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Markossian S, Grossman A, Arkin M, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004-.
Terry L Riss , PhD, Richard A Moravec , BS, Andrew L Niles , MS, Sarah Duellman , PhD, Hélène A Benink , PhD, Tracy J Worzella , MS, and Lisa Minor .
Terry L Riss , PhD, 1 ,* Richard A Moravec , BS, 1 Andrew L Niles , MS, 1 Sarah Duellman , PhD, 1 Hélène A Benink , PhD, 1 Tracy J Worzella , MS, 1 and Lisa Minor 2 ,† .
Published May 1, 2013 ; Last Update: July 1, 2016 .
This chapter is an introductory overview of the most commonly used assay methods to estimate the number of viable cells in multi-well plates. This chapter describes assays where data are recorded using a plate-reader; it does not cover assay methods designed for flow cytometry or high content imaging. The assay methods covered include the use of different classes of colorimetric tetrazolium reagents, resazurin reduction and protease substrates generating a fluorescent signal, the luminogenic ATP assay, and a novel real-time assay to monitor live cells for days in culture. The assays described are based on measurement of a marker activity associated with viable cell number. These assays are used for measuring the results of cell proliferation, testing for cytotoxic effects of compounds, and for multiplexing as an internal control to determine viable cell number during other cell-based assays.
Cell-based assays are often used for screening collections of compounds to determine if the test molecules have effects on cell proliferation or show direct cytotoxic effects that eventually lead to cell death. Cell-based assays also are widely used for measuring receptor binding and a variety of signal transduction events that may involve the expression of genetic reporters, trafficking of cellular components, or monitoring organelle function. Regardless of the type of cell-based assay being used, it is important to know how many viable cells are remaining at the end of the experiment. There are a variety of assay methods that can be used to estimate the number of viable eukaryotic cells. This chapter will provide an overview of some of the major methods used in multi-well formats where data are recorded using a plate reader. The methods described include: tetrazolium reduction, resazurin reduction, protease markers, and ATP detection. Methods for flow cytometry and high content imaging may be covered in different chapters in the future.
The tetrazolium reduction, resazurin reduction, and protease activity assays measure some aspect of general metabolism or an enzymatic activity as a marker of viable cells. All of these assays require incubation of a reagent with a population of viable cells to convert a substrate to a colored or fluorescent product that can be detected with a plate reader. Under most standard culture conditions, incubation of the substrate with viable cells will result in generating a signal that is proportional to the number of viable cells present. When cells die, they rapidly lose the ability to convert the substrate to product. That difference provides the basis for many of the commonly used cell viability assays. The ATP assay is somewhat different in that the addition of assay reagent immediately ruptures the cells, thus there is no incubation period of reagent with a viable cell population.
A variety of tetrazolium compounds have been used to detect viable cells. The most commonly used compounds include: MTT, MTS, XTT, and WST-1. These compounds fall into two basic categories: 1) MTT which is positively charged and readily penetrates viable eukaryotic cells and 2) those such as MTS, XTT, and WST-1 which are negatively charged and do not readily penetrate cells. The latter class (MTS, XTT, WST-1) are typically used with an intermediate electron acceptor that can transfer electrons from the cytoplasm or plasma membrane to facilitate the reduction of the tetrazolium into the colored formazan product.
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay was the first homogeneous cell viability assay developed for a 96-well format that was suitable for high throughput screening (HTS) (1). The MTT tetrazolium assay technology has been widely adopted and remains popular in academic labs as evidenced by thousands of published articles. The MTT substrate is prepared in a physiologically balanced solution, added to cells in culture, usually at a final concentration of 0.2 - 0.5mg/ml, and incubated for 1 to 4 hours. The quantity of formazan (presumably directly proportional to the number of viable cells) is measured by recording changes in absorbance at 570 nm using a plate reading spectrophotometer. A reference wavelength of 630 nm is sometimes used, but not necessary for most assay conditions.
Viable cells with active metabolism convert MTT into a purple colored formazan product with an absorbance maximum near 570 nm (Figure 1). When cells die, they lose the ability to convert MTT into formazan, thus color formation serves as a useful and convenient marker of only the viable cells. The exact cellular mechanism of MTT reduction into formazan is not well understood, but likely involves reaction with NADH or similar reducing molecules that transfer electrons to MTT (2). Speculation in the early literature involving specific mitochondrial enzymes has led to the assumption mentioned in numerous publications that MTT is measuring mitochondrial activity (3, 4).
