What is the protocol of real-time viability assay?

Real-time viability assay is the only cell viability technique that allows researchers to monitor cell viability in real time. 

In this procedure, cells are split into individual wells and treated with a medium containing the desired compound and a real-time viability assay buffer. The cells are then incubated at a temperature of 37°C, and luminescence measurements are taken at regular intervals, typically every 30 minutes or every 60 minutes, depending on the experimental setup. The real-time viability assay buffers are designed to remain stable at 37°C for up to 72 hours, but an optimization step is required for each specific type of cell in order to determine the optimal time frame in which the limiting factor, pro-substrate, is completely consumed.

The real-time viability buffer is added to the wells after the treatment period for end-point experiments. In this procedure, cells are incubated with the buffer at a temperature of 37°C for a period ranging from 10 minutes to 60 minutes, and the luminescent signal is then measured. Either percentage cell growth or a standard ATP measurement can be used to calculate cell viability.