Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Many researchers who study cultured cells need to measure the rate at which those cells divide. Measuring the rate of growth (or cell division) of the population provides valuable information about basic health and cell maintenance as well as the responses to particular drugs.
Cell-proliferation assays monitor such parameters as DNA content, DNA synthesis, metabolic activity, protease activity and tracking-dye intensity. Each requires different instrumentation, and all have pros and cons. But careful consideration can help you decide which one is best for you.
Measuring by DNA
One common method of assessing cell proliferation looks at the entire DNA content in cells, either over time or as an endpoint assay. “DNA content assays give a convenient snapshot of cell number at a given time point to provide an easy proliferation assay,” says Stephen Oldfield, senior market development manager at Life Technologies. For example, the CyQUANT® assays from Life Technologies are designed for measuring cell proliferation in microplates using a fluorescent readout.
Researchers who want to measure new DNA synthesis (i.e., recently synthesized DNA, as opposed to the entire DNA content), might turn to Life Technologies’ Click-iT® EdU assays, which work in many platforms, including flow cytometry, microplate assays, imaging and in vivo assays, and in tissue samples. “New DNA synthesis assays provide a precise marker of proliferation at the population level or the individual cell level,” says Oldfield. And, he adds, “the assay can be multiplexed with other cellular markers like mitochondrial function or cell morphology.”
DNA-based assays also are available from BioCat, Cell Signaling Technology, Clontech, EMD Millipore, GE Healthcare Life Sciences, New England Biolabs, Roche Applied Science, Sigma-Aldrich, Thermo Fisher Scientific and others. These assays bind to DNA regardless of metabolic state, however, so if metabolism is crucial to your research, read on.
Measuring by metabolic activity
A tried-and-true method of measuring proliferation by metabolism is using tetrazolium salts such as MTT, MTS or XTT. The salts are reduced by metabolically active cells to a colored formazan, which is then detected using a spectrophotometer.
The tetrazolium method is especially convenient for researchers who need to perform multiple assays using a plate-based spectrophotometer. However, notes Pam Guthmiller, strategic marketing manager at Promega, which offers such kits, users should be aware that the salts themselves can be cytotoxic when used for extended periods, which can lead to a loss of signal. Furthermore, the assay’s sensitivity “can be limiting for higher throughput applications due to interferences with the wavelengths detected.”
Kits using this method are also available from Abcam, Cyprotex, EMD-Millipore, Life Technologies, Roche Applied Science, Sigma-Aldrich and others.
Other colored indicators used in cell-proliferation assays, such as the indicator dye alamarBlue®, are derived from the compound resazurin. The dye enters all cells, but only in metabolically active cells is it reduced to become fluorescent. Resazurin-based assays also can be read with plate readers to increase throughput, and they are available from AbD Serotec (a Bio-Rad Company), BioVision, Cell Signaling Technology, Life Technologies, Pierce Biotechnology, Promega, R&D Systems, Sigma-Aldrich and others.
Finally, determining ATP as a measure of viable cells in a sample is also well documented in the literature, especially for high throughput screening applications where scalability and high sensitivity are desirable. The method uses ATP from viable cells in a luciferase reaction, which produces light. The light is detected with a luminometer, with the amount of light generated correlating directly with the number of cells in the sample. The method is simple, fast and highly sensitive, and though it requires cell lysis, can be multiplexed with other fluorescent cell-based assay chemistries, Guthmiller says.
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ATP-based assays are available from BD Biosciences, BioVision, Lonza, Perkin Elmer, Promega, Roche Applied Science and others.
Measuring by staining dividing cells
For following the division of cells through successive generations, one commonly used reagent is the succinimidyl ester of carboxyfluorescein diacetate, also known as CFSE. CFSE binds to proteins and is split evenly between daughter cells upon mitosis. This conveniently labels the cells’ generations in that each generation’s fluorescence intensity is half that of its parents. Labeled cells can be detected in various ways, such as using a plate reader, fluorescence microscope or flow cytometer. This reagent is available from Life Technologies as the CellTrace™ Violet Stain; similar assays are available from Abcam, BioLegend, Cayman Chemical, PromoKine and others.
Life Technologies’ Vybrant® DyeCycle™ stains also let researchers examine the distribution of cell-cycle phases in a population of cells. For studying cell cycles up close in single cells, the company’s Premo™ FUCCI Cell Cycle Sensor lets you watch labeled cells progress through the cell cycle using fluorescence microscopy or high-content imaging.
Measuring by high-content imaging
High-content imaging is one of the newest tools for monitoring cell proliferation, and it generally offers higher sensitivity and signal-to-noise ratios than other methods.
The technique also offers a higher degree of information. For instance, the Thermo Scientific™ High Content Platforms can be used for everything from simple cell counting to more complex assays that measure progression through the cell cycle, mitosis, and bromodeoxyuridine incorporation, according to technical product manager Audra Ziegenfuss.
Researchers can use high-content platforms to work with different types of reagents, either individually or in multiplexed assays, including antibody-based reagents, dye-based reagents, and fluorescent proteins.
Life Technologies’ Premo™ FUCCI Cell Cycle Sensor, for instance, lets researchers visualize the progression of individual, live cells through the cell cycle. FUCCI is a molecular sensor that uses both green and red fluorescent proteins. Each fluorescent protein is fused to a different cell cycle regulator (either geminin or Cdt1). When an individual cell transitions from one phase of the cell cycle to another, the FUCCI indicator changes color – for example, from red to green – and the switch is observable by fluorescence microscopy.
Other high-content analysis tools for cell proliferation are available from Cell Signaling Technology, Essen BioScience, Life Technologies, Molecular Devices, Revvity and others.
One of the most important factors in choosing a method to measure cell proliferation is for researchers to have a clear idea of the questions they want to ask, Ziegenfuss says. This helps them stay focused when presented with the myriad points of interest that tend to emerge from high-content analysis. “Cell proliferation is complex and monitoring the progress of how cells divide, maintain themselves, or even die requires a multifaceted approach,” says Ziegenfuss.
As with any new laboratory endeavor, one should also take into consideration the instrumentation requirements when choosing an assay. “What type of detection instrument [is] available? Luminometer, fluorometer, spectrophotometer, fluorescence imaging, flow cytometry?” asks Guthmiller. In addition, she says, think about potential future needs, such as ability to increase throughput, while also accounting for the difficulty of the workflow and the level of sensitivity required.
Given all the options, making a choice can seem daunting. Just remember, the right system is out there. You just need to do some homework to find it.
Image: A 96-well MTT assay. (Source: Wikimedia Commons)