Background

Alamar blue has been commonly used for cell viability and cytotoxicity studies in various biological and environmental systems. There are several other commercially available tetrazolium salts that are frequently used as an indirect measure for viable cells. Some of them includes 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium, 3’-[1-phenylamino)-carbonyl]-3,4-tetrazolium]-bis (4-methoxy-6-nitrobenzene) sulfonic acid hydrate (XTT), and water-soluble tetrazolium salt (WST-1). Besides, Alamar blue and MTT are considered to be the most appropriate, reliable and economic methods. These offer certain advantages including ease of use, increased sensitivity and accuracy, rapid indication of cell toxicity. Both are employed on colorimetric detection of the number of viable cells based on the cellular metabolism activity.

Resazurin, an active component in Alamar blue assay monitors the reducing environment of living cell. The dye acts as an electron acceptor in electron transport chain without disrupting the normal transfer of electrons. As soon as the dye accepts the electrons, it gets reduced to pink color, fluorescent resorufin state. The change in the reduced state can be quantitatively measured spectrophotometrically by recording the absorbance at 570 and 600 nm wavelengths (Rampersad, 2012). Fluorescent signals can also be determined at an excitation wavelength of 530-560 nm and emission wavelength of 590 nm. In the current protocol, anticancer activity of silver nanoparticles was evaluated in A549 lung carcinoma cell lines through Alamar blue assay (Figure 1 A; Kumari et al., 2016; Arya et al., 2018).

In MTT assay (Figure 1 B), the tetrazolium salt is reduced to insoluble formazan dye by dehydrogenase enzyme present in the viable cells at 37 °C. Further, the insoluble formazan salt is dissolved by the addition of solubilizing agents, and the colored product is quantitatively measured spectrophotometrically. The dead cells lose the ability to reduce tetrazolium salts and fail to convert into colored formazan products. Thus, the intensity of the colored product is directly proportional to the number of viable cells present in the culture (Präbst et al., 2017).


Figure 1. Schematic representation of general procedure for cytotoxicity assays using either Alamar blue (A) or MTT reagent (B)