Determining the exact cause/effect relationship between a treatment and a cellular outcome is not a simple matter, but is critical for really understanding how therapeutic treatments affect target cells or exercise any off-target effects.
Four key factors are critical for determining whether or not a particular treatment or compound is toxic.
- Dosage (usually addressed by a dilution series)
- Exposure time
- Mechanism of Action
- Cell Type
In a recent Promega Webinar, A Cytotoxicity Assay That Fits Your Timeline, Promega scientist Dr. Andrew Niles presented the CellTox™ Green Cytotoxicity Assay—a new tool that gives researchers more power to answer the question “Is my compound or treatment toxic?”
Cell-based models are critical for drug discovery, medical research and basic research in the life sciences. Determining the nature cytotoxic effects resulting from test compounds or other cell treatments is an essential research activity. Many enzyme assays are available to measure cytotoxicity, but because biomarkers of cytotoxicity and apoptosis are transient, these assays may underestimate cytotoxicity if the window of biomarker activity is missed, or they may simply fail to detect an effect such as cell cycle arrest (1,2). Viability assays can be used to report the number of viable cells after a treatment, but even if a decrease in viability is observed, these assays provide no information on the actual mechanism of cytotoxicity.
The CellTox™ Green Dye is a cell-impermeant probe that labels DNA from cells that have lost membrane integrity. The brightness of the dye increases dramatically upon DNA binding, producing a fluorescent signal that can be measured using the “standard” green fluorometry wavelengths (485nmEX/530mnEM). Unlike other compounds that are cell impermeant and that fluoresce when they bind DNA (e.g. propidium iodide), the CellTox™ Green Dye is non-toxic to cells and extremely stable. These characteristics allow you the flexibility of adding the dye at your endpoint, at seeding or at dosing—giving you the ability to measure cytotoxicity in real time or at a convenient endpoint.
Importantly the dye is also compatible with luminescent measurements or other spectrally distinct fluorescent assays, allowing same-well assessment of multiple parameters to determine mechanism of cell death (3). The CellTox™ Green Dye has also been tested for its potential for multiplexing with reporter assays as a control for cytotoxic compound effect on cell viability (4); the ability to add the CellTox™ Green Dye at dosing, seeding or plating allows you to monitor cytotoxicity throughout your experiment, giving you the ability for more meaningful interpretation of your reporter assay data.
For more information about the CellTox™ Green Assay or to request a sample, visit Promega.com.
- Sundquist, T. et al. (2006) Timing Your Apoptosis Assays Cell Notes 16, 18–21.
- Riss, T. and Moravec, R.A. (2004) Use of multiple assay endpoints to investigate effects of incubation time, dose of toxin and plating density in cell-based cytotoxicity assays. Assay Drug Dev. Technol. 2, 51–62.
- Niles A, Worzella T, Zhou M, McDougall M and Lazar D. Measuring Cytotoxicity in Real Time with a Highly Stable Green Dye. [Internet] December 2012. [cited: year, month, date]. Available from: http://www.promega.com/resources/articles/pubhub/measuring-cytotoxicity-in-real-time-with-a-highly-stable-green-dye/
- Bratz, M., Hook, B. and Schagat, T. Multiplexing Assays to Improve Data Interpretation. [Internet] November 2012. [cited: year, month, date]. Available from: http://www.promega.com/resources/articles/pubhub/making-sense-of-your-genetic-reporter-data-through-assay-multiplexing/
About the Webinar Series
www.promega.com/webinars/ provides a schedule of upcoming webinars. In addition, there are links to previous webinars, which allow you to view the recording or download a pdf of the presentation. There is also a pdf of additional material available for each past webinar.
To register for a webinar, use the Registration link at: www.promega.com/webinars/ This allows you to view a live webinar and participate in the live chat.
Need a reminder? You can also sign-up for monthly invitations to webinars at the webinars page (see link above). Note: Live chat is only available for live webinars, not recorded webinars.