Selecting the Best Method for Measuring Cell Proliferation

Cellular proliferation is the fundamental biological process by which cells undergo division to produce new cells. There are five key methods for measuring cell proliferation—indirect measures, cell cycle markers, dye dilution assays, DNA synthesis methods, and live-cell imaging—which we covered in an article last year. This year, we asked experts for some additional tips to help researchers choose the right cell proliferation measurement technique. Here is what they had to say.

Indirect Measures

Fang Tian, Director of Biological Content at the American Type Culture Collection (ATCC), notes that the easiest and most common cell proliferation techniques involve indirect methods that detect viable cells. “Although cell viability differs from cell proliferation, these methods can indirectly measure cell proliferation with the appropriate experimental design and proper controls to confirm the effects on metabolism.”

For experiments investigating cell response to chemicals, cytokines, or other perturbations, Tian recommends indirect cell proliferation measurements based on metabolic activity. “These indirect methods provide the benefits of high throughput, sensitivity, and ease of use.” As examples, she highlights tetrazolium dye assays (including MTT, MTS, WST-1, and XTT) and CellTiter-Glo assays.

These measurements can be performed in 96-, 384-, or even 1,536-well plates. The assay readouts include colorimetric, fluorescent, and luminescent signals, and a regular microplate reader can be used.

Robin Clark, Global Product Manager of Advanced Cell Culture at MilliporeSigma, agrees that tetrazolium dye assays are convenient because they can typically be measured using a spectrophotometer, a very accessible instrument. “However, remember that MTT, XTT, MTS, and WST-1 measure proliferation indirectly, and readings are interpreted as evidence of increased cell titer.” From a quality perspective, Clark also advises that users check the product claims for chemical entities like MTT and XTT to see if they are tested by the vendor for cell culture applications.

Cell cycle markers

Tian notes that the analysis of cell cycle markers is another standard and easy-to-use method for measuring cell proliferation. “This technique measures the expression levels of cell cycle proteins and other proteins required for proliferation,” she explains.

Multiple proteins can be used as cell cycle markers, including Ki67, proliferating cell nuclear antigen (PNCA), and phospho-histone H3. Tian notes that a fluorescence microscope and imaging analysis software are typically required for these assays. However, Clark warns that while most labs can access fluorescence microscopy, fluorescence detection methods are harder to quantify.

Tian explains that cell cycle markers are commonly used in research and pathology labs for cancer diagnosis. “For example, ki67 is a great marker to choose if a researcher is trying to verify tumor tissue samples or is looking for the co-localization of proliferation markers.” Although Clark agrees that approaches based on cell cycle markers are high content, she notes that they typically do not allow for high-throughput analyses.

Dye dilution

For labs that have access to flow cytometry equipment and expertise, Clark recommends cytoplasmic dye dilution methods because they are accurate and give very clear results. The most popular assay uses carboxyfluorescein succinimidyl ester (CFSE), a non-toxic fluorescent dye that can be stably and evenly incorporated into cells to trace multiple rounds of cell division. “Several generations of cells can therefore be monitored, as the dye is halved with each cell division. The number of cells in each generation is then identifiable as a population,” explains Clark.

Clark emphasizes that flow cytometry is highly sensitive and quantitative. “It is one of the few detection instruments that can quantify proliferation with single-cell precision.”

However, Tian notes that dye dilution may be more labor- and time-intensive than indirect methods and cell cycle markers. For instance, dye dilution requires researchers to analyze CFSE fluorescence intensity in regular time intervals after staining.

DNA synthesis methods

Tian notes that these methods―which measure proliferation during the synthesis (S) phase of the cell cycle―are recommended if a precise cell proliferation measurement is needed in a study of cell cycle regulation and cell division. “These are the most direct, reliable, and accurate methods to measure cell proliferation,” she says. However, DNA synthesis methods typically only provide endpoint readouts.

Tian warns that DNA synthesis approaches can be very intensive in terms of labor and equipment. She explains that these techniques—including bromodeoxyuridine (BrdU) and ethynyl deoxyuridine (EdU) assays—incorporate radioactive isotopes or non-radioactive nucleoside analogs during new DNA synthesis. Live cells are typically incubated with a thymidine analog and analyzed using flow cytometry, imaging, or a microplate reader.

Tian also emphasizes the importance of a well-prepared sample for accurate results. “When using a DNA synthesis method, there will be a series of steps involving sample preparation, labeling, and washing,” she adds.

Clark notes that BrdU assays requiring immunodetection are typically low throughput. As an alternative, she recommends EdU, an alternative thymidine analog that crosslinks the analog to a small, freely diffusible fluorescent azide. “This eliminates the need for DNA denaturation, simplifying the detection protocol.”

Live-cell imaging

Tian explains that live-cell imaging is an emerging method to monitor cell proliferation. “The imaging and tracking of individual cells enable the measurement of cell proliferation in a controlled incubation environment over time.”

Nathalie Opdam-van de Laar, Application Scientist at Axion BioSystems, notes that label-free, live-cell analysis methods offer a simple and very convenient option to monitor proliferation over time. Live-cell imaging also allows for high-throughput analyses. She recommends impedance-based platforms such as the Maestro Z. Alternatively, newer whole-well imaging systems, such as the Omni platform, can sensitively measure proliferation at a wide range of densities.

Tian explains that current live-cell imagers usually generate cell growth curves by measuring cell count (cells/cm2) or cell confluence percentage. However, she warns that the setup for cell imaging and analysis parameters could impact the accuracy of the overall readout. “For instance, adherent cells are much more suitable than suspended cells for live-cell imaging proliferation assays. This is because adherent cells attached to the culture plate enable more accurate imaging and cell count.”

Opdam-van de Laar points out that cell proliferation, by its very nature, has a time component. “Therefore, continuous live-cell assays that deliver results in real-time offer advantages over endpoint assays by providing rate information over the entire course of the experiment.”

Final recommendations

When selecting a cell proliferation method from the five listed above, Opdam-van de Laar emphasizes the importance of understanding the type of data you hope to collect. Furthermore, she notes that it is essential to consider the culture conditions and characteristics of your cells to ensure the assay will be sensitive and robust enough. Finally, you should evaluate reagent costs and the hands-on time required for the throughput you will need. “While the biology of proliferation can be complex, measuring it doesn’t have to be,” she concludes.

Helpful reading

Romar GA, Kupper TS, Divito SJ. Research Techniques Made Simple: Techniques to Assess Cell Proliferation. J Invest Dermatol. 2016;136(1). https://pubmed.ncbi.nlm.nih.gov/26763463/