by Dr. Sonal Patel

Cell proliferation plays a vital role during the lifespan of an organism, from the time of embryogenesis throughout development. Cell proliferation helps maintain homoeostasis by recycling out old cells, and introducing new cells. Abnormal cell proliferation can lead to unusual and unnatural cell growth, which may result in disease or cancer. For this reason, there are many available methods and techniques for measuring cell proliferation across fluorescent, colorimetric, and luminescent platforms. In general, four main techniques are used to assess cell proliferation which measure the:

Overall, the chosen cell proliferation assay depends on the available laboratory resources, the types of cells or tissues to be studied, and your specific experimental goals. This may sound like a daunting task; however, a fundamental understanding of each assay type will help you easily select the right assay, most suited to your needs. Refer to the table below for a quick overview of the different cell proliferation assays mentioned in this article, including their pros and cons.

A cell spends most of its life in the interphase where it grows and prepares for division. The interphase is divided into three stages: Gap 1 (G1), Synthesis (S), and Gap 2 (G2). During the S phase of interphase, the cell's DNA is replicated to ensure that each daughter cell will have a complete set of genetic instructions after cell division. DNA polymerases play a crucial role in this process by adding nucleotides to the growing DNA strand. Cell proliferation assays that measure DNA synthesis play on this inherent quality of cells and work by incorporating nucleoside-analogs into newly synthesized DNA during the S phase, which is later quantified.

One example of this type of cell proliferation assay uses BrdU (Bromodeoxyuridine), a thymidine analog. Once BrdU is incorporated into new DNA during synthesis, it remains attached to the DNA and is passed down to daughter cells after mitosis. One way to accomplish this involves incorporating the thymidine analog, BrdU (17030), into newly replicated DNA during the S phase of the cell cycle. Proliferating BrdU-labeled cells can then be measured using fluorescently labeled anti-BrdU antibodies. BrdU staining is suitable for various applications, including IHC, ICC, and ELISA, and is amenable to staining with other biomarkers of interest in flow cytometry. We offer fluorescent conjugates of the mouse monoclonal anti-BrdU antibody clone Bu20a labeled with PE (V103305) and unlabeled mouse monoclonal anti-BrdU antibody clone Bu20a (V103300) and clone MoBu-1 (V103295) that can be labeled with our brightest and most photostable iFluor® dyes. Note that the BrdU cell proliferation assay does not measure the number of cells in a well, but the rate of proliferation.

The downsides of the BrdU assay helped in the creation of another thymidine analog, EdU (5-ethynyl-2'-deoxyuridine) to monitor cell proliferation through DNA synthesis. EdU is incorporated into live cells, followed by fixation, permeabilization, and click chemistry. Click chemistry is based on a copper-catalyzed reaction that adds a fluorescent azide to an alkyne group onto EdU, which incorporates into the DNA. This fluorescent tag is small enough to easily penetrate native tissues to reach DNA, allowing for easy identification of proliferating cells.

This copper-catalyzed azide-alkyne cycloaddition (CuAAC) provides a bio-orthogonal system for monitoring cell proliferation that does not non-specifically label other biomolecules. Click chemistry requires only mild aqueous conditions, eliminating concerns about unwanted molecular byproducts. Unlike the BrdU assay, no DNA denaturation step is required, allowing for further identification and classification of cellular subpopulations. Tissues used in the EdU assay can also be used for additional analyses, such as further immunostaining. As click chemistry methods continue to advance, research suggests that cell populations can be labeled in the presence of fluorescent proteins and phycobiliproteins without excessive quenching by copper. Compared to BrdU, the EdU assay takes less time to perform and better preserves cellular, nuclear, and chromosomal morphologies.

Unlike BrdU assays, Bucculite™ XdU Cell Proliferation Assays do not rely on antibodies to quantify nascent DNA, nor do they require DNA denaturation needed to facilitate antibody detection of BrdU-labeled nucleosides. Rather, Bucculite™ XdU Cell Proliferation Assays use a proprietary mixture of alkyne-containing thymidine analogs, XdU, that is incorporated into newly synthesized DNA.

Because of the mild reaction conditions, Bucculite™ XdU Cell Proliferation Assays preserve cell morphology, antigen-binding sites, and sample integrity, affording the user an opportunity for multiplexing analysis with cell cycle dyes, antibodies against cell surface markers, and fluorescent proteins. After XdU incorporation, the reaction and subsequent wash steps can be completed in less than 60 minutes, and nascent DNA can be quantified using standard imaging systems or a flow-cytometer. Also available are environmentally safe copper-free Bucculite™ FdU Cell Proliferation Assays in green, red, and deep red fluorescence. These kits utilize our patent Buccutite™ labeling chemistry to facilitate the detection of newly synthesized DNA in actively proliferating cells.

