The drug discovery arena, especially in oncology, demands precise and reliable assays. Gauging the efficacy of anti-cancer drugs involves understanding their impact on cell proliferation, viability, and the mechanism by which cell death occur. The ability to measure cell viability, cytotoxicity and apoptosis events robustly and accurately is paramount. These parameters serve as the bedrock of understanding cellular health and drug efficacy. Here we present two renowned assays commonly used to assess cell health, the CellTiter-Glo™ (CTG) and ApoTox-Glo™ Triplex.

What method is used to assess cell viability?

Most viability assays rely on several cell functions such as cell membrane activity, enzyme activity, nucleotide uptake activity and adenosine triphosphate (ATP) production. Measuring of ATP, which is only synthesized in metabolically active cells, is one of the most common methods to detect viable cells thus acting as a surrogate marker for cell viability. The CellTiter-Glo™ assay allow to quantify accurately the amount of ATP content.

Assessing Potential Chemotherapeutics on Cell Growth and Viability

The CTG assay rely on a stabilized luciferase and luciferin substrate to measure ATP using a bioluminescent assay. Briefly, using ATP from viable cells, luciferase uses luciferin to generate luminescence, which is detected with a luminescence spectrophotometer. The resulting signal is proportional to the number of viable cells. Therefore, this method can indicate the number of viable cells post-drug treatment. A decrease in luminescence suggests reduced cell viability, implying a drug’s potential cytotoxic effect. This method is great for high-throughput applications.

Probing Cytotoxicity or Apoptosis Events in Cell-Based Assays Post-Drug Treatment

While cell viability assay offers insights into overall cell health, understanding the mechanisms of drug-induced cell death is equally crucial. This is where the ApoTox-Glo™ Triplex shines. By concurrently evaluating viability, cytotoxicity, and caspases activation (a hallmark of apoptosis), this assay provides a holistic view of cellular responses.

The method used takes advantage of the loss of membrane integrity. Indeed, the cytotoxicity marker relies on the activity of enzyme marker that leaked from the cytoplasm of cells with damaged membrane while the viability marker depends on the activity of enzymes present in intact cells. Both viability and cytotoxicity markers generate fluorescence which is measured at two wavelength sets. Caspases activity is then measured through the cleavage of a marker resulting in the production of luminescence.

Through this trifocal lens, researchers can discern whether cell death is primarily due to necrosis, apoptosis, or a combination of both.

Conclusion

While both CellTiter-Glo™ and ApoTox-Glo™ offer valuable insights, their utility depends on the specific research questions posed. A comparative study, available on our website, delves deeper into the nuances of these assays, guiding researchers towards informed decisions.

 

References

CellTiter-Glo Luminescent Cell Viability Assay Technical Bulletin, Promega Corporation.

ApoTox-Glo Triplex Assay Technical Manual, Promega Corporation.

Cory S, Adams JM. The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer. 2002;2(9):647-56.

Méry B, Guy JB, Vallard A, Espenel S, Ardail D, Rodriguez-Lafrasse C, Rancoule C, Magné N. In Vitro Cell Death Determination for Drug Discovery: A Landscape Review of Real Issues. J Cell Death. 2017 Feb 24;10:1179670717691251.

Shamas-Din A, Brahmbhatt H, Leber B, Andrews DW. BH3-only proteins: Orchestrators of apoptosis. Biochim Biophys Acta. 2011;1813(4):508-20.