6.3.1 Alamar Blue (AB) assay
The resazurin reduction assay is also termed as alamar Blue assay. The mitochondrial enzymes convert the blue nonfluorescent dye resazurin into the pink fluorescent resoruin in this experiment(O’Brien et al., 2000). Resazurin is an aphenoxazin-3-one dye and a cell permeable redox indicator that can be used to estimate the number of viable cells. It is well known that the electron transport chain uses molecular oxygen as an intermediate electron acceptor between the final reduction of oxygen and cytochrome oxidase(Ansar Ahmed et al., 1994). This material is non-fluorescent and blue in colour. Resazurin is converted to resorufin, a red-colored and highly luminous molecule, when it penetrates cells(Page et al., 1993). The viable cells transform resazurin into resorufin, which brightens and changes the colour of the cell culture medium. The quantity of viable cells directly correlates with the production of resorufin. The ratio of living cells can be determined using a microplate reader fluorimeter and a 560 nm excitation/590 nm emission filter combination. Resorufin can also be found using changes in absorbance, but this technique is less accurate than fluorescence detection. The metabolic activity of the cells, the number of cells in each well, as well as additional elements, such as the type of growth media, all affect how long it takes to obtain a meaningful fluorescence signal above the background.(Riss et al., 2004). Compared to tetrazolium tests, the alamar blue (resazurin reduction) assay has multiple advantages, including lower cost and higher sensitivity. It can also be used with other approaches to understand more about the cytotoxicity process, such as identifying caspase activity. Fluorescence interfering from the test substances, as well as the commonly missed direct harmful effects on the cells generated by this fluorescence interference, are also drawbacks of the assay(Riss et al., 2004).