6.1.1 Trypan blue dye exclusion assay
A common technique for estimating the percentage of alive and/or dead
cells in a cell suspension is trypan blue dye exclusion. Trypan blue has
a large, 872.88 molecular weight and is negatively charged. The premise
behind this is that intact cell membranes are a characteristic of living
cells. Cells that are adherent or non-adherent are cultured in this test
using various dilutions of the test substance for varying lengths of
time. After the chemical treatment, the cells are washed and resuspended
in medium. To find out whether cells take up or reject dye, dye is added
to the cell suspension, loaded onto cell counters
(haemocytometers/automated counters), and microscopically analysed. The
living cells have a clear cytoplasm, whereas the cytoplasm of dead cells
is blue. As a result, the dye is entering the cytoplasm through the
damaged cell membrane (Johnston, 2010)(Wiley, 2005). The amount of
active & dead cells/ unit volume is compared to untreated control cells
using light microscopy.
This method is advantageous since it is simple to apply, inexpensive,
and an excellent indicator of membrane integrity. If deceased cells are
exposed to the dye, they become blue in seconds(Stone et al., 2009).The
drawbacks of this technique include counting mistakes, as cell counting
is often performed with a haemocytometer[20]. The poor cell
dispersion, cell loss during cell dispersion, inaccurate cell dilution,
insufficient chamber filling, and the presence of air bubbles in the
chamber can all result in counting errors (Stone et al., 2009).However,
the staining method is simpleĀ and can handle a number of samples if it
is regained to evaluate cytotoxic effects escalate. The black trypan
also distinguishes between healthy and damaged cells(Yip and Auersperg,
1972).As a result, it lacks the sensitivity needed to detect in vitro
cytotoxicity. Trypan blue also has the disadvantage of having negative
side effects on mammalian cells when exposed for extended periods of
time(Kim et al., 2016).