Cell Health Assays

What Are Cell Health Assays?Detection Methods Common Types of Cell Health Assays 1. Cell Proliferation and Cell Viability Assays Promega CellTiterGlo Viability Detection Assay Calcein AM Cell Viability Assay Tetrazolium Salt Cell Viability Assays CCK-8: Impact of Drug Treatment on HeLA Viability 2. Cellular Apoptosis, Cytotoxicity, and Cell Death Assays CaspaseGlo 3/7 Apoptosis Assay NucView488: Apoptosis in HeLa Cells Considerations for Cell Health Assays

What Are Cell Health Assays?

Cell health assays are a broad category of laboratory techniques for monitoring cellular events, populations, environments, and conditions. These assays can be used to study or predict disease states, screen drug candidates for cytotoxicity, analyze cellular responses to treatments, and further elucidate basic indicators of health and our fundamental understanding of cell biology. Cell health assays are some of the most diverse and flexible applications performed by cell biologists today.

Researchers generally analyze cell health assays with the help of dedicated plate readers or imaging systems. These instruments are specifically designed to measure light produced in micro-well plates via chemical or enzymatic reactions. However, plate readers detect light well-by-well, and they often only provide a numerical readout of the signal produced in each well, leaving researchers without a holistic visual image of their entire plate.

The Odyssey® M Imaging System solves this issue. With up to 19 available channels and a large scanning bed, it not only fills the role of Western blot imaging system and slide scanner, but it can also measure many signals produced via chemical and enzymatic reactions in micro-well plates, much like a plate reader or luminometer. The Odyssey M can quickly analyze up to 4 individual micro-well plates at the same time, producing crisp visual readouts of almost any cell health assay at up to 5 µm resolution. When combined with Empiria Studio^® Software or Image Studio™ Software, the Odyssey M Imaging System enables visualization and quantification of plate-based cell health assays without the need for a dedicated plate reader or luminometer.

The Odyssey F Imaging System can also image and quantify fluorescent cell health assays. It features up to 10 channels and the same 25 x 18 cm scanning bed as the Odyssey M, allowing it to quickly scan up to 4 micro-well plates at once. The Odyssey F is also compatible with both Empiria Studio Software and Image Studio Software, and since many cell health assays don’t require high-resolution scanning, the Odyssey F Imager is beyond capable of quantifying many fluorescent cell health assays.

Detection Methods

Cell health assays can be performed using a wide range of detection methods. These include fluorescent, chemiluminescent, and colorimetric detection. All the following assays can be reliably performed using at least one channel of the Odyssey M Imaging System, and the following fluorescent cell health assays can also be performed using an Odyssey F Imaging System. Refer to the table below to find the right wavelength to analyze your particular cell health assay on an Odyssey M or Odyssey F Imaging System.

Common Types of Cell Health Assays

Most cell health assays fall into one of two categories: endpoint assays or real-time assays. Endpoint assays only provide a cellular readout at a single point in time, often at the end of the experiment. By contrast, real-time assays take repeat measurements throughout the process, providing ongoing insights into cell populations and conditions over time. Here we briefly describe two of the most common applications of endpoint and real-time cell health assays: cell proliferation or cell viability and cell death.

1. Cell Proliferation and Cell Viability Assays

Cell viability assays measure the proportion of healthy, living cells within a given population. Cell proliferation is a common indicator of cell viability, and cell viability assays play a central role in drug discovery research. Some common markers of cell proliferation or cell viability are membrane integrity, metabolic activity, and certain enzymatic processes.^[1] The following assays were performed by LICORbio researchers and are compatible with at least one channel of the Odyssey M Imaging System. The Odyssey F Imaging System also supports some fluorescent cell health assays such as the calcein AM cell viability assay.