Metabolic activity assays use enzymatic activity as a marker for cell viability. Specific enzymes produce either a colored or fluorescent product that can be easily measured with a standard plate reader. These assays indirectly measure cell proliferation, so it is important to consider that the metabolic activity of non-proliferative cells also affects the results.

Tetrazolium assays rely on the bioreduction of tetrazolium salts (e.g., MTT, XTT, MTS, and WST-1). The tetrazolium salt is first cleaved by living and metabolically active cells. The samples then undergo a mild reduction which reduces the tetrazolium salt, disrupting the chemical stability of the tetrazole ring. This reaction turns the colorless-to-pale yellow substrate into a rich blue formazan product. Through the creation of a standard curve, this color can be quantified. A deeper color indicates that more formazan was generated in the reaction, which is directly proportional to metabolic activity. The convenient Cell Meter™ Colorimetric MTT Cell Proliferation Kit (22768) simplifies the task of counting cells to an easy mix and read format without the need for solubilization. The concentration of formazan determined by optical density at 560 nm is directly proportional to the number of live cells.

Among the other tetrazolium salts, the water-soluble WST-8 offers the highest sensitivity. Reduction of WST-8 by cellular dehydrogenases yields water-soluble orange formazan crystals that do not require solubilization prior to quantification. The concentration of formazan product can be determined by measuring absorbance at 460 nm and is proportional to the number of living cells. The Cell Meter™ Colorimetric WST-8 Cell Quantification Kit provides a sensitive assay with exceptional linearity of up to ~106 cells per well. No pre-mixing of components is required, as the WST-8 substrate is conveniently provided in a solution that can be directly added to the cells. Each kit provides sufficient reagents for either ~1000 (22770) or ~5000 tests (22771) using a standard 96-well microplate. WST-8 is also available as a stand-alone reagent in 25 mg (15705), 100 mg (15706), and 1 g (15707) unit sizes supplied as 50 mM aqueous solutions.

Resazurin is a water-soluble non-toxic dye that allows for the continuous monitoring of cell proliferation in cultures over time. Like the aforementioned tetrazolium salts, the oxidation-reduction indicator resazurin (15700) can be useful for quantifying cell proliferation. First, the oxidized form of the resazurin reagent, which is non fluorescent and blue in color, is incubated with the cell sample. Resazurin is then converted to Resorufin by mitochondrial enzymes which transform the sample color into a highly fluorescent, pink, product. The product can then be measured colorimetrically or fluorometrically. Compared to tetrazolium salt-based assays, the resazurin assay offers fewer steps, and subsequent assays can be performed on the same sample. Resazurin is also stable, ready-to-use, and minimally toxic to cells in culture. The assay is highly sensitive, and only a very small population of cells is needed to provide accurate results.

Proteolytic enzymes, or proteases, are involved in many cellular processes and regulate the fate, localization, and interaction of many proteins. In research, pharmaceutical, and clinical fields, proteases are commonly used as diagnostic and prognostic biomarkers. A common protease activity assay uses glycyl-phenylalanyl-aminofluorocoumarin (GF-AFC), a fluorogenic cell-permeable substrate that selectively detects protease activity in viable cells. When GF-AFC enters live cells, the glycine and phenylalanine amino acids are released, generating a fluorescent signal directly proportional to the number of viable cells. After cell death, a second protease is released into the medium. This second protease can be detected by the non-permeable fluorogenic substrate AAF-R110, which serves as a biomarker for non-viable cells. Typically, GF-AFC and AAF-R110 are used in tandem. After a 30-minute to one-hour incubation, fluorescent signals can be quantified. Both substrates have low levels of toxicity and are compatible with other reagents. Additional parameters can be monitored without interference with downstream applications.

CFSE and CytoTell® indicators are fluorescent tracking dyes useful for monitoring cell proliferation in living cells by flow cytometry. As cells divide, the fluorescent dye is uniformly distributed between daughter cells, resulting in the progressive halving of fluorescence intensity with each successive generation. These proliferation dyes can be used to track cell proliferation long-term both in vitro and in vivo for several generations.

CFSE (Carboxyfluorescein diacetate, succinimidyl ester, 22022) and ReadiUse™ CFSE (22028) are cell-permeable green fluorescent proliferation indicators that emit a fluorescence signal at 517 nm when excited by the 488 nm argon-ion laser. Both dyes passively diffuse across uncompromised cell membranes, whereby enzyme reactions with intracellular esterases cleave acetate groups from CFSE to yield an amine-reactive carboxyfluorescein, SE that covalently binds to proteins within the cell. While CFSE remains widely used as a green fluorescent proliferation indicator excited at 498 nm, it is, however, not without caveats. CFSE is highly toxic to cells, susceptible to dye leakage, and is known to spill over into the PE and PE-Texas Red channels.

