3.8. PrestoBlue Assay

PrestoBlue is a resazurin-based membrane permeable solution, which, upon reduction by living cells, forms a red fluorescent compound called resorufin via mitochondrial enzymes of viable cells in the tested systems. Consequently, the reagent exhibits a change in color, and a shift in its fluorescence, which is quantitatively measurable providing information on the cell viability. For the experiment, 5000 GICs, GIC7 and PG88, were seeded in each well of a 96 multi-well plate in complete culture medium. The cells were incubated 48 h at 37 °C under 5% CO₂ and 5% O₂. Next, the medium was aspirated and Na[o-COSAN] in fresh medium was added to achieve the final tested concentrations [µM]: 50, 75, 100, 125, 150, 175, 200, 250, and 300, added to each well in triplicates. Control cells that contained only cells in medium without Na[o-COSAN] were also incubated at 37 °C under 5% CO₂ and 5% O₂ for 48 h. Next, 10 µL of PrestoBlue 10X (Life Technologies, Carlsbad, CA, USA) in 90 µL of fresh NSC medium was added and incubated at 37 °C for 2 h. Absorbance (Abs) was recorded at wavelength of 570 nm and 600 nm by the Synergy (BioTek, Agilent Technologies). Data was normalized according to the manufacture protocol.