Promega CellTiterGlo Viability Detection Assay

In this luminescence endpoint cell viability assay, luciferase reacts with cellular ATP to produce a quantifiable luminescent reaction. The presence of cellular ATP is a primary indicator of cell health and metabolism, providing a reliable means to quantify viable cells.^[1] Therefore, the strength of this luminescent reaction’s signal is proportional to the number of metabolically active (viable) cells.

EC₅₀ = 148.5 nM

Figure 1. Cell viability was monitored in HeLa cells using a CellTiterGlo Cell Viability Kit. HeLa cells were treated with increasing concentrations of staurosporine (STS), a protein kinase inhibitor, for 3 hours. Cell viability was determined through the quantification of ATP levels, a key marker of metabolically active cells. Images were acquired using the chemiluminescence channel of the Odyssey M Imaging System using 2-minute acquisition times. Data was obtained in Image Studio Software, and the EC50 value was determined using GraphPad Prism 10.

Calcein AM Cell Viability Assay

Esterases play a key role in regulating various cellular functions including metabolism and gene expression.^[2] In this fluorescence endpoint cell viability assay, non-fluorescent calcein AM is taken up by live cells and hydrolyzed by intracellular esterases to produce fluorescent calcein. Live cells hold this fluorescent calcein in their cytoplasm, and only viable cells with intact membranes exhibit fluorescence as a result. The strength of this fluorescent reaction’s signal is proportional to the number of metabolically active (viable) cells.

EC₅₀ = 265.9 nM

Figure 2. Cell viability was monitored in HeLa cells using a Calcein AM Cell Viability Kit. HeLa cells were treated with varying concentrations of staurosporine (STS), a protein kinase inhibitor, and cell viability was monitored after 24 hours. Non-fluorescent calcein AM is taken up by live cells and hydrolyzed by intracellular esterases to produce fluorescent calcein detected in the 488 channel (green) of an Odyssey M Imaging System. Data was obtained in Image Studio Software and the EC50 value was determined using GraphPad Prism 10.

Tetrazolium Salt Cell Viability Assays

In these endpoint cell viability assays, tetrazolium salts (e.g., MTT, XTT, MTS, CCK-8) are reduced to chromogenic formazans in the presence of metabolically active cells. XTT, MTS, and CCK-8 produce water-soluble formazans, while MTT produces a water-insoluble formazan, which must be solubilized before data acquisition.^[2] Refer to the table below to find the right wavelength(s) to analyze your particular tetrazolium salt cell viability assay on an Odyssey M Imaging System.

CCK-8: Impact of Drug Treatment on HeLA Viability

In this colorimetric endpoint cell viability assay, water-soluble WST-8 is reduced to an orange formazan by the dehydrogenases in live cells. Dehydrogenase activity provides a direct measurement of cell viability. Thus, the strength of this colorimetric reaction’s signal is proportional to the number of metabolically active (viable) cells. Since it’s nontoxic, the CCK-8 assay is suitable for further studies on treated cells.^[3] It’s also stable enough for long incubation times and features a higher detection sensitivity than other tetrazolium salts like MTT.^[4]

EC₅₀ = 164.1 nM

Figure 4. Cell viability was monitored in HeLa cells using a CCK-8 Cell Viability Kit. HeLa cells were treated with varying concentrations of staurosporine (STS), a protein kinase inhibitor, and cell viability was monitored after 18 hours. After a 3-hour incubation period with WST-8, the metabolic conversion of WST-8 to the chromogenic formazan, an indicator of metabolically active cells, was detected in the 470 trans channel of an Odyssey M Imaging System. Empiria Studio Software was used to transform transillumination signal to absorbance, and the EC50 value was determined using GraphPad Prism 10.

