4.5.2. Alamar Blue Assay

Assessment of cell viability after incubation with testing agents was performed with resazurin reduction experiments, using Alamar Blue viability assay. Cell viability at each time point (24, 48, 72 h) was quantified by adding Alamar Blue (Gibco, Invitrogen Inc. Carlsbad, CA, USA) to each well. Treated cells were supplemented with 10% alamar blue reagent and incubated for 6 h at 37 ◦C. Wells that contained only alamar blue were considered as blank while positive control were considered those wells that contained the untreated cells. Percent viability was calculated according to the following formula:

$$\text{V (\%)}=\text{(OD1}-\text{OD2}-\text{Blank)}/(\text{OD3}-\text{OD2})\times 100\tag{3}$$

where OD1 stands for the optical density in nm for treated with the chemical cells, OD2 stands for the optical density in nm for those well containing nutrient medium and the chemical and OD3 stands for the cells that were not exposed to the chemical. Optical density was read at 570 nm. Results were expressed as IC50 values; thus, the concentration of the chemical that causes 50% inhibition with respect to the untreated cells.
