A Growing Issue: Cell Proliferation Assays

Cell Proliferation Assays Suppliers of Cell Proliferation Assays Courtesy of Zymed LaboratoriesMouse anti-PCNA (PC10)-stained colonic mucosa. Scientists often require rapid and accurate measurement of viable cell number and cell growth. These researchers traditionally assess cell viability via membrane integrity (e.g., trypan blue exclusion), and cell proliferation via the incorporation of labeled nucleotides (e.g., [3H]-thymidine) into newly synthesized DNA during cell division. Work to im

By | June 25, 2001

Courtesy of Zymed Laboratories

Mouse anti-PCNA (PC10)-stained colonic mucosa.

Scientists often require rapid and accurate measurement of viable cell number and cell growth. These researchers traditionally assess cell viability via membrane integrity (e.g., trypan blue exclusion), and cell proliferation via the incorporation of labeled nucleotides (e.g., [3H]-thymidine) into newly synthesized DNA during cell division. Work to improve viability detection methods is yielding assays that enumerate cells more rapidly, precisely, and safely. Likewise, new technology to detect proliferating cells based on metabolic activity is producing rapid, safe, and reliable alternatives to DNA-related methodologies.

One of the most familiar and widely used methods for quantifying cell proliferation is the measurement of tritiated thymidine ([3H]-thymidine) incorporation. Cells incorporate the labeled DNA precursors into newly synthesized DNA, such that the amount of incorporation, measured by liquid scintillation counting, is a relative measure of cellular proliferation. Unfortunately, this technique is time-consuming and labor-intensive, and exposes the researcher to scintillation fluid and tritium, both of which are toxic.

Amersham Pharmacia Biotech of Piscataway, N.J., offers both a [14C]-thymidine and a [3H]-thymidine uptake assay system, each designed to decrease radioisotope handling. The Thymidine Uptake [14C] Cytostar-T™ Assay measures DNA synthesis by [14C]-thymidine incorporation using 96-well Cytostar-T scintillating microplates without the need to separate labeled DNA onto filters. The system is designed for high-throughput (HT) screening and provides real-time measurement of DNA synthesis. The company's [3H]- Thymidine Uptake Assay Kit incorporates the scintillation proximity assay (SPA). Considered the gold standard for HT screening applications, the SPA system is based on a polyvinyltoluene bead-containing scintillant whose outer surface is modified to enable [3H]-DNA binding. Tritiated thymidine and its metabolites do not bind to the bead, enhancing the signal-to-noise ratio. This system is highly sensitive and precise, eliminates liquid scintillant handling, and is amenable to automation.

Some researchers use the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) instead of [3H]-thymidine in proliferation assays. BrdU is incorporated into cellular DNA in a manner similar to thymidine. However, unlike [3H]-thymidine, researchers can easily quantify BrdU incorporation using anti-BrdU monoclonal antibodies (mAbs) in ELISAs, eliminating the use of radioisotopes. Currently available kits based on this technology are adapted to 96-well microtiter plate formats. Roche Molecular Biochemicals of Indianapolis offers three such assay kits with a choice of detection systems (ELISAs with colorimetric or chemiluminescent detection), as well as immunohistochemical (IHC) systems for use with tissue sections and in vivo incorporation studies. Roche is now offering second generation systems that involve fewer steps and offer higher sensitivity and faster results than did the original versions. Companies such as Oncogene Research Products of San Diego, Zymed Laboratories of South San Francisco, Molecular Probes of Eugene, Ore., and Amersham Pharmacia Biotech offer similar ELISA- and IHC-based proliferation assay systems.

Courtesy of Zymed Laboratories

Mouse anti-BrdU (ZBU30)-stained, BrdU-labeled colonic epithelium.

