstrated viable cells and was measured by a PowerWave XS microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA), and cell viability was expressed as a percentage of control cells.