Real-Time Analysis for Cell Viability, Cytotoxicity and Apoptosis: What Would You Do with More Data from One Sample?

You are studying the effects of a compound(s) on your cells. You want to know how the compound affects cell health over a period of hours, or even days. Real-time assays allow you to monitor cell viability, cytotoxicity and apoptosis continuously, to detect changes over time.

Why use a real-time assay?
A real-time assay enables you to repeatedly measure specific events or conditions over time from the same sample or plate well. Repeated measurement is possible because the cells are not harmed by real-time assay reagents. Real-time assays allow you to collect data without lysing the cells.

Advantages of  Real-Time Measurement
Real-time assays allow you to:

  • Detect kinetic changes during extended incubations, because cells remain viable when using a real-time cell viability assay. Traditional endpoint assays lyse cells in order to collect data, thus only allow sampling of cells at one time point.
  • Perform an extended time study with a single plate, instead of the need to have a separate plate for each time point.
  • Use less reagents. A single-plate assay uses fewer cells and plates, also less culture media and other cell culture tools than multiple plates.
  • Multiplex your cell viability data with other assays to gain more data from a single plate well. Multiple assays from the same cells provides an internal control and less variability than working with multiple, replicate plates.

Here are some examples of data generated using real-time assays.

Real-Time Cell Viability
To measure cell viability in real time using a simple, plate-based, bioluminescent method, we used the RealTime-Glo™ MT Cell Viability Assay . RealTime-Glo™ determines the number of viable cells in culture by measuring the reducing potential of cells and thus metabolism. The nonlytic nature of this assay allows the cells to be used for additional downstream applications, including multiplexing with a variety of assay chemistries.

Five hundred A549 cells were plated in medium containing the RealTime-Glo Reagents in 384-well plates with indicated concentrations of bortezomib. Luminescence was monitored from each well every hour for 72 hours on a Tecan Infinite® 200 Multimode Reader with Gas Control Module (37°C/5% CO2).

For more details on how the RealTime-Glo™ MT Cell Viability Assay works, watch this animation.

Real-Time Cytotoxicity
You can detect the accumulation of dead cells in culture via fluorescence, using a plate reader or microscope and the CellTox™ Green Cytotoxicity Assay. In the following figure, accumulation of dead cells was monitored over 72 hours.

K562 cells were treated with log dilutions of terfenadine (or vehicle control) in the presence of CellTox™ Green Reagent. Fluorescence data was collected every 15 minutes for 72 hours. These cytotoxicity traces reveal the relationship between dose, exposure time and magnitude of response.

Coming Soon: Real-Time Apoptosis Assays
We are preparing to roll out a new real-time , plate-reader based, apoptosis assay based on annexin V binding paired with NanoBiT® Technology. Stay tuned for more information on this new real-time apoptosis assay.

Are you using real-time assays for viability or cytotoxicity analysis? How would you use a real-time apoptosis assay? Tell us about the advantages you’ve gained using real-time assays in your research.

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Kari Kenefick

Kari has been a science writer/editor for Promega since 1996. Prior to that she enjoyed working in veterinary microbiology/immunology, and has an M.S. in Bacteriology, U of WI-Madison. Favorite topics include infectious disease, inflammation, aging, exercise, nutrition and personality traits. When not writing, she enjoys training her dogs in agility and obedience. About the practice of writing, as we say in DNA purification, spin, rinse and repeat.

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