Some systems that use anti-BrdU mAbs to detect BrdU incorporation denature the cellular DNA into single strands in order to expose the BrdU epitope to the antibody, either by heating to greater than 90°C or by acid (2-4N HCl) treatment. This treatment makes these protocols incompatible with a number of downstream assays, because many cellular proteins are lost or degraded during the denaturation process. Some kits, however, employ Strand Break Induced Photolysis (SBIP), a technique that bypasses the need for DNA denaturation and preserves antigens and other cellular features. In the SBIP assay, cells are grown in the presence of a BrdU photolyte to label the DNA of proliferating cells. An enhancer photolyte is added to sensitize the genome- incorporated BrdU to subsequent photolysis. After the cells are harvested and washed, they are UV-irradiated to induce strand breaks at BrdU-incorporation sites. Scientists then assess cell proliferation by labeling the break sites with bromolated deoxyuridine triphosphate (Br-dUTP) in a TUNEL assay-like reaction catalyzed by terminal deoxynucleotidyl transferase (TdT), which adds deoxyribonucleotides in a template-independent manner. Chemicon International of Temecula, Calif., offers a kit that includes a fluorescein-labeled anti-BrdU antibody for identifying incorporated BrdU. The ABSOLUTE-S Cell Proliferation Assay Kits, from Phoenix Flow Systems of San Diego, in conjunction with Molecular Probes, features an Alexa Fluor 488-labeled anti-BrdU antibody, which has excitation and emission criteria similar to those of fluorescein. These kits are optimized for the coupling and simultaneous measurement of BrdU incorporation and cell cycle-marker detection using flow cytometry.

BD Biosciences of San Jose, Calif., offers a variant of this technique that uses DNase to expose incorporated BrdU. Recommended for use with individual peripheral blood mononuclear cells (PBMCs), the FastImmune Anti-BrdU with DNase reagent can be used for the phenotypic identification of cells undergoing proliferation as well as for the study of activation markers and intracellular cytokine production. The kit includes a fluorescently labeled, anti-BrdU antibody to identify dividing cells. The inclusion of DNase within the reagent causes cellular DNA to be denatured for exposure to the anti-BrdU antibody while preserving the structure of intracellular proteins and the fluorescent properties of the fluorochrome.1-3 BD Biosciences' Pharmingen subsidiary offers the BD BrdU Flow Kit, for flow cytometric detection of BrdU incorporation coupled with surface markers or intracellular proteins.

Some companies have adopted nucleotide staining techniques to quantify both the number of viable cells, as well as cell proliferation. The Quantos™ Cell Proliferation Assay Kit, from Stratagene of La Jolla, Calif., includes Quantos dye reagent that fluoresces only when bound to DNA. Similarly, the TACS Hoechst Cell Proliferation Assays I and II, offered by Trevigen Inc. of Gaithersburg, Md., for live and fixed cells, respectively, feature dyes that noncovalently interact with A:T base pairs, causing the dyes to fluoresce. Molecular Probes' CyQUANT Cell Proliferation Assay Kit uses the CyQUANT-GR dye, whose green fluorescence is enhanced when bound to cellular nucleic acids.

Other companies have adopted membrane-staining techniques. For example, the Cell Census Plus System provided by Sigma-Aldrich of St. Louis, includes a patented fluorescent aliphatic reporter molecule that acts as a plasma membrane dye. The novel dye becomes equally distributed into the membranes of daughter cells, providing a monitor for up to 10 generations of cell divisions. Researchers can measure the relative abundance of each daughter generation, as well as the fraction of nonproliferating cells within a population. Perhaps one of the most attractive uses of the Cell Census Plus System is in cell-tracking studies when measuring the proliferative effect of an agent or treatment upon a select subset of cells within a culture population.

Both Dojindo Molecular Technologies of Gaithersburg, Md., and BioVision Inc. of Palo Alto, Calif., offer staining kits to quantify live and dead cells. The Live-Dead Cell Staining Kit from BioVision features "Cyto-dye", a live cell-permeable green fluorescent dye as well as non-cell-permeable propidium iodide (PI), which stains dead cells and fluoresces red. Dojindo's Cellstain Double-Staining Kit also includes PI for dead cell staining, but features the fluorogenic esterase substrate, calcein-AM, to label live cells.

