The reasons you might need to measure cell proliferation are many—testing the effects of pharmacological agents or growth factors, assessing cytotoxicity or investigating circumstances of cell activation. In a cell proliferation assay, you measure the number of cells, or the change in the proportion of cells, that is dividing. There are four main types of cell proliferation assays, and they differ according to what is actually measured: DNA synthesis, metabolic activity, antigens associated with cell proliferation and ATP concentration. The option you choose will depend in part on your particular cell type and protocol.
DNA synthesis cell proliferation assays
One of the most reliable and accurate assay types is measurement of DNA synthesized in the presence of a label. Traditional cell proliferation assays involve incubating cells for a few hours to overnight with 3H-thymidine. Proliferating cells incorporate the radioactive label into their nascent DNA, which can be washed, adhered to filters and then measured using a scintillation counter. Besides the length of the experiment, the obvious downsides to this method are the hazards and hassle of using and disposing of radioactive materials. If that’s not your thing, you can perform a similar protocol using 5-bromo-2'-deoxyuridine (BrdU), which also becomes incorporated into newly made DNA. This adds a few steps because you must incubate with a BrdU-specific monoclonal antibody, sometimes followed by a secondary antibody as a reporter, before you can measure a colorimetric, chemiluminescent or fluorescent reporter signal. On the other hand, you don’t need to work with radioactivity. This is suitable for immunohistochemistry, immunocytochemistry, in-cell ELISAs, flow cytometry analysis and high-throughput screening.
Metabolic cell proliferation assays
Another measure of cell proliferation is the metabolic activity of a population of cells. Tetrazolium salts or Alamar Blue are compounds that become reduced in the environment of metabolically active cells, forming a formazan dye that subsequently changes the color of the media. This is caused by increased activity of the enzyme lactate dehydrogenase during proliferation. The absorption of the media-containing dye solution can be read using a spectrophotometer or microplate reader in low- or high-throughput configurations.
Four types of tetrazolium salts are most common:MTT,XTT, MTS and WST1. A disadvantage of MTT is that it is insoluble in standard culture medium, and the formazan crystals produced during reduction must be dissolved in DMSO or isopropanol. Because of this, MTT is mainly an endpoint assay. The other salts, as well as Alamar Blue, are soluble in culture media and are nontoxic. They can be used for continuous monitoring, to follow dynamic changes in proliferation over time. XTT reduces less efficiently and may need additional factors added. WST1 is more sensitive, reduces more efficiently and shows faster color development compared to the other salts. Alamar Blue is also sensitive, capable of detecting as few as 100 cells in a well of a microtiter plate. The tetrazolium salts and Alamar Blue redox dyes can be quantified with a range of instruments for conventional or high-throughput studies using, for example, standard spectrophotometers or spectrofluorometers or plate readers for spectrophotometric or spectrofluorometric microtiter well plates.
Detecting proliferation markers
A third way to measure cell proliferation is to detect an antigen present in proliferating cells, but not nonproliferating cells, using a monoclonal antibody to the antigen. For example, in human cells, the antibody Ki-67 recognizes the protein of the same name, expressed during the S, G2 and M phases of the cell cycle but not during the G0 and G1 (nonproliferative) phases. Using this assay with tissue slices precludes high-throughput methods. On the other hand, this method enjoys an advantage for cancer researchers because it’s suitable for assaying tumor cell proliferation in vivo. Other common markers for cell proliferation and/or cell cycle regulation, targeted by antibodies, include PCNA (proliferating cell nuclear antigen), topoisomerase IIB and phosphohistone H3.
A final type of cell proliferation assay takes advantage of the tight regulation of intracellular ATP within cells. Dying or dead cells contain little to no ATP, so there is a tight linear relationship between cell number and the concentration of ATP measured in a cell lysate or extract. The bioluminescence-based detection of ATP, using the enzyme luciferase and its substrate luciferin, provides a very sensitive readout. In the presence of ATP, luciferase produces light (proportional to the ATP concentration) that can be detected by a luminometer or any microplate reader capable of reading luminescent signals. This approach is also well suited to high-throughput cell proliferation assays and screening.
Which approach should you choose?
The method to choose depends partly on your cell type and protocol and partly on what road you prefer to travel to proliferation measurement. If you have a population of cells (such as plated or in suspension) and are interested in changes in metabolic activity, you might use tetrazolium salts with colorimetric detection, for example. Alternatively, if you are more interested in changes in DNA synthesis, use BrdU with colorimetric, chemiluminescent or fluorescent detection. If you are studying single cells, you might choose to measure DNA synthesis by labeling with BrdU, tagging it with a fluorescently labeled antibody and then using FACS (fluorescence-activated cell sorting) to examine single cells by flow cytometry. Regardless of the type of cell proliferation assay you choose, rest assured that all of these dependable methods have been replicated many times and are virtually guaranteed to be reliable.
The image at the top of the page is from Life Technologies' CyQUANT® Cell Proliferation Assays.