What are the disadvantages of real-time viability assay?

One limitation of real-time viability assay is that metabolically active cells eventually deplete the pro-substrate. The luminescent signal produced typically correlates with the number of metabolically active cells. However, the linear relationship between the luminescent signal and cell number is influenced by factors such as the number of cells per well and their metabolic activity. To ensure accurate results, it is advisable to optimize maximum incubation time very carefully for each cell type.