2. Cellular Apoptosis, Cytotoxicity, and Cell Death Assays

Cellular apoptosis, or programmed cell death, is the natural end stage of a cell’s life cycle. However, apoptosis can also be induced by toxicity, abnormal cellular conditions, or hostile environments, such as those produced by drug treatments and candidates. Some common markers of cell apoptosis or cytotoxicity include cell stress, cell shrinkage, loss of membrane integrity, nuclear fragmentation, and chromatin condensation, among other indicators like enzymatic activity.^{[5, 6]} Like cell proliferation and cell viability assays, cellular apoptosis assays play a central role in drug discovery research. The following assays were performed by LICORbio researchers and are compatible with the Odyssey M Imaging System.

CaspaseGlo 3/7 Apoptosis Assay

This real-time luminescence cell apoptosis assay quantifies caspase-3 and -7 activity in cell cultures. Because caspases are primary regulators of apoptosis and inflammation, the strength of this luminescent reaction’s signal is proportional to the number of apoptotic cells in a sample.

EC₅₀ = 155.4 nM

Figure 2. Apoptosis was monitored in HeLa cells using a Caspase-Glo 3/7 Kit. HeLa cells were treated with increasing concentrations of staurosporine (STS), a protein kinase inhibitor, and caspase 3/7 activity was monitored after 3 hours. Data was acquired using the chemiluminescent channel of an Odyssey M Imaging System using 2-minute acquisition times. Data was obtained in Image Studio Software, and the EC50 value was determined using GraphPad Prism 10.

NucView488: Apoptosis in HeLa Cells

This endpoint fluorescence cell apoptosis assay quantifies intracellular caspase-3 activity in cell cultures. The strength of this fluorescent reaction’s signal is proportional to the number of apoptotic cells in a sample. This assay can be reliably performed using either an Odyssey F or Odyssey M Imaging System.

EC₅₀ = 231.7 nM

Figure 12. Cellular apoptosis was monitored in HeLa cells using a NucView 488 Caspase-3/7 substrate. HeLa cells were treated with varying concentrations of staurosporine (STS), a protein kinase inhibitor, and cell viability was monitored after 3 hours. After a 30-minute incubation period with the labeled caspase-3/7 substrate, the enzymatic release of the fluorescent DNA dye was detected in the 488 channel (green) of an Odyssey M Imaging System. Data was obtained in Empiria Studio Software, and the EC50 value was determined using GraphPad Prism 10.

Considerations for Cell Health Assays

Whether you’re measuring cell viability or cellular apoptosis, it’s important to understand a few crucial factors before choosing and designing your cell health assay. Proper preparation in the early phases of assay development will save your lab valuable time, budget, and samples down the line.

Always consider your target of interest, the sensitivity required to image and quantify it, and the instrumentation and software you plan to use for analysis. Is your instrument sensitive enough for accurate cell quantification? Does it support your detection method(s) of choice? Will your analysis software help or hinder your lab’s progress? These questions may seem like common sense, but simply overlooking any of them could set you back drastically.

Also, thoroughly consider the ideal assay format and readout for your treatment. If your treatment’s response time is already well known and you’re less concerned about conserving samples, then a basic endpoint assay format may be your best option. However, real-time cell health assays are often ideal for uncovering the time course of a response while conserving cell samples, though they can be more involved than endpoint formats.

Finally, keep in mind that cell cultures may perform differently than cells analyzed in vitro or in vivo. As such, 3D cell cultures can add unexpected challenges and increased complexity to your experimental design. For example, cultured cells often interact with each other quite differently than those grown on plates. Plus, traditional cell health assay reagents may not penetrate large 3D tissues as deeply or evenly as cells plated in wells. In these cases, we suggest optimizing your assay format, reagents, and detection method(s) for 3D cell culture systems.

LICORbio Supports Cell Health Assays

Our goal is to be your constant amid the uncertainty of biological research. This is why our industry-leading instruments support dozens of research applications including various cell health assays. In fact, we specifically designed the Odyssey M and Odyssey F to be the fastest, most sensitive systems in the world for imaging and quantifying gels, membranes, plates, and slides. If you have any questions about the Odyssey M, Odyssey F, or cell health assays in general, then we encourage you to contact us for more information today.