6.4.2 Real-time viability assay
A novel technique for instantly counting viable cells was recently
created. In this experiment, a small molecule pro-substrate and an
altered marine shrimp luciferase are used. The pro-substrate and
luciferase are immediately combined in the cell culture medium. The
pro-substrate is converted into a substrate by living cells with an
active metabolism, and luciferase uses this substrate to create a
powerful signal. The test offers continuous reading and endpoint
measurement as its two options. To count the number of cells in ”real
time,” the bright signal from the sample wells can be repeatedly
recorded over a long period of time using the continuous read
format(Riss et al., 2004).
This assay is advantageous because it is the only one that can quickly
determine cytotoxicity and cell viability. This test can be multiplexed
with other luminous assays because the luminous signal rapidly decreases
after cell death in these procedures. To avoid interference, future
luminous experiments must account for the brightness decline following
cell death.
The pro-substrate would eventually run out due to metabolically active
cells, which is a drawback of the real-time test. The total number of
metabolically active cells determines the strength of the signal that is
produced. How many cells are in each well and how active they are
metabolically affect how long the brilliant signal is linear with cell
number. Therefore, for each cell type and seeding density, the maximum
incubation time required to maintain linearity should be determined
empirically(Riss et al., 2004).