CytoTell® dyes are a series of cell-permeable fluorescent tracers designed to monitor cell proliferation in living cells without any of the drawbacks of CFSE. Upon passive diffusion across cell membranes, CytoTell® dyes are hydrolyzed by intracellular esterases to yield highly fluorescent amine-reactive or thiol-reactive dyes that irreversibly bind to amine or thiol groups on intracellular proteins. Cytotoxicity is minimal, and the dyes are well-retained within cells through several generations of division. Visualize up to 9 generations using CytoTell® UltraGreen (22240). CytoTell® dyes are available in 7 different fluorescence emission colors offering greater flexibility in multicolor analysis. They may be fixed and permeabilized to analyze intracellular targets using standard formaldehyde-containing fixatives and saponin-based permeabilization buffers.

Adenosine 5'-triphosphate (ATP), primarily produced in a cell's mitochondria, is the main energy source of most living organisms. The energy supply in ATP comes from its high-energy phosphate bonds, and ATP-fueled reactions are multipurpose across the microenvironment, often linking catabolic and anabolic processes. Since all cells require a healthy supply of ATP to carry out specialized cellular functions, ATP is a useful tool for investigating the integrity of living cells. The quantitative measurement of total ATP levels within a population can not only provide information on cell viability but also reveal the cytotoxic effects of different agents or drugs. In this way, ATP assays are an indirect method of assessing cellular proliferation.

ATP assays commonly use bioluminescent detection methods instead of fluorescent or colorimetric techniques. In experimentation, cells are first lysed to release ATP, which then reacts with the firefly enzyme luciferase and its substrate luciferin. The light emitted from this reaction can be measured by a luminometer, with quantitation directly proportional to the total ATP content within a sample. The bioluminescent ATP assay offers a rapid, high-throughput, and easy-to-follow protocol with minimal hands-on steps. It is also more sensitive and stable compared to other methods, such as fluorometric assays, as the light signal emitted is stable for longer periods. Importantly, the fluctuation in ATP levels is influenced by many factors, including cell number, cell death, and various metabolic activities. Therefore, while the ATP assay provides valuable information on cell viability and overall cellular activity, it does not differentiate between cytostatic (growth-inhibiting) and cytotoxic (cell-killing) effects when used in isolation.

To better understand the effects on cell proliferation, ATP assays can be combined with other assays, such as cell viability assays (e.g., MTT or EdU assays), which provide more detailed insights into cellular health and proliferation dynamics.

Different proteins are generated at each phase of the cell cycle (G1, S, G2, M, and G0) as cells mature and develop. Monitoring these phase-specific proteins offers yet another way to measure cell proliferation. Cell proliferation marker assays play on this inherent functionality of cells and utilize antibodies specific to the desired target proteins as modes of detection. Some examples of protein markers and their associated cell cycle phases include:

The major advantage of using cell proliferation marker assays is the versatility in detecting antigen-antibody interactions using various techniques, such as ELISA, flow cytometry, Western blot, immunofluorescence (IF), immunocytochemistry (ICC), chromatin immunoprecipitation (ChIP), and immunoprecipitation (IP). This versatility allows for the use of different types of biological samples as starting material. For instance:

Additionally, multiple markers can be used simultaneously to assess various stages of proliferation within a single sample.

A minor disadvantage of this technique is that once cells are fixed and permeabilized, subsequent downstream assays cannot be performed on the same sample. Therefore, careful planning is required to ensure that the sample is used optimally for the intended analyses. Consideration should also be given to the potential for cross-reactivity of antibodies, which might affect the specificity of detection and require optimization and validation for accurate results.

With so many types of cell proliferation assays to choose from, ranging in complexity and providing different advantages and disadvantages, it is important to make an informed decision. Ultimately, choosing the correct cell proliferation assay for your experiment depends on the aim of your research, available equipment, the number of samples to be analyzed, the desired level of sensitivity, and if you need continuous monitoring. To streamline this process and help you making a decision, we have created a detailed flowchart designed to guide you through the selection process.

Some more points to consider:

Choosing an appropriate cell proliferation assay may seem daunting, but understanding your laboratory's capabilities and the specific needs of your experiment will guide you in making an informed decision. Having a clear plan for your experimental goals will help establish the best approach. Fortunately, many commercial kits are available, offering simple, cost-effective, and rapid solutions for measuring cell proliferation. If needed, performing multiple assays can help verify accuracy and provide a more comprehensive understanding of your cell population from different perspectives.

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