Proliferation is one of the most fundamental qualities to be measured in cell cycle research. But with so many different cell proliferation assays available, which one should you choose?
Why measure cell proliferation?
There are many reasons for researchers to measure cell proliferation. According to Fang Tian, Director of Biological Content at ATCC, these include the study of biological processes such as stem cell renewal, tissue repair, and immune responses, as well as assessment of drug efficacy and toxic effects. “Cell proliferation is also measured during routine cell culture workflows,” she says. “Not only does it allow for verification of cell health, but it can also flag whether a given cell culture has encountered a genetic drift, phenotype change, or potential contamination.”
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Dan Lazar, Senior Research Scientist at Promega, adds that measuring cell proliferation is also important in immuno-oncology research, for both tumor cells and immune cells. “Researchers want to understand changes in proliferative markers, including cell cycle regulators, as a prognostic indicator of cancer progression and guide to potential treatment options,” he reports. “In addition, T cell proliferation is a useful marker to understand if T cells are activated and ready to expand their numbers to attack cancer cells.”
Types of cell proliferation assays
Assays for measuring cell proliferation can be broadly divided into direct and indirect methods. Measuring DNA synthesis is a direct method, usually based on flow cytometry, imaging, or the use of a microplate reader. “These types of assays involve incubating live cells with a thymidine analog such as BrdU or EdU and monitoring its incorporation into newly synthesized DNA using fluorophore-labeled antibodies,” explains Marc van Vijven, Ph.D., Application Scientist at CytoSMART Technologies. “While highly precise, they are limited by a complex, multi-step protocol and the fact that they typically provide an end-point readout.”
Another direct method involves measuring the expression of cell cycle markers, again via immunostaining. Common markers include phospho-histone H3 (Ser10), which is specific for mitosis; Ki67, which is expressed only by proliferating cells; and PCNA, a sliding clamp protein involved in DNA replication. Van Vijven also highlights the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI), which utilizes the mitotic phase-dependent expression of geminin and Cdt1 to provide a red fluorescent signal during G1 and a green fluorescent signal in S, G2, and M, and is popular for live-cell imaging studies.
Measuring dye dilution is an indirect means of tracking cell proliferation. “Carboxyfluorescein succinimidyl ester (CFSE) is a fluorescent dye that has been used for assessing immune populations for decades,” reports Robin Clark, Ph.D., Global Product Manager, Advanced Cell Culture at MilliporeSigma. “When cells proliferate, the CFSE present in the parent cells is effectively halved in each of the daughter cells, so each new generation is half as bright as its parent. Analysis by flow cytometry then reveals clear population generations based on diminishing fluorescence intensity, as well as the number of cells in each generation.”
Proliferation and viability should not be confused
Most of the indirect methods for measuring cell proliferation are based on viability, which refers to the number of live, healthy cells in a population. It is worth noting that not all viable cells will necessarily be proliferating—instead, proliferative cells represent a subset of the viable cell population.
“Tetrazolium reduction, resazurin reduction, and protease activity assays all involve incubating a reagent with live cells and monitoring its conversion to a colored or fluorescent product,” explains Tian. “When cells die, they rapidly lose this ability, therefore the signal measurement is proportional to the number of cells present. While these types of assays reflect viable cell metabolism and not specifically cell proliferation, they offer the advantages of high sensitivity and ease of use.”
The number of viable cells in culture can also be determined by measuring ATP content, such as with the CellTiter-Glo 2.0 Assay or the BioTracker ATP-Red Live Cell Dye. “ATP assays are easily miniaturized to 384- and 1,536-well formats, enabling screening of many anti-proliferative compounds at once,” notes Lazar. “They can also be multiplexed with other cellular parameters to help understand proliferation versus altered viability—for example, multiplexing with a DNA binding dye to measure total DNA content, or with a marker of cell death.”
Ultimately, choosing between the different assay formats available will depend on the aim of your research, as well as what instrumentation is available, how many samples need to be analyzed, what level of sensitivity is required, and whether you want to measure the sample over time. “If a direct measurement of changes that are specific to the rate of mitosis is needed, then detection of phospho-histone H3 could be a good choice,” says Clark. “For high-throughput assessment of viable cells with proliferative potential, you may instead decide to use a viability assay based on a tetrazolium salt like MTT or XTT. In situations where tracking cell division over time is required, a dye dilution assay may be preferred.”
Live-cell imaging of cell proliferation
The use of live-cell imaging for measuring cell proliferation is burgeoning, due to the advantages it affords. Critically, live-cell imaging systems circumvent the need to remove cells from the controlled incubator environment and can eliminate inter-sample inconsistency as a source of variation when comparing time points. Tracking cell proliferation over time also minimizes the number of samples that must be prepared.
“CytoSMART live-cell imaging devices such as the CytoSMART Omni FL have an integrated Confluency Module, which enables users to measure cell proliferation over time using the brightfield and/or fluorescence channels,” says Van Vijven. “Besides improving the accuracy in quality control, the quantitative analysis mediated by live-cell imaging systems is seeing increased uptake for oncology research, where the aim is to assess the reduced proliferative capacity of cancer cells when exposed to a potential drug candidate or cellular therapy.”
Original brightfield image (left) and corresponding overlay of the CytoSMART algorithm for confluency quantification (green; right) of 3T3 fibroblasts which were monitored over time. Images from the start and the end (t = 30 hours) of the experiment are shown.