Structures of MTT and colored formazan product.
The formazan product of the MTT tetrazolium accumulates as an insoluble precipitate inside cells as well as being deposited near the cell surface and in the culture medium. The formazan must be solubilized prior to recording absorbance readings. A variety of methods have been used to solubilize the formazan product, stabilize the color, avoid evaporation, and reduce interference by phenol red and other culture medium components (5-7). Various solubilization methods include using: acidified isopropanol, DMSO, dimethylformamide, SDS, and combinations of detergent and organic solvent (1, 5-7). Acidification of the solubilizing solution has the benefit of changing the color of phenol red to yellow color that may have less interference with absorbance readings. The pH of the solubilization solution can be adjusted to provide maximum absorbance if sensitivity is an issue (8); however, other assay technologies offer much greater sensitivity than MTT.
The amount of signal generated is dependent on several parameters including: the concentration of MTT, the length of the incubation period, the number of viable cells and their metabolic activity. All of these parameters should be considered when optimizing the assay conditions to generate a sufficient amount of product that can be detected above background.
The conversion of MTT to formazan by cells in culture is time dependent (Figure 2).
Direct correlation of formazan absorbance with B9 hybridoma cell number and time-dependent increase in absorbance. Note: there is little absorbance change between 2 and 4 hours. Adapted from CellTiter 96 ® Non-Radioactive Cell Proliferation Assay (more. )
Longer incubation time will result in accumulation of color and increased sensitivity up to a point; however, the incubation time is limited because of the cytotoxic nature of the detection reagents which utilize energy (reducing equivalents such as NADH) from the cell to generate a signal. For cell populations in log phase growth, the amount of formazan product is generally proportional to the number of metabolically active viable cells as demonstrated by the linearity of response in Figure 2. Culture conditions that alter the metabolism of the cells will likely affect the rate of MTT reduction into formazan. For example, when adherent cells in culture approach confluence and growth becomes contact inhibited, metabolism may slow down and the amount MTT reduction per cell will be lower. That situation will lead to a loss of linearity between absorbance and cell number. Other adverse culture conditions such as altered pH or depletion of essential nutrients such as glucose may lead to a change in the ability of cells to reduce MTT.
The MTT assay was developed as a non-radioactive alternative to tritiated thymidine incorporation into DNA for measuring cell proliferation (1). In many experimental situations, the MTT assay can directly substitute for the tritiated thymidine incorporation assay (Figure 3).
A comparison of using the MTT and 3 [H]thymidine incorporation assays of hGM-CSF-treated TF-1 cells. A blank absorbance value of 0.065 (from wells without cells but treated with MTT) was subtracted from all absorbance values. Adapted from CellTiter 96 (more. )
However, it is worth noting that MTT reduction is a marker reflecting viable cell metabolism and not specifically cell proliferation. Tetrazolium reduction assays are often erroneously described as measuring cell proliferation without the use of proper controls to confirm effects on metabolism (10).
Shortly after addition of MTT, the morphology of some cell types can be observed to change dramatically suggesting altered physiology (11 and Figure 4).
Change in NIH3T3 cell morphology after exposure to MTT (0.5 mg/ml). Panel A shows a field of cells photographed immediately after addition of the MTT solution. Panel B shows the same field of cells photographed after 4 hours of exposure to MTT. Panel (more. )
Toxicity of the MTT compound is likely related to the concentration added to cells. Optimizing the concentration may result in lower toxicity. Given the cytotoxic nature of MTT, the assay method must be considered as an endpoint assay. A recent report speculated that formazan crystals contribute to harming cells by puncturing membranes during exocytosis (12). The observation of extracellular formazan crystals many times the diameter of cells that grow longer over time make it seem unlikely that exocytosis of those large structures was involved (Figure 4 and 5).
U937 cells incubated with MMT tetrazolium for 3 hours showing formazan crystals larger than the cells. Image was captured using an Olympus FV500 confocal microscope. Scale bar = 20 µm
Growing crystals may suggest that marginally soluble formazan accumulates where seed crystals have begun to deposit.
