Cell Viability

To measure cell viability, AlamarBlue assay was performed at days 0, 3, 5, 7, 10, and 14. The AlamarBlue Assay incorporates an oxidation-reduction (REDOX) indicator that changes color in response to chemical reduction of growth medium resulting from cell growth. As cells being tested grow, innate metabolic activity results in a chemical reduction of AlamarBlue (resazurin) to resorufin. AlamarBlue assay was performed according to the manufacturer's protocol (AlamarBlue® Cell proliferation assay Kit, BIORAD). Briefly, the culture medium was completely aspirated from the wells and cells were washed twice with sterile PBS. One milliliter of fresh medium was added along with 100 μl of AlamarBlue reagent. Additionally, wells containing medium and AlamarBlue reagent only (no cells) were used for blank measurements. Cells and blank samples were incubated for 4 h (37°C, 5% CO₂). After 4 h incubation, three aliquots (100 μl) of each sample were pipetted in a 96 well plate and absorbance was measured at 570 and 600 nm using plate reader (Infinite 200PRO Tecan, München, Germany). Absorbance mean values of triplicates for each sample were calculated and the percentage of Alamar blue reduction was calculated using a formula described in the manufacturer's protocol (AlamarBlue® Cell proliferation assay Kit, BIORAD).