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Section12:Methods for Evaluating Cell Proliferation
From Assay Guidance Wiki
Methods for Evaluating Cell Proliferation
Methods of measuring cell proliferation include the detection of proliferation associated antigens by immunohistochemistry, quantitation of DNA synthesis by measuring tritiated thymidine (3H-thymidine) or bromodeoxyuridine (BrdU) uptake or propidium iodide staining, quantitation of reduction of the intracellular environment by tetrazolium salt reduction or AlamarBlue reduction and quantitation of intracellular ATP concentration.
Detection of Antigens Associated with Proliferation
This method detects an antigen that is present in proliferating cells but absent in non-proliferating cells. The appearance of several antigens during cell proliferation has been noted. A frequently cited antigen used to monitor proliferation of human cells is the antigen recognized by the monoclonal antibody Ki-67. Because this antigen is expressed during S, G2, and M phases of the cycle but absent in G0 and G1, this monoclonal antibody is also suitable for examination of the cell cycle.
The use of monoclonal antibody Ki-67 requires that the cells under examination be processed for immunohistochemical staining. Because of this requirement for processing, the method cannot be used for rapid high volume screen. However, this method is very useful to assess in-vivo tumor cell proliferation, as other methods are not as feasible with tumor samples.
3H-Thymidine Incorporation
Historically, researchers have used the incorporation of 3H-Thymidine as a measure of cell proliferation. This method employs incubation of cells with 3H-thymidine for 16-24 hours following treatment with compounds or growth factors or testing sample. During this incubation the 3H-thymidine is incorporated into nascently synthesized DNA. Incorporated 3H-thymidine is usually quantitated by scintillation counting of labeled cells collected by aspiration upon membrane filters. As only proliferating cells incorporate 3H-thymidine, this method is an accurate indicator of DNA synthesis and is highly regarded as a gold standard for measuring the cell proliferation. Limitations of this assay for some nucleoside analogs are discussed by Drach and Shipman in Science, 1981, 212(4494):549-551.
Bromodeoxyuridine Incorporation
Like 3H-thymidine, 5-bromo-2’-deoxyuridine (BrdU) is incorporated into nascently synthesized DNA and provides a measurement of cell proliferation. The incorporation of BrdU must be detected by an indirect method, most commonly with BrdU-specific antibody conjugated to a reporter system such as a fluorochrome or enzyme suitable for use in immunohistochemistry, immunocytochemistry, in cell ELISA and flow cytometry analysis. As with 3H-thymidine, the use of BrdU requires extensive processing of samples.
Propidium Iodide Staining
This method quantitates the DNA content of cells as a measure of cell proliferation as propidium iodide binds to DNA. Cells are lysed by a freeze-thaw cycle in the presence of 1% propidium iodide. A fluorescence plate reader measures fluorescence intensity. The fluorescence is directly proportional to the DNA content in the samples. DNA content values higher than controls indicate proliferation while cultures with DNA content values lower than controls indicate cytotoxicity. This assay has several advantages as it can be used for both cells in suspension or grown as a monolayer, it does not require excessive sample processing, is cheaper and more rapid compared to 3H-thymidine and BrdU incorporation assays.
Tetrazolium Salt Reduction
The internal environment of the proliferating cell is more reduced than that of non-proliferating cells. Specifically, the ratios of NADPH/NADP, FADH/FAD, FMNH/FMN, and NADH/NAD, increase during proliferation. Compounds such as tetrazolium salts and AlamarBlue, which can be reduced by these metabolic intermediates, can be used to monitor cell proliferation. Their reduction is accompanied by a measurable shift in wavelength with a corresponding change in color.
An expansion in the number of viable cells results in an increase in the overall activity of mitochondrial dehydrogenases in the sample. This augmentation in the enzyme activity leads to an increase in the amount of formazan dye formed, which directly correlates to the number of metabolically active cells in the culture. The formazan dye produced by metabolically active cells is quantified by a scanning multiwell spectrophotometer (ELISA plate reader) by measuring the absorbance of the dye solution at appropriate wavelength.
The most frequently used of these are MTT (3-(4,5-dimethyethiazol-2-yl)-2,5-diphenyltetrazolium bromide), XTT (sodium 3’-[1-phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate, and MTS. Tetrazolium salt reduction, as an indicator of cell growth, has been used in models for screening cytocidal chemical agents or cell growth promoting factors and cytokines. There are several disadvantages to the use tetrazolium salts. They are generally cytotoxic. MTT is not soluble in culture medium and the formazan crystals that are the product of the reduction of MTT must be solubilized with DMSO or HCl/isopropanol. This treatment destroys the cells under investigation allowing only a single time point measurement. MTT is best suited for use with adherent cell lines. XTT has the advantage over MTT in that it is soluble in culture medium, and is therefore suitable for use with non-adherent as well as adherent cell lines. Because it is soluble, continuous monitoring can be performed using XTT. Since XTT is not efficiently reduced, the use of XTT requires the presence of PMS (phenozine methosulfate) for efficient reduction. The requirement of the presence of PMS has led to the questioning of the validity of results obtained with this method. MTS is a recently developed, alternative tetrazolium salt, which also has utility in cell proliferation assays. As with XTT, MTS has the advantage over MTT in that the formazan that results from its reduction is soluble in culture medium but also has the disadvantage of requiring the presence of PMS for effective reduction. WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) is member of the tetrazolium salt family which when reduced produces a water soluble formazan product. In contrast to XTT and MTS, WST-1 is more stable, has a wider linear range and shows accelerated color development.