Reducing compounds are known to interfere with tetrazolium reduction assays. Chemicals such as ascorbic acid, or sulfhydryl-containing compounds including reduced glutathione, coenzyme A, and dithiothreitol, can reduce tetrazolium salts non-enzymatically and lead to increased absorbance values in assay wells (13-17). Culture medium at elevated pH or extended exposure of reagents to direct light also may cause an accelerated spontaneous reduction of tetrazolium salts and result in increased background absorbance values. Suspected chemical interference of test compounds can be confirmed by measuring absorbance values from control wells without cells incubated with culture medium containing MTT and various concentrations of the test compound.
Commercial kits containing solutions of MTT and a solubilization reagent as well as MTT reagent powder are available from several vendors. For example:
CellTiter 96 ® Non-Radioactive Cell Proliferation Assay. Promega Corporation Cat.# G4000, Cell Growth Determination Kit, MTT based. Sigma-Aldrich Cat.# CGD1-1KT, and MTT Cell Growth Assay Kit. Millipore Cat.# CT02. Thiazolyl Blue Tetrazolium Bromide (MTT Powder). Sigma-Aldrich Cat.# M2128.The concentration of the MTT solution and the nature of the solubilization reagent differ among various vendors. The amount of formazan signal generated will depend on variety of parameters including the cell type, number of cells per well, culture medium, etc. Although the commercially available kits are broadly applicable to a large number of cell types and assay conditions, the concentration of the MTT and the type of solubilization solution may need to be adjusted for optimal performance.
Dissolve MTT in Dulbecco’s Phosphate Buffered Saline, pH=7.4 (DPBS) to 5 mg/ml.
Filter-sterilize the MTT solution through a 0.2 µM filter into a sterile, light protected container.
Store the MTT solution, protected from light, at 4°C for frequent use or at -20°C for long term storage.
Choose appropriate solvent resistant container and work in a ventilated fume hood.
Prepare 40% (vol/vol) dimethylformamide (DMF) in 2% (vol/vol) glacial acetic acid.
Add 16% (wt/vol) sodium dodecyl sulfate (SDS) and dissolve.
Adjust to pH = 4.7
Store at room temperature to avoid precipitation of SDS. If a precipitate forms, warm to 37°C and mix to solubilize SDS.
Prepare cells and test compounds in 96-well plates containing a final volume of 100 µl/well.
Incubate for desired period of exposure.
Add 10 µl MTT Solution per well to achieve a final concentration of 0.45 mg/ml.
Incubate 1 to 4 hours at 37°C.
Add 100 µl Solubilization solution to each well to dissolve formazan crystals.
Mix to ensure complete solubilization.
Record absorbance at 570 nm.
More recently developed tetrazolium reagents can be reduced by viable cells to generate formazan products that are directly soluble in cell culture medium. Tetrazolium compounds fitting this category include MTS, XTT, and the WST series (18-23). These improved tetrazolium reagents eliminate a liquid handling step during the assay procedure because a second addition of reagent to the assay plate is not needed to solubilize formazan precipitates, thus making the protocols more convenient. The negative charge of the formazan products that contribute to solubility in cell culture medium are thought to limit cell permeability of the tetrazolium (24). This set of tetrazolium reagents is used in combination with intermediate electron acceptor reagents such as phenazine methyl sulfate (PMS) or phenazine ethyl sulfate (PES) which can penetrate viable cells, become reduced in the cytoplasm or at the cell surface and exit the cells where they can convert the tetrazolium to the soluble formazan product (25). The general reaction scheme for this class of tetrazolium reagents is shown in Figure 6.
Intermediate electron acceptor pheazine ethyl sylfate (PES) transfers electron from NADH in the cytoplasm to reduce MTS in the culture medium into an aqueous soluble formazan.
In general, this class of tetrazolium compounds is prepared at 1 to 2mg/ml concentration because they are not as soluble as MTT. The type and concentration of the intermediate electron acceptor used varies among commercially available reagents and in many products the identity of the intermediate electron acceptor is not disclosed. Because of the potential toxic nature of the intermediate electron acceptors, optimization may be advisable for different cell types and individual assay conditions. There may be a narrow range of concentrations of intermediate electron acceptor that result in optimal performance.
Commercial kits containing solutions of MTS, XTT, and WST-1 and an intermediate electron acceptor reagent are available from several vendors. For example:
