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).