Assessment of cell health is crucial in basic biomedical research, assay development and drug discovery. You want to know: How does a cellular pathway or chemical compound affect cell viability over time? When does cytotoxicity occur and why?
It can be frustrating when using MTT assays, imaging methods or flow cytometry to measure cell health. The complicated procedures, expensive cell culture and equipment, difficult data analysis and ambiguous results… There’s got to be a better way!
We hear you. Promega has developed a series of real-time plate-based assays that are capable of measuring either
live cells, dead cells or apoptotic cells. Learn how real-time plate-based assays are a better way to quantitatively measure cell health.
What are real-time plate-based assays?
Real-time plate-based assays are homogeneous assays with reagents that are added directly at the time of cell plating or treatment to continuously monitor cell health in real-time. Continual analysis is possible because the assay chemistry is highly optimized, nonlytic and nontoxic to cells. This is in contrast to traditional endpoint assays, such as MTT assays, which can be toxic to cells. Endpoint assays are also performed at potentially arbitrary time points. The general protocol for these real-time plate-based assays are similar: add assay reagent at the time of cell plating or combined with compound treatment (or after treatment), incubate for the desired exposure time, and continuously measure luminescent or fluorescent signal with a plate-reader at any time points needed.
How can real-time plate-based assays help resolve current challenges in drug discovery?
Drug discovery has always been like searching for a needle in a haystack. You hope to find good candidate compounds in a cost-effective and efficient manner. This is a complex task because any effect a drug may have on cell health (e.g., cytotoxic, apoptotic) is highly dependent on dosage and timing. Endpoint assays, such as MTT or flow cytometry, can only collect one time point data for every sample setup. So which time points do you choose? Missing a critical time point means you might miss the next blockbuster drug. Real-time assays take out the guess work. You can collect data over the entire drug exposure period—one sample well can produce data for multiple time points. This means less work, less time and less money spent on running parallel endpoint assays. Real-time assays effectively maximize the amount of data that can be collected and minimize your investment per data point.
Missing a critical time point means you might miss the next blockbuster drug. Real-time assays take out the guess work.
Real-Time Assay vs. Endpoint Assay You can get more data from one real-time assay plate than seven endpoint assay plates!
Figure 1. (Left) Real-time Assay: Data for each dose was obtained at multiple time points using a single assay plate with one RealTime-Glo™ Annexin V Apoptosis Assay reagent addition. (Right) Endpoint assay: Seven separate plates were used to collect parallel data using endpoint assay.
Why would someone using MTT assays to measure cell health switch to real-time plate-based assays?
MTT assays have been popular since the 1980s. Today, however, MTT assays are rarely the best choice for assessment of cell health due to their many limitations. There’s a better way. Here’s why you’ll want to switch from MTT assays to real-time plate-based assays:
Fast, simple detection
MTT assays require hours of waiting for signal development and a second solubilization step before absorbance can be measured. Real-time plate-based assays are faster because luminescent or fluorescent signal can be detected within minutes and no second solubilization step is needed.
MTT assays use absorbance for detection, which is generally less sensitive than luminescent or fluorescent methods. Under optimal conditions, MTT assays can typically detect around 1,000 cells per well; while our real-time plate-based assays can detect 10–100 cells per well.
No chemical interferences
Many reducing compounds interfere with the MTT assay by causing non-enzymatic reduction of MTT to formazan. This affects the accuracy of the assay. In contrast, the luminescent or fluorescent signal from real-time plate-based assays are much less affected by chemical interference.
MTT assays are toxic due to spiky substrate accumulation. Real-time plate-based assays, on the other hand, are non-toxic and do not disrupt normal cell function.
Why would someone using imaging to measure cell health switch to real-time plate-based assays?
Measuring cell health using imaging is based on cell morphology. Sure, we all like to see cool images of cells. However, quantifying cell health using morphology may not be necessary and could even complicate analysis. There’s a better way. Here’s why you might want to switch from imaging to real-time plate-based assays:
Cell morphology is easy to see but difficult to define. In order to determine cell health using imaging techniques, every cell type and apoptotic/cytotoxic effect has to be subjectively characterized using mathematical parameters. This process often leads to ambiguous data. Real-time plate-based assays do not rely on subjective analysis. Instead, you get quantitative signal values directly proportional to the number of viable, cytotoxic or apoptotic cells.
Easy data analysis
Imaging is fun—until you have to export and analyze all the data! Analyzing cell health using morphology is challenging and requires a steep learning curve. In contrast, the readout from real-time assays are definitive, objective luminescent or fluorescent signal values. Cell health analysis is easily done by graphing the average signal values of sample wells over time.
No expensive equipment
Imaging requires sophisticated instrumentation and software, which are expensive and challenging to learn and maintain. Real-time assays only require a plate-reader capable of detecting luminescence or fluorescence. No need to worry about software updates or hard-drive failures.
The readout from real-time assays are definitive, objective luminescent or fluorescent signal values.
Why would someone using flow cytometry to measure cell health switch to real-time plate-based assays?
Flow cytometry is a powerful way to identify certain cell populations. However, when it comes to detecting cell health, there’s a better way. Here’s why you might want to switch from flow cytometry to real-time plate-based assays:
Preparing cells for flow cytometry is hard work. Trypsinization, fixing, staining, washing…all before you can run an experiment. Real-time plate-based assays do not require any of these steps: just add reagent during cell plating or combined with compound treatment into sample wells, incubate for the desired exposure time and measure signal with a plate-reader from the same sample wells throughout treatment.
Quantitative, nonsubjective data
A common criticism of flow cytometry is that data processing relies on manual and subjective gating strategies. Small manipulations in the parameters can profoundly affect the distribution of cell populations, giving you ambiguous results. With real-time plate-based assays, you get definitive, quantitative data. The signal either increases or decreases. No more ambiguity.
Because of the tedious procedures involved in flow cytometry, it is extremely difficult to scale up when dealing with hundreds or thousands of samples. The simple protocol and high sensitivity of real-time plate-based assays make them perfect for assay development and high-throughput applications.
No complicated instruments
To perform flow cytometry, you obviously need a flow cytometer. You’ll need to learn how to use it or have dedicated operating staff. A lot of time and effort is spent on training, instrument maintenance and troubleshooting. In contrast, real-time plate-based assays only require a luminescent or fluorescent plate-reader. And plate-readers never clog.
Real-Time Plate-Based Assay vs. Flow Cytometry Real-time plate-based assays give you quantitative, nonsubjective data that is easy to interpret compared to flow data.
Figure 2. (Top Panel) Cells were harvested, washed, and labeled with Alexa Fluor® 488 (green fluorescence, PtdSer:AnxV binding) and 7-AAD (red fluorescence, membrane integrity) and analyzed by flow cytometry (10,000 events). (Bottom Panel) K562 cells (10,000/well) were exposed to serial dilutions of bortezomibin the presence of the RealTime-Glo™ Annexin V Apoptosis Assay reagent. Luminescence (PtdSer:AnxV binding) and fluorescence (membrane integrity) were recorded kinetically. The 16 hour incubation with bortezomib is shown.