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Chapter 3:
Bioluminescence- and Fluorescence-based Cell Viability and Proliferation Assays

Originally, the bioluminescence assays developed to measure levels of different intracellular metabolites were used in conjunction with destructive sampling methods to provide information on intracellular processes. Among the best known bioluminescence methods are the ATP assay, based on the use of firefly luciferase, and the Ca2+ assay, based on the photoprotein aequorin. The intensity of the bioluminescence produced in the presence of these bioluminescent proteins is proportional to ATP and Ca2+ concentrations, respectively. The reported sensitivity of the assays is in the attomole (10-18 mole) range. There have been many publications in which these methods were used to assess intracellular concentrations of many important metabolites either directly or through the coupling of bioluminescent reactions with other enzymes (for review, see Refs. 1 and 2).

From the 1990s on, it became clear that bioluminescence could be detected and quantified directly from inside a living cell. This gave rise to numerous possibilities for noninvasive in vivo monitoring of intracellular processes using bioluminescent molecules as reporters. Bioluminescent viability assays are widely used in environmental testing and for both anti-tumor and bacteriocidal drug development, as well as in transplantation research.

3.1 Principles of Bioluminescent Cell Viability and Proliferation Tests

Bioluminescent cell viability and proliferation tests are based on the assumption that at stable physiological conditions, and in the presence of bioluminescent enzymes in cells, the intensity of the emitted light per cell is constant. Thus, the measured total bioluminescence intensity is indicative of the number of live cells present in the sample. The time course of the bioluminescence intensity after the application of certain physical, chemical, or biological treatments shows the respective growth or decline in the number of live cells. Because of the direct correlation between viability and cell number in light emission, the effect of various treatments can be determined very rapidly using a high-throughput format. For this application, the luciferases from firefly, bacteria, Gaussia, and Renilla have been used.

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