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

Posted on by Kari Kenefick

Originally posted May 25, 2017. Updated 2022

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.

Measuring Cell Viability with Real-Time Assays

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.

Data figure showing measurement of cell viability using real time assays
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.

Measuring Cytotoxicity in Real Time

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.

Data figure showing cytotoxicity measured using the CellTox™ Green Assay. Real-time assays
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.

Measuring Immunogenic Cell Death in Real Time

The RealTime-Glo™ Extracellular ATP Assay is a bioluminescent assay designed for kinetic monitoring of ATP released from dying, stressed or activated cells. Extracellular ATP can function as a damage associated molecular pattern (DAMP) and is a key biomarker for determining whether a treatment induces immunogenic cell death, a specialized form of cell death that results in an immune response.

Data figure showing immunogenic cell death detected using the RealTime-Glo™ Extracellular ATP Assay. Real-time assays
U937 cells were treated with a dilution of mitoxantrone, an anthracycline shown to induce immunogenic cell death (ICD). RealTime-Glo™ Extracellular ATP Assay Reagent was added, and the assay plate was placed in a plate reader set at 37°C. Luminescence was collected every 10 minutes for 24 hours.

Measuring Apoptosis Using Real-Time Assays

The RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay is a simple, plate-based assay for real-time apoptosis detection. The RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay measures the real-time exposure of phosphatidylserine (PS) on the outer leaflet of cell membranes during the apoptotic process, and it can be scaled for high-throughput applications.

Data figure showing secondary necrosis detected using the RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay. Real-time assays
Measurement of Annexin V binding (luminescence) and loss of membrane integrity (fluorescence) repeatedly from the same sample well after exposure to a test compound allows resolution of the effects of cytotoxic stimuli and determination of mechanism of action. Here, the time delay between the emergence of phosphatidylserine : Annexin V binding and the loss of membrane integrity indicates an apoptotic phenotype that leads to secondary necrosis. 

Want more information on the value of real-time assays for your work? We have blogs covering applications of real-time assays right here at Promega Connections.

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Kari Kenefick
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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 for cell-based assays, "add-mix-measure".

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