Assays to assess the proliferative activity of cells grown in culture or harvested from tissue samples are a core tool for monitoring the health and growth rate of a cell population. Historically, cell proliferation assays relied on the detection of tritiated thymidine 3H uptake. However, the divergence of trends away from the use of radioactivity and toward assay platforms compatible with automated sample handling, high-throughput screening in microtiter plates, and, more recently, high-content screening (HCS) using live cell assays to image cell function, metabolism, and signaling at the level of the individual cell has led to an expanded range of assay formats for measuring cell proliferation. These include fluorescent, luminescent, and colorimetric assays that can determine cell count, detect DNA synthesis, or measure metabolic activity.
Some of these assays require cell lysis or disrupt DNA duplication events, whereas others are nondestructive and allow for multiplexing and simultaneous or sequential combinations of biomarker detection assays to be performed on the same cell population. Intrinsic features of these assays that can be used to distinguish one from another are their ease of use (for example, the number of steps and need for wash steps), compatibility with high-throughput assay formats, dynamic range, and reproducibility.
Key trends in this area include the demand for assays amenable to automated high-throughput screening. Growing interest in systems biology and imaging of live, intact cells to study biological pathways and how they interact and respond to external stimuli is driving the development of assays and reagents appropriate for use in high-content, cell-based screening. Furthermore, applications of cell proliferation assays in drug discovery are increasing demand for related assays that can be used to verify study results and provide additional information on cell viability and toxicity.
To meet the growing demand for assays designed for use in automated cell imaging studies in microplate formats, Invitrogen (www.invitrogen.com) recently introduced the Cyquant NF cell proliferation assay kit. This new NF, or no freeze, version of the companys assay eliminates the freeze/thaw cell lysis step.
The main advantage of the Cyquant assay is its linear dynamic range of about 200-fold, from 50 cells to 10,000 cells per well, says Brett Williams, business segment director for imaging and microscopy at Molecular Probes, a business unit of Invitrogen. It improves workflow by allowing the combination of cell proliferation determination with other assays to interrogate multiple markers of cell health, adds Williams. The assay utilizes a new cell-permeant, DNA-binding dye together with a plasma membrane permeabilization reagent.
The trend now is to do cell-based assays in drug discovery, which are faster and more economical than animal studies, says Melissa Stolow, director of marketing for functional biology at Stratagene (www.stratagene.com). The companys Quantos cell proliferation assay lends itself to high-throughput screening in 96- or 6-well formats.
Cellomics (www.cellomics.com) portfolio of assays for HCS and imaging applications all yield cell count or cell density information as part of the assay output. When customers buy an assay for some other target, like apoptosis or transcription factor activation, they have the ability to look at proliferation or changes in cell count at the same time, says Sarah Burroughs Tencza, Ph.D., product manager for bioapplications at Cellomics. The company does not market a cell proliferation assay per se, but includes cell counta direct measure of cell number and cell proliferative activityas a core component of all of its HCS assays. For a more detailed assessment of what might be causing an increase or decrease in proliferation, Cellomics offers a cell viability assay, a cell cycle assay, and a mitotic index assay.
Cellomics reagents are validated for use with HCS, and now that the company is part of the Fisher Biosciences group, Judy Masucci, Ph.D., director of marketing and sales support at Cellomics, expects to see growth in the number of assay kits produced for HCS applications. Although users require only a fluorescence microscope to perform these assays, the ability to use them on a HCS platform allows for automation and multiplexing.
We are hearing from our end-users in pharma/biotech of a need for flexibility to scale up assays in terms of throughput for high-throughput screening applications, says Jeffrey Emch, product manager at Roche Applied Science (www.roche-applied science.com). In the future, we will see the market moving toward molecular markers for cell proliferation, as well as cell death and toxicity. To meet that emerging need, Roche recently introduced the LightCycler 480 platform for high-throughput, real-time PCR.
The most recent generation of ELISA BrdU assays are compatible with highthroughput screening formats. They offer good sensitivity down to 5,00010,000 cells/well and detection down to a 20% increase or decrease in DNA synthesis, says Michelle Moore, applications and technical consultant at Roche.
Cambrex (www.cambrex.com) ViaLight HS (high sensitivity) BioAssay is a one-step assay, scalable for high-throughput applications in 96- or 384-well formats. The assay is based on bioluminescent detection of cellular ATP and can measure down to 10 adherent or non-adherent mammalian cells per microwell. ViaLight has two key advantages; the ability to measure over a dynamic range of six decades and its long signal durationthe signal retains its strength and quality for six hours, according to D.G. Ferneyhough, segment manager for research products at Cambrex Biosciences Walkersville.
