Measurement of cell proliferation is essential for labs that routinely use cell cultures as an experimental system—and not only for routine maintenance of cell health. Accurately assessing the effect of various drugs or drug candidates on cell proliferation, for example, is an important way to unlock secrets of anticancer weapons, and to learn about the origins of cancer itself.
Generally, cell proliferation assays measure the number of cells (or the change in the proportion of cells) that are dividing. The four main types of cell proliferation assays differ in the nature of their endpoint measurements, whether it be metabolic activity, DNA synthesis, ATP concentration, or the presence of markers for cell proliferation. All of these assay types are adaptable to microplate readers and multiwell plates and are available as convenient assay kits from multiple vendors, allowing researchers to take advantage of automation and higher-throughput technology.
Assaying metabolic activity
Cells that are actively proliferating have higher metabolic rates than those that are not. A convenient and widely used assay for cell proliferation measures the metabolic activity of cells using tetrazolium salts (for example, MTT, MTS, XTT, and WST-1; and similar resazurin redox dyes such as Alamar Blue). Tetrazolium salts become reduced to formazan in the presence of cells that are metabolically active. In MTT assays, for example, the chemical reduction produces a purple formazan crystal when dehydrogenase enzymes cleave the tetrazolium ring. After solubilizing the crystals, colorimetric signals —proportional to cell number and to metabolic activity—are easily read by microplate spectrophotometers and are effective tools for measuring the effects of proliferation inhibitors.
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Learn more about the different methods available to characterize cell proliferation as well as their advantages and disadvantages. For example, BioTek recently demonstrated the cytotoxic effect of thiostrepton on mesothelial cells using Invitrogen’s VybrantⓇ MTT Cell Proliferation Assay Kit and BioTek’s EpochTM Microplate Spectrophotometer.
Other tetrazolium salts such as XTT and MTS are similar to MTT, but their reduced product is soluble in culture media, eliminating the need for the extra solubilization step. For example, in XTT assays, healthy cells convert XTT to a water-soluble, orange-hued product of formazan in a complex bioreduction reaction. Alamar Blue redox dye is a type of resazurin that enters live, viable cells and then becomes reduced intracellularly. The subsequent cell signals can be detected using microplate readers.
A similar reagent, known as water soluble tetrazolium salts (WSTs), are tetrazolium dyes that do not enter cells but become reduced extracellularly by electron transport at the cell membrane to produce soluble formazan. For example, in WST-1 assays, the WST-1 tetrazolium salt is reduced to a soluble form of formazan in a reaction that is linked to the glycolytic production of NAD(P)H and is, therefore, directly related to metabolic activity of the cells.
Assaying ATP concentration
Proliferation can also be assayed by taking advantage of the tight regulation of the intracellular concentration of ATP. Healthy cells synthesize ATP, whereas unhealthy or dead cells contain little or no ATP. The firefly enzyme luciferase uses ATP to oxidize the substrate D-luciferin, resulting in light production. This bioluminescent system makes for a sensitive assay of the detection of ATP in cell cultures.
The assay usually begins with lysing the cells to release the intracellular stores of ATP, while also inhibiting ATPases to prevent the degradation of the released ATP. The amount of ATP is then measured by adding a combination of luciferase and luciferin to the lysed cell sample. As the enzyme luciferase uses the available ATP to oxidize the substrate, it produces light proportional to the ATP concentration, allowing a sensitive measurement of healthy cells in a cell sample using microplate readers.
Assaying DNA synthesis
Some workflows may benefit from readouts of DNA synthesis as indicators of proliferation. Proliferating cells are characterized in part by increased DNA synthesis activity. Traditionally, cells were incubated with radioactive [3H]-thymidine, which was incorporated into DNA. However, today, nonradioactive alternatives exist for researchers who don’t want to undertake the extra time, effort, and health risks involved in using radioactive reagents and then dealing with radioactive waste disposal.
An alternative method of assaying DNA synthesis is to incubate cells with the nucleoside analog 5-bromo-2'-deoxyuridine (BrdU), which becomes incorporated into newly made DNA in the place of thymidine during the S phase of the cell cycle. Because BrdU is not radiolabeled, it must be subsequently detected by incubating with a BrdU-specific monoclonal antibody and then with a secondary antibody that acts as a reporter by emitting a signal detectable by microplate readers (whether by fluorescence or chemiluminescence).
Similar to BrdU, another nucleoside 5-ethynyl-2'-deoxyuridine (EdU) becomes incorporated into newly synthesized DNA, but EdU is detected using fluorescence rather than immunodetection, which can eliminate some protocol steps. A chemical reaction catalyzed by copper enables the attachment of fluorescent probes to the EdU nucleoside to facilitate subsequent detection.
Detecting antigens in proliferating cells
Proliferating cells express marker antigens that nonproliferating cells do not, so immunologic detection of these markers with monoclonal antibodies can prove a useful method for assaying proliferation. These distinct antigens are usually expressed in the nuclear or perinuclear regions of proliferating cells. The most well-known and widely used proliferative marker is Ki67, but it is mainly used in fixed cell samples. However, knowing how its staining overlaps with that of other proliferative markers may prove to be informative when comparing to the body of already published studies that feature Ki67.
Other common markers for proliferating live cells include PCNA (proliferating cell nuclear antigen), which detects cells mainly in late G1 and S phases of the cell cycle, and MCM-2, which detects cells in G1, S, G2, and M phases of the cell cycle. Both PCNA and MCM-2 are also showing promise for use as prognostic markers in some types of cancers.
All of the types of cell proliferation assays discussed here are routinely adapted for microwell plates so that their signals (whether colorimetric, fluorescent, or luminescent) can be measured by microplate readers. Microplate readers provide a powerful tool for accomplishing many cell proliferation assays in a short amount of time. The particular type of assay a researcher chooses depends partly on their cell type, experimental system, and protocols, and partly on what aspect of cell proliferation is of interest. Regardless of the type of cell proliferation assay chosen, rest assured that all of these dependable methods have been replicated many times and are virtually guaranteed to be reliable.
If you want to discover more about measuring cell proliferation, download our free eBook The Many Ways to Measure Cell Proliferation and learn about the types of cell proliferation assays, quantification of cell numbers from proliferation studies, label-free cell counting, and more.