Cell health assays are an essential component of any good drug discovery screening cascade. They are used to identify off-target cytotoxic effects of test compounds and/or to confirm on-target cell killing where this is the aim.
Achieving Reproducible and Consistent Assays
The choice of assay will be project specific and can involve specific measurements of a particular mechanism of cell death or be broader readouts which infer loss of cell viability via surrogate mechanisms.
In early drug discovery bioassays not only reveal whether a compound acts on a biological target but also how effective it is and if it has any potentially off target effects. In turn, this can influence decisions about whether the candidate warrants further study.
The importance of bioassays in early drug discovery may be clear, yet what is less clear is how they should be developed to ensure they are optimised for their specific purpose.
Assay design and development is a complex process, involving many considerations that can each determine the specificity, sensitivity, and reliability of a compound’s action on a target. As such, if you develop your own assays, what you produce can mean the difference between valid and invalid results that help or hinder your drug discovery research.
Choosing the Right Assay
A common cell health assay utilizes CellTiter-Glo® reagents to measure the amount of ATP present within a well. As the concentration of ATP is proportional to the number of cells, this can provide an excellent general assay for the effects of compounds on cell health.
Whilst a reduction in the number of cells can indicate toxicity, impacts on the cellular growth rate cannot be ruled out and as such additional controls are used to identify cytostatic vs cytotoxic effects.
Figure 1: (Left) Diagram depicting how CellTiter-Glo® is used to measure cell viability. (Right) Example data generated from a CellTiter-Glo® cell viability assay.
FAQs of Assay Development
Where a specific mechanism of cell-killing is desired or known then using assays that directly measure that process is recommended.
Apoptosis is a regulated process within the cell with well-known mechanisms and markers of activation, this enables multiple opportunities to measure apoptosis using the method most applicable for the project.
Apoptosis measurements include (but are not limited to):
- Annexin V staining to identify cells with externalised phosphatidyl Serine
- Caspase-3/7 substrate dyes to measure activation of executioner caspases
- TUNEL assays to measure apoptosis-induced DNA cleavage
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