New Approaches

Courtesy of Phoenix Flow Systems

HL-60 cells stained with Phoenix FlowSystems' ABSOLUTE-S Kit.

In 1993, two novel approaches to the measurement of cellular proliferation were published that have become widely accepted alternatives to the [3H]-thymidine incorporation method. One method, based on studies originally presented in 1983 by Mosmann, repeats and confirms experiments studying the use of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrasodium bromide (MTT) tetrazolium salt in cell proliferation assays.4,5 Mosmann's study measured both lymphocyte proliferation and the lytic/growth inhibitory activities of cytotoxic lymphokines on normal cells using MTT.4 The later work showed that the MTT assay is a viable alternative to the [3H]-thymidine incorporation assay.5 The second method, published by Crouch and coworkers, demonstrates the use of adenosine triphosphate (ATP) bioluminescence as an indicator of increased cell number.6

MTT salts are commonly used in cell proliferation assays. The tetrazolium ring of MTT is reduced to formazan, which is blue in color, by the succinate- tetrazolium reductase system active only in viable cells.7 The intensity of the resulting color change indicates the enzymatic activity of living cells. In actively proliferating cells, MTT conversion increases, whereas in senescent and dying cells, the rate of MTT conversion slows. Comparison of this value to an untreated control provides a measure of the change in cellular proliferation.

Many companies, including Promega Corp. of Madison, Wis., R&D Systems of Minneapolis, Roche Molecular Biochemicals, Trevigen, Chemicon, and Sigma-Aldrich, offer colorimetric cell proliferation assay kits employing MTT reduction to formazan. To use these kits, researchers add the MTT solution directly to adherent or suspension cells cultured in 96-well plates. Viable cells convert MTT to a water-insoluble formazan dye within this incubation period. The dye is then solubilized within the well and quantified using an ELISA plate reader. The absorbance measured correlates directly with cell number. Scientists using these systems can quantify proliferation experiments directly and rapidly within tissue culture plates. These assays are also amenable to HT screening for drug-discovery programs.

Various manufacturers have streamlined MTT-based kits by substituting MTT with other tetrazolium salts: WST-1, WST-8, XTT, and MTS. Reduction of these tetrazolium salts produces water-soluble formazan salts in the presence of an electron-coupling reagent supplied in the kits. Therefore, cell proliferation assay kits employing MTS, XTT, WST-1, or WST-8 avoid the solubilization step, making quantification even more rapid. In addition, multiple time points can be taken in a single assay when using these new tetrazolium compounds, which is not possible when using MTT. Companies including Roche, Oncogene Research Products, BioVision, Chemicon, Promega, Sigma-Aldrich, and Dojindo all offer different XTT, MTS, WST-1 or WST-8 assay kits (visit our Web site for a detailed listing of kit names and components).

While such kits based on cellular metabolic activity are rapid, safe, and easy to use, it is important to include detailed, well-thought-out controls in studies using these kits, as results could be misleading. For example, the metabolic activities of certain cell lines may be far different than those of others. Cells that have a particularly low metabolic activity, such as lymphocytes, must be used in higher numbers than more metabolically active cells, so direct cell-to-cell comparisons are difficult. Also, the assays may not be linear over a broad logarithmic range of cell proliferation because of the use of ELISA plate readers with these kits.7 Finally, despite their widespread use, the details of tetrazolium salt cellular bioreduction are still not well understood.7

An alternative technology used for quantifying cell proliferation is based on the generation of ATP in metabolically active cells. Cellular ATP levels are closely regulated, since ATP is necessary for survival. This molecule therefore provides an accurate measurement of the number of living cells within a population. The most successful technique for ATP measurement is the luciferin-luciferase bioluminescent assay, as it is highly sensitive and accurate over six orders of magnitude.6 The reaction involved is catalyzed by firefly luciferase, yielding a light intensity linearly related to ATP concentration:6