Alamar Blue Reduction
As with tetrazolium salts, AlamarBlue monitors the reducing environment of the proliferating cell. AlamarBlue is soluble, stable in culture medium and is non-toxic. The continuous monitoring of cells in culture is therefore permitted. Proliferation may be monitored with AlamarBlue using a standard spectrophotometer, a standard spectrofluorometer, a spectrophotometric microtiter well plate reader, or spectrofluorometric microtiter well plate reader.
The oxidation-reduction potential of AlamarBlue is +380 mV at pH 7.0, 2°C. AlamarBlue , therefore, can be reduced by NADPH (Eo=-320 mV), FADH (Eo=-220 mV), FMNH (Eo=-210 mV), NADH (Eo=-320 mV), as well as the cytochromes (Eo=290 mV to +80 mV). The oxidation-reduction potential of MTT is +10 mV. MTT can only be reduced by NADPH, FADH, FMNH, and NADH, and not by the cytochromes. Because it can be reduced by the cytochromes, AlamarBlue substitutes for molecular oxygen as an electron acceptor while tetrazolium salts such as MTT do not. As the tetrazolium salts accept electrons from these compounds, they change in color from yellow to purple, a change that is easily monitored spectrophotometrically. As AlamarBlue accepts electrons from these compounds, it changes from the oxidized indigo blue, non-fluorescing state to the reduced fluorescent pink state.
ViaLight Cytotoxicity/Cell Proliferation Assay
ViaLight assay is intended for use with mammalian cells grown either in suspension or on the surface as a monolayer. ViaLight assays use bioluminescence catalyzed by the luciferase enzyme to measure intracellular ATP. Since ATP is central o the metabolism of all cells the intracellular concentration is very precisely regulated. When a cell dies, the ATP levels drop instantly. The combination of these two observations means that the number of viable cells in a culture can be measured by measuring the concentration of ATP in that culture. Bioluminescent detection of ATP is very simple and sensitive. Reagent containing luciferase and its substrate is simply added to the cell extract and the light emission is measured by microplate luminometer. The light emission is directly proportional to the ATP concentration in the samples. ATP values higher than controls indicate proliferation and cultures with ATP concentrations lower than controls indicates cytotoxicity.
Oxygen Biosensor System
The BD ViaSante Oxygen Biosensor System incorporates an oxygen-sensitive ruthenium-based fluorophore into a gas-permeable silicone matrix. Under ambient dissolved oxygen concentrations, fluorescence is largely quenched. As the concentration of oxygen is depleted in the polymer matrix, through cellular respiration or enzymatic reaction, the fluorescence increases. The signal correlates directly to the extent of cellular or enzymatic oxygen consumption.
Table of Detection Methods
| Detection Method | Pros | Cons |
|---|---|---|
| Ki-67 Antigen Detection – Immunohistochemical staining for DNA-binding protein antigen associated with cell cycle which reaches maximum levels during G2 and M | Can be used for tissue samples (frozen or paraffin imbedded); Correlation with morphology; Double labeling for immunophenotyping; Flow cytometry use | Limited throughput and labor intensive |
| 3H-Thymidine Incorporation – newly synthesized DNA measured | Direct measure of DNA synthesis; High sensitivity; Good for slow growing cells | Radioactivity and its waste; Moderately labor intensive; Does not measure percentage of cell proliferating |
| BrdU labeling – indirect measure of newly synthesized DNA | No radioactivity; Good sensitivity; Suitable for animal studies | Labor intensive with many washing steps; Indirect method to measure DNA synthesis |
| Propidium Iodide Staining – measures total DNA content in sample | Direct measure of DNA content; For adherent cells and cell suspensions; Rapid | Low to moderate labor intensity; Includes intact dead cells |
| Metabolic Proliferation Assay – Tetrazolium salt reduction by reducing environment of proliferating cells; (Alamar Blue>WST>XTT>MTS>MTT in ease of use) | Easiest method; Measures viability of cells | Some require addition of PMS to achieve reduction; All except Alamar Blue are toxic to cells |
| Metabolic Proliferation Assay – ViaLight measure [ATP] in cells ATP levels in cell | extracts measured enzymatically with luciferase. No wash steps, easy read-out on luminometer (Wallac, TriLux) | Expensive; May not be suitable for ATP competitive kinase inhibitors |
| Metabolic Proliferation Assay – Oxygen Biosensor; Measures oxygen consumption by proliferating cells | O2 sensitive fluorophore embedded in plate measures increase or decrease in O2 consumption. No reagent addition, read on 96 well fluorescence reader, can follow activity in real time and signal is reversible. Suitable for HTS. | Expensive; Difficult to visualize cell directly; Best for non-adherent cells |