Promegas (www.promega.com) CellTiter-Glo Luminescent Cell Viability Assay measures ATP in a high-throughput micro-well format. ATP assays are capturing the market from tetrazolium assays, says Terry Riss, director of project management, R&D at Promega. They provide an immediate endpoint and minimize the risk of chemical interference, associated with tetrazolium or rosazurin reduction assays.
The CellTiter-Glo assay can detect 10 cells or fewer in a well and has been miniaturized for use in 1,536-well plates. According to Riss, it contains an engineered, highly stable form of luciferase, enabling the use of large amounts of detergent to stop any ATPase activity. The luminescent signal has a half-life typically greater than five hours. The use of luminescence eliminates problems of background noise associated with fluorescent assays that may be compromised by fluorescence interference from library compounds.
Combining Related Assays
Most researchers will combine cell proliferation with measures of toxicity and apoptosis, says Moore. If a compound appears to up-regulate cell survival, they will need to support that finding by demonstrating a change in multiple markers, such as a down-regulation of cytotoxicity or apoptosis. Roches Cell Death Detection ELISAPlus measures DNA fragmentation, which is a reliable endpoint measure of cell commitment to an apoptotic pathway. The company will launch its Cytotoxicity Detection KitPlus [LDH], a one-plate, four-step absorption assay that rapidly assesses cytotoxicity.
Customers are interested in combining these assays with other assay technologies to multiplex and make screening more efficient, says Riss. This requires a clear understanding of the assay chemistries to ensure that they are compatible.
Promega is developing the MultiTox assay that will enable simultaneous measurement of live and dead cells in a single well. The assay measures markers of protease activity, differentiating between protease activity within live cells (using a cell-permeable protease substrate) and proteases that have escaped from lysed dead cells (using a noncell-permeable substrate), with the two substrates fluorescing at different wavelengths.
Molecular Probes offers a variety of kits for measuring cell count, proliferative activity, DNA content, and live versus dead cells. CountBright absolute counting beads incorporate a calibrated suspension of fluorescent microspheres and measure cell concentration using flow cytometry. Vybrant DyeCycle stains for flow cytometry, available in violet, green, and orange, enable DNA content analysis in live cells. The stains permeate the cell membrane and bind to the DNA, emitting a fluorescent signal that is proportional to the DNA mass, thereby indicating cell cycle phase.
The companys Live/Dead dead cell stain kit assesses mammalian cell viability using an amine-reactive fluorescent dye that crosses the leaky membranes of dead cells, creating a detectable difference in fluorescence intensity between live and dead cell populations.
Drug Discovery Research
Cell proliferation assays have long been a workhorse tool for cell biologists. The proliferative state of a cell population has also become an important parameter in drug discovery research, particularly in evaluating cancer therapeutics and in assessing the health of a cell population as part of ADME/tox studies.
In drug discovery, cell proliferation often is one component of a group of related assays that assess not only proliferative activity but also cell viability, metabolic activity, cell cycle phase, cell toxicity, and apoptosis. Together, the information derived from these assays can indicate whether a cell population that has been exposed to an experimental stimulus is healthy or dying, actively dividing or in stasis, or has committed to an apoptotic pathway.
Ferneyhough is seeing a growing number of applications for cell proliferation and cell toxicity assays in drug discovery. Using cancer therapeutics as an example, he describes the utility of a product, such as Cambrexs ToxiLight Cytotoxicity Assay, to ensure that a cancer drug that successfully targets epithelial cells, for example, is not also harming nontarget cells, such as neurons or mesenchymal stem cells, two cell types that are highly sensitive to cell stress. In addition to such ADME/tox types of applications, cell proliferation assays might also be used to test the effectiveness of antiangiogenesis drugs in blocking the ability of cells to go through a proliferative cycle.
Cellomics recently introduced its Micronucleus BioApplication for the ArrayScan HCS Reader, which looks at a variety of measures of cell proliferation and reports on both the cell count and proliferative index. Although the main purpose of this assay is to detect genotoxicityby determining the frequency of micronucleus formation in mononucleate or binucleate cellsit is also able to detect toxic events related to proliferation that are not genotoxic or cytotoxic and do not necessarily cause cell death.
You can detect subtle changes in the health of cells, and determine whether an experimental compound is subtly or grossly cytotoxic, explains Tencza. Were seeing broader applicability of these assays, with particular interest in neuroscience research for the study of survival and proliferation of neurons.
Another recent addition to the Cellomics line of HCS tools is the Cell Cycle BioApplication, a multichannel software module that enables users to measure the cell cycle phase of individual cells within a population by measuring their DNA content and correlating this measurement with the activity of multiple other cell cycle-associated proteins in each cell.
