Fluorogeneviabiliteitsassays

Fluorogeneviabiliteitsassays are fluorescence-based methods used to quantify cell viability by exploiting enzymatic activities or metabolic processes present in living cells. These assays rely on substrates that are nonfluorescent or weakly fluorescent until converted by cellular enzymes, producing a fluorescent product that correlates with the number of viable cells or their metabolic activity. They are commonly applied in mammalian and microbial systems and are suitable for high-throughput formats.

Common examples include calcein-based assays, where calcein-AM enters cells and is cleaved by intracellular esterases to yield fluorescent calcein, accumulating in live cells. Another class uses redox-based reporters such as resazurin, which is reduced to the fluorescent resorufin in metabolically active cells. Fluorogenic live/dead staining, using combinations of dyes that distinguish live cells from compromised or dead cells, is also used. Readouts are typically obtained with a fluorescence plate reader, measuring emission at characteristic wavelengths after an incubation period.

Advantages of fluorogeneviabiliteitsassays include compatibility with high-throughput screening, rapid results, and avoidance of radiolabels. Limitations include potential interference from test compounds that are intrinsically fluorescent or that affect fluorescence signals, variability in esterase activity or metabolism across cell types, and the need for appropriate controls and calibration to relate fluorescence to viability.

Applications span cytotoxicity testing, drug discovery, microbiology, and environmental toxicology. Best practices involve optimizing substrate concentration and incubation time, including positive and negative viability controls, and validating fluorescence data with an independent viability measure when feasible.