Luciferase + ATP + Luciferin + O2®
Oxyluciferin + AMP + PPi + CO2 + Light

LumiTech's ViaLight kits from BioWhittaker of Walkersville, Md., the Cytotoxicity and Cell Proliferation Kit from Thermo Labsystems of Franklin, Mass., the CytoLux Assay Kit from PerkinElmer Life Sciences of Boston, and the CellTiter-Glo™ Assay from Promega all measure ATP bioluminescence in metabolically active mammalian cells. Promega's CellTiter-Glo Assay uses a genetically engineered, stable form of luciferase to produce a luminescent signal half-life of several hours. BioWhittaker offers three different versions of LumiTech's ViaLight Kit: a high-sensitivity (HS) version capable of detecting as few as 10 cells/well for use with all mammalian (adherent and nonadherent) cells, a HT version with slightly lower sensitivity, and the ViaLight-MDA Kit for use with noneukaryotic cells.

Each of these assays are safe, involve no radioisotopes or toxic materials, and are ideally suited for HT applications, since a 96-well plate can be processed in approximately 10 minutes. LumiTech has shown through extensive studies that the level of intracellular ATP per cell is highly regulated and remains essentially constant in a cell population even during a metabolic burst. The amount of ATP per cell is a function of cell size and does not increase when stimulated metabolically. The cell responds to the increased demand for ATP by increasing the production or turnover rate of ATP. Increasing levels of ATP is therefore a reflection of cell proliferation and an increase in the number of viable cells per well. In addition, controlled studies have shown that ATP measurements correlate well with traditional tritiated thymidine incorporation methods.6

Populations vs. Single-cell Measurements

Some companies offer kits designed to assay cellular proliferation in individual cells in addition to their systems designed for quantifying cell growth in cell populations. Roche's In Situ Cell Proliferation Kit, FLUOS, as well as the BrdU Labeling and Detection Kits I & II, are designed for single cell measurement of BrdU incorporation using immunocytochemical and immunohistochemical detection.

Cell cycle regulators can also be used as proliferative indices. For example, cyclin-dependent kinase (CDK) assays can be used to measure activity changes, and methods such as Western blot, ELISA, or immunocytochemistry can quantify expression levels. Zymed Laboratories features the PCNA Kit, designed for detection of proliferating cell nuclear antigen. Roche Molecular Biochemicals offers a wide range of mAbs directed against various cell cycle regulators. Monoclonal antibodies directed against cell cycle antigens can identify actively cycling cells and can sometimes mark cell cycle phase.7 These assays provide more indirect measures of cell proliferation than does DNA content, and caution must therefore be used in experimental design and interpretation. Nevertheless, it is clear that with so many options available, researchers will be able to find the right proliferation assessment tool to fit their individual needs.

Barbara A. Cunningham (Barbara.A.Cunningham@Hitchcock.org) is a freelance writer in Lyme, N.H.
1.N.J. Gonchoroff et al., "S-phase detection with an antibody to bromodeoxyuridine. Role of DNase pretreatment," Journal of Immunological Methods, 93:97-101,1998.

2.P. Carayon, A. Bord, "Identification of DNA-replicating lymphocyte subsets using a new method to label the bromo-deoxyuridine incorporated into the DNA," Journal of Immunological Methods, 147:225-30, 1992.

3.S. Takagi et al., "Detection of 5-bromo-2-deoxyuridine (BrdUrd) incorporation with monoclonal Anti-BrdUrd antibody after deoxyribonuclease treatment," Cytometry,14:640-8, 1993.

4.T. Mosmann, "Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays," Journal of Immunological Methods, 65:55-63, 1983.

5.R.F. Hussain et al., "A new approach for measurement of cytotoxicity using colorimetric assay," Journal of Immunological Methods, 160:89-96, 1993.

6.S.P. Crouch et al., "The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity," Journal of Immunological Methods, 160:81-8, 1993.

7.Apoptosis and Cell Proliferation 2nd Ed. (1998) Boehringer Mannheim GmbH, Biochemica, pp. 64-113.

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