Choosing an Assay Strategy
A variety of assay approaches can provide information on a cell populations proliferative activity and capability. The most direct means of measuring cell proliferation is to count the number of cells present.
For analysis of cell proliferation in primary cell cultures, Guava Technologies (www.guavatechnologies.com) offers its CellGrowth assay that contains a cell-tracking dye that diffuses into cells and is retained within the cell without affecting cellular function. With each round of cell division the relative fluorescence intensity of the dye decreases by half. The CellGrowth assay is capable of cell proliferation analysis at the single cell level and can assess proliferation out to five generations.
For screening compounds that inhibit or stimulate cell proliferation, the CellGrowth Assay can tell you the exact number of cells that have been affected, says Kim Mulcahy, director of product marketing at Guava. CellGrowth is one of three nonradioactive cell tracking assays offered by Guava, all of which use CFSE dye to track cells without affecting cell function. The other two are the CellToxicity Assay and the CellPaint Assay that tracks target cells in a mixed cell population.
Miltenyi Biotecs (www.miltenyibiotec.com) Blood Dendritic Cell Enumeration Kit specifically identifies and counts dendritic cells and dendritic cell subsets in whole blood or peripheral blood mononuclear cells (PBMCs) by flow cytometry. For the detection of dead or apoptotic cells the company offers its Annexin V-FITC kit and the Fixation and Dead Cell Discrimination kit.
Other strategies for assessing proliferative activity involve measuring the DNA content of cells or their metabolic activity. One traditional approach involves measuring DNA content via bromodeoxyuridine (BrdU) incorporation into growing DNA strands. Invitrogens Absolute-S SBIP (strand break induction by photolysis) cell proliferation assay kit offers a twist on the conventional BrdU assay, which typically requires cell lysis and DNA denaturation to detect BrdU. Incorporation of the companys BrdU Photolyte and exposure to UV light creates a nick in the DNA, and these strand breaks are then detected using an Alexa Fluor 488 dye-labeled anti-BrdU antibody conjugate.
Stratagenes Quantos cell proliferation assay measures total double-stranded DNA with high sensitivity over a broad linear range, according to Stolow. It detects the fluorescence emitted by a DNA/dye complex without perturbing cell function or causing cell death. It can thus be used on a HCS platform in combination with other assays to measure cell response to an experimental compound.
Assessing Metabolic Activity
Whereas cell count is a direct measure of proliferation, measurements of DNA content or metabolic activity are correlates that can be altered by factors other than cell count. A classic approach to assessing metabolic activity involves the use of tetrazolium salts, such as MTT or XTT, which are cleaved in the mitochondria of metabolically active cells to form formazan and then solubilized to generate a soluble orange dye that can be measured by absorbance.
Simplifying the traditional tetrazolium MTT assay, Promega developed its CellTiter 96 AQueous One Solution Cell Proliferation Assay that uses an MTS reagent instead of MTT. The MTS tetrazolium compound is reduced to form an aqueous soluble formazan product, thereby eliminating the need for a formazan solubilization step and facilitating assay optimization. The assay incorporates phenazine ethosulfate, an electron coupling reagent.
The colorimetric change that results when tetrazolium salts are cleaved to form soluble formazan dye by mitochondrial dehydrogenase in viable cells is the basis for Takara Mirus Bios (www.takaramirusbio.com) Premix WST-1 cell proliferation kit. The amount of formazan dye produced is directly proportional to the number of metabolically active cells.
The main advantage of the MTT and XTT tetrazolium salt-based assays for measuring the metabolic activity of cells is the strong body of literature that supports them. The WST-1 assay is a popular and sensitive assay capable of measurements down to 1,000 cells/well, and detection of a 5% up- or down-regulation, says Moore.
MTT and XTT assays have the advantage of being relatively inexpensive and supported by an extensive literature base, according to Ferneyhough, but, he says, they are not very sensitive when the cell number drops to the 105,000 cell-per-well range. There might be good data that you are missing. By the very nature of putting in the tetrazolium dye, you interfere in the cell metabolism, and the changes that result may or may not indicate whether the cell is happy and proliferating.
Many researchers accept metabolic activity as a proxy for proliferation, but that may not necessarily be the case, says Williams. Whereas cell number is an absolute measure of cell proliferation, metabolic activity is more a measure of cell health. Combining these assays can yield a more detailed view of cell activity. In cancer drug discovery, for example, the goal may not be to cause cell death but simply to knock down the metabolic and proliferative activity of a cell with cell stasis being the desired outcome.