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).