by Catherine Shaffer
There are two main types of assays that most people associate with the term cellular proliferation. One is, of course, a pure cellular proliferation assay, which is a measurement of the number of cells that are dividing in a culture. Often, however, cell viability—or the number of healthy cells in a sample—falls under the umbrella of cellular proliferation. These assays have applications in many areas of research, including toxicity, cancer, neuroscience, and stem cell research. Being able to quickly assess the viability or proliferation of cells in vitro or in vivo is important in a fast-paced research program.
The classical method for measuring cellular proliferation is by treating cells with tritiated thymidine, and then measuring the amount of tritium taken up in the newly synthesized DNA. This method has been used recently and to good effect to show neuronal recruitment in adult male and female zebra finches as they memorize the vocalizations of their fledglings.1
A more common method for assaying cellular proliferation is the BrdU (5'-bromo-2'-deoxyuridine) assay. Cells incorporate BrdU into DNA in the place of thymidine during cell division, and it persists and is passed down to daughter cells. Detection of the BrdU label involves fixation of the cells and denaturation of the DNA. Once this step has been accomplished, the BrdU label can be detected immunologically (using, for example, mouse anti-BrdU, followed by goat anti-mouse conjugated to horseradish peroxidase (HRP) and a colorimetric or fluorogenic HRP substrate). This results in a fairly straightforward, sensitive assay that can be done in less than twenty-four hours to measure proliferation of cells.
Calbiochem/EMD offers a BrDU assay kit in microtiter plate format that combines the washing, fixation, and denaturation steps in a single reagent. The colorimetric readout follows application of mouse anti-BrdU antibody and anti-mouse-HRP conjugate, giving a readout at 450 nm with a total assay time of three hours. This is more sensitive than most BrdU assays on the market, with detection levels of 1000 cells after a two-hour incubation, or just 100 cells with overnight incubation. Says Mo Saedi, PhD, group leader for Calbiochem, "With respect to other competitors, for two hour and overnight incubations, in both cases our assay is very sensitive."
In January's issue of the Journal of Neuroscience Methods, Virginia researchers used a BrdU assay to study neural stem cells and progenitor cells in rats. These cells have increased mitotic activity, and so they are both more likely to take up BrdU and also more susceptible to damage by radiation. After treatment with BrdU, and then a single 15Gy X-ray exposure, they showed that 85% of the proliferating cell population of the rat CNS was depleted for as long as three months.2
One drawback to a BrDU assay is that it requires the fixation/denaturation process, and that necessitates destruction of the DNA. For certain applications, a scientist may wish to simultaneously detect total DNA in addition to cell proliferation. However, once
the DNA is denatured and no longer double stranded, many common nucleic acid stains including DAPI or Hoechst 33342 are able to detect it. The Click-iT™ EdU assay from Molecular Probes (Invitrogen) bypasses the denaturation step by using a unique chemistry (click chemistry) to detect the incorporation of the thymidine analog, EdU (5-ethynyl-2'-deoxuridine) in intact double stranded DNA. This is because instead of a bulky antibody for detection, the detection molecule is a small azide connected to a fluorophore, such as an Alexa Fluor® dye.
Kathy Free, product manager for Invitrogen, explains: "With the BrdU method, you had to be very careful how you denatured the DNA to get the BrdU antibody into the cells, but still retain enough double stranded DNA so you could simultaneously look at cell cycle. With EdU, because you no longer have to denature DNA, you no longer have to be an expert to run this type of assay ... furthermore it's possible that HCl, commonly used to denature the DNA, can damage an antigen recognition site, and other antibodies that they were trying to use at the same time might not have been able to bind as well in part due to the BrdU protocol. With EdU, you have an assay that is easier to perform that easily produces great imagery in 30-90 minutes."
In some cases, a simple cell viability assay could serve the same purpose as a cell proliferation assay. Methods for assessing cell viability are much more diverse, and in many cases less invasive, giving them applicability to a wider range of experiments. These assays often depend on measuring signposts of metabolic activity using reagents such as Alamar Blue, MTT, and other tetrazolium salts. They detect proliferation primarily by measuring redox activity. Cells with decreased respiration have less energy for proliferation, so this gives an indirect measurement of cellular proliferation, and that may be perfectly adequate for some applications.
Calbiochem's rapid cell proliferation kit is, strictly speaking, a cell viability kit. It uses the (tetrazolium salt) reagent WST-1 for a quick colorimetric assay of viability in cell populations, in microplate format. Mitochondria cleave the WST-1 reagent, yielding water-soluble formazan salt, so this is a fairly reliable measure of the health of the cell. A similar but more sensitive assay is Calbiochem's Ultrasensitive Cell Proliferation Assay Kit, using calcein-AM (a fluorescent dye) to stain viable cells. The increased sensitivity comes with an extra assay step, as the medium or serum should be replaced with PBS to minimize background.
Invitrogen's offerings in cell viability/proliferation assays include an assay that measures ATP using the enzyme luciferase. Emission of light by luciferin gives a sensitive readout on the health of the cells with very little background. The absence of signal can be
interpreted to mean that the mitochondria are no longer producing ATP. Good applications for indirect cellular proliferation assays would include those where negative data are sought, such as apoptosis or drug toxicity.
Choices in cellular proliferation depend heavily on the specific application. Considerations include speed and sensitivity, as well as other assays that may be desired alongside cellular proliferation. In some cases, a surrogate assay for cell viability is a good substitute.
References
1Barkan S, et al. “Neuronal recruitment in adult zebra finch brain during a reproductive cycle.” Dev Neurobiol. May; 67 (6): 687-701, 2007.
2 McGinn MJ, et al. “Utilizing X-irradiation to selectively eliminate neural stem/progenitor cells from neurogenic regions of the mammalian brain.” J Neurosci Methods. Jan 4, 2008.
