Methods to measure cell proliferation are essential to many areas of cell-based research. There is no one best method; instead, several methods available today feature different strengths. This article looks at the main methods of measuring cell proliferation, their advantages and disadvantages, and shares advice from experts on choosing the best fit.
Proliferation measured by DNA synthesis
The most accurate assay for cell proliferation measures newly synthesized DNA. Lindy O’Clair, head of bioanalytics reagents and consumables product management at Sartorius, notes that DNA synthesis is a relatively specific metric for proliferation, because DNA replication must occur prior to cell division. “Although highly accurate and reproducible, it is not a simple technique, as it often incorporates radioactive isotopes,” she says. The protocol can be time-consuming, and is typically an endpoint assay; today, nonradioactive alternatives to the traditional 3H-thymidine labels are available.
How it works
DNA synthesis assays work by incorporating labeled nucleosides into newly synthesized DNA, using the older BrdU or the newer Click-iT EdU systems. “The Click-iT technology, which uses very small and highly specific binding moieties, has been demonstrated to be a superior alternative to traditional BrdU-based proliferation assays,” explains Brian Almond, senior manager in product management at Thermo Fisher Scientific. “In the Click-iT EdU proliferation assay, the modified thymidine analogue EdU is efficiently incorporated into newly synthesized DNA and fluorescently labeled with a bright, photostable Alexa Fluor® dye in a fast, highly specific click reaction.”
Uses
Kits available today can measure proliferation with better throughput and in more assay types than ever before. “Newer formulations of BrdU and EdU have expanded the use of thymidine analogs by allowing fluorescent labeling of the incorporated analogs for multiplexing,” says Peter Brescia, applications scientist at BioTek (part of Agilent Technologies). “In vivo models can benefit from labeling methods that can freely diffuse into native tissues and DNA such as whole-animal studies, where labeling moieties can be injected at the site of interest.” BioTek offers instrumentation supporting workflows for biochemical and image-based proliferation assays.
Mahesh Dodla, global product manager for research monitoring tools at MilliporeSigma, believes that DNA synthesis assays are most useful when analyzing large numbers of cells by flow cytometry, but also notes that they “can be used to study cell cycle kinetics at single-cell resolution.”
Proliferation measured by marker immunostaining
How it works
Cell proliferation can be assayed by immunostaining cells or tissues, using antibodies specific for markers of cell proliferation. Common markers of proliferating cells include Ki-67, PCNA, MCM2, Topoisomerase II alpha5, and phospho-Histone H3.
Uses
Immunostaining is mainly used for tissue slices or FFPE tissue samples, but can also be used for cells analyzed by flow cytometry or lysates analyzed by western blotting. It is a lower throughput endpoint assay that usually requires some skill (though this is changing with automated plate readers). However, it can also be multiplexed to look for co-localization with other markers of interest.
Proliferation measured by metabolic indicators
Metabolic assays are an indirect measurement of proliferation because they can also reflect the biochemical activity of healthy cells, not necessarily just cells that are actively dividing. Assay types include reduction of tetrazolium salts such as MTT; reduction of water-soluble tetrazolium salts (WTSs); and reduction of the resazurin dye AlamarBlue. Assays using these colorimetric indicators are sensitive, easy to use, and amenable to high-throughput screening.
How it works
When the indicator is added to cell culture medium, enzymatic activity within the cell reduces the tetrazolium salts or redox dye, resulting in a color change. MTT is insoluble in cell culture media and first needs to be dissolved in DMSO or isopropanol, so is better used for endpoint assays. Other indicators, such as WST1, XTT, and AlamarBlue, are soluble and non-toxic, making them suitable for live cell assays. “Some reagents are compatible with cellular imaging allowing correlation with metrics such as morphological changes, as well as apoptotic and necrotic indicators, during drug treatment,” says Brescia.
Uses
This method is especially suited to “high-throughput assays for analyzing large number of experimental conditions or treatments using plate readers,” says Dodla. “However, metabolic activity might not be directly correlated with proliferation, and the sensitivity and signal/noise ratio could be low for some assays.”
Thermo Fisher Scientific recently released high sensitivity versions of the indicators AlamarBlue and PrestoBlue, “created using an innovative process that removes contaminants, resulting in dramatic changes in the signal to background ratio,” says Almond. “Since these reagents are non-toxic and sterile, after the fluorescent signal is detected the media can be replaced and the cells returned to the incubator.” He adds that a proliferation assay that measures cell health without killing cells is unique.
Counting nuclei and DNA
Measuring cell proliferation by means of simply counting nuclei is benefiting from recent advances in label-free technology. For example, high contrast brightfield imaging allows direct quantification of cell nuclei. “This can be highly advantageous when cells become crowded and a measurement of confluence can mask total cell number as cell volume decreases with crowding,” says Brescia.
Label-free and fluorescent options are available in Sartorius’s Incuycte®, an imaging-based system that can monitor cell proliferation of cultures in real time. Incucyte can use “label-free fit-for-purpose software tools, labeling of the nucleus via fluorescent cell labeling strategies, as well as analysis cell-cycle progression continuously over multiple cell divisions,” says O’Clair.
A related method of assessing proliferation involves dyeing cell DNA and counting the fluorescence associated with it. “Because the amount of DNA per cell is highly regulated, an increase in the amount of DNA corresponds to an increase in cell number,” says Almond. Thermo Fisher Scientific’s CyQUANT® Direct assay and CyQUANT™ Direct Red Cell Proliferation Assays use a cell-permeant DNA-binding dye introduced along with a cell-impermeant masking dye reagent. The latter blocks the staining of dead or unhealthy cells, so that only healthy cells emit a stained DNA signal. “Using a dilution series of known cell numbers one can use the fluorescent signal from the CyQUANT assays to determine cell numbers and proliferation,” says Almond. The CyQUANT Direct Cell Proliferation assays work well for assessing proliferation of cells of all cell types including suspension and adherent cells.
For generation tracking, or studying the number of generations or proliferation events within a cell population, Thermo Fisher Scientific offers the CellTrace™ stains. These are improved over previous dyes such as CFSE, resulting in lower toxicity, greater brightness, and multiple colors. Combining CellTrace stains with flow cytometry can be powerful because “with this technique you are able to see differences in the rate of proliferation by measuring cell divisions over a particular time period,” Almond adds.
Further considerations
In measuring cell proliferation, perhaps most effective has been the combination of advances in instrumentation and biochemistry. “This includes both the advancement in specific and sensitive reagents that allow direct cell-specific measurements in complex models, as well as instrumentation that enables long-term analysis of these models,” notes O’Clair. These advances enable multiparametric analysis that includes proliferation. “Now we can monitor cell proliferation while investigating morphological changes simultaneously with indicators of additional cellular events,” says Brescia. “These tools help to advance efforts from basic academic research to drug discovery and development.” Clarifying which parameters you need to measure along with proliferation can help you decide the right cell proliferation method for your work.