Briefly, cell viability is the number of live, healthy cells in a sample². Calculated as a percentage of control, 80-95% cell viability indicates a healthy culture. This number may be slightly lower in suspension cultures as dead cells do not get washed away during trypsinizing.

Overview of cell viability assays

The following assays can be used to measure cell viability:

• Metabolic assays
• Cytotoxicity assays
• Cell proliferation and cell cycle assays

These assays rely on metabolic activity, ATP content or cell proliferation as indicators of cell health. Loss of membrane integrity and other markers of cell damage or death can be used to measure cell viability indirectly.

See below to learn more about these assay methods, or review our most popular assay kits here:

• Cell viability assays: MTS , resazurin, TMRE, calcein violet, and ATP luminescence.
• Cytotoxicity assays: LDH assay, DRAQ7®, and our combined dye live:dead cell assay.
• Cell proliferation and cell cycle assays: EdU, propidium iodide, and CFSE

What’s the difference between a viability assay and a cytotoxicity assay?

Although metabolic assays, cytotoxicity assays and cell proliferation and cell cycle assays can all be used to assess viability, they each provide different perspectives.

Cell viability assays identify markers of healthy cell function, such as metabolism, DNA synthesis and cell division, and whilst these are used to measure the number of living cells, they can also be used as an indication of cytotoxicity because they can tell you if the level of metabolic activity and healthy function has decreased. Viability assays only tell you the number of remaining viable cells in a sample, and not whether this number is the result of cytotoxic or antiproliferative effects.

In contrast, cytotoxicity assays measure the toxicity of a chemical on cells by detecting markers of severe cell damage, such as loss of membrane integrity. It’s possible to use cytotoxicity assays to measure cell viability indirectly.

Combining a viability assay with a cytotoxic assay may be a good solution if you’re looking for a fuller picture of what’s happening in your cells.

Metabolic assays

Metabolism is a complex process that lies at the core of biology. Changes to metabolism are involved in a huge range of outcomes, from cancer to neurodegeneration, and more.

Within the cell, metabolism encompasses multiple enzymatic reactions that produce energy and maintain life. Measuring these processes can be a critical part of research, whether it’s to inform future experiments or to assess disease progression.

Metabolic assays indicate cell viability by quantifying the presence of enzymes and proteins involved in metabolism. Cells undergoing proliferation increase their metabolic rate³, therefore metabolic assays can also be used to assess proliferation.

Factors to consider when selecting a metabolic assay include:

• Assay sensitivity
• Signal-to-noise ratio
• Ease of use
• Reagent stability⁴

Generally, luminescence-based assays are more sensitive than fluorescence or absorbance-based assays.

Metabolism assays are also used for purposes other than to assess viability, such as to measure metabolites and metabolic enzymes. See our guide to measuring metabolism.

Dye reduction assays

Dye reduction assays are broadly split into tetrazolium assays and resazurin assays. These assays incubate a reagent with the cell sample to discover the proportion of viable cells in a population. The compounds in the reagent form a dye in a metabolically active environment, and the resulting color change can be quantified to indicate the extent of cell viability (see Figure 1).

Figure 1. Dye reduction assay overview.

Tetrazolium assays

Tetrazolium cell viability assays rely on cellular dehydrogenases to form a colored formazan product, measured by absorbance.

The most commonly used tetrazolium assays are split into two groups:

• MTT assay – a positively charged tetrazolium salt that readily penetrates cells.
• MTS, XTT and WST-1 assays – negatively charged tetrazolium salts that do not penetrate cells easily⁵.

MTT assay

The MTT assay was developed to provide a non-radioactive alternative to the tritiated thymine incorporation assay. However, the MTT assay is a measure of cell viability and not cell proliferation.

MTT is converted into formazan via an electron transfer reaction with substrates in the cell, such as NADH and NADPH (Figure 2). However, the resulting formazan crystals are insoluble, and form as precipitate inside the cells and culture medium. For the color change to be detected via a spectrophotometer, the crystals must be solubilized. Solutions such as DMSO and SDS can be used to dissolve the formazan crystals however this, along with the toxic nature of MTT, means that the MTT assay must be used as an endpoint assay. MTT is also light-sensitive, therefore, must be kept and used in the dark.

Advantages:

• Simple
• Easy to use
• Widely used

Disadvantages:

• Toxicity
• Susceptible to chemical interference
• Less sensitive than fluorescent and luminescent cell viability assays

Figure 2. Reduction of MTT in metabolically active cells to form insoluble formazan.

MTS, XTT and WST-1 assays

The MTS, XTT and WST-1 assays produce products that are soluble in culture medium. Although this removes the solubilization step, these tetrazolium salts do not easily penetrate cell membranes. Therefore, an intermediate electron acceptor reagent is needed. This intermediate reagent enters the cell, is itself reduced, and then exits the cell, where it can then transfer electrons to the tetrazolium salt. The salt is then converted to a soluble formazan product (Figure 3). However, the intermediate reagent may be toxic to the cells, so it is important to optimize the reaction depending on your sample and assay conditions.

As the product is soluble, multiple readings can be taken from the same plate across a time-course, although incubations longer than 4 h should be avoided. Additionally, background readings for MTS, XTT and WST-1 assays are generally higher than those for MTT assays, with absorbance readings of 0.3 and 0.05 respectively. However, these readings are dependent on culture medium and pH.

Advantages:

• Easy to use
• No DMSO dissolution required
• More sensitive and accurate than MTT (WST-1 being the most sensitive)

Disadvantages:

• Intermediate reagent required

• Figure 3. Reduction of MTS to form soluble formazan via the intermediate electron acceptor pheazine ethyl sulfate (PES).

Advantages:

• Easy to use
• No DMSO dissolution required
• More sensitive and accurate than MTT (WST-1 being the most sensitive)

Disadvantages:

• Intermediate reagent required

Resazurin assays

Resazurin assays are based on the same principle as tetrazolium assays and use electron transfer to convert one compound into another (Figure 4). In this case, resazurin forms a dark-blue solution when dissolved in physiological buffers, which is converted to resorufin.

Resorufin is a pink, fluorescent product, and, as fluorescence is generally more sensitive than absorbance measurements, this is an advantage over tetrazolium assays. Additionally, resazurin can penetrate cells, meaning that an intermediate electron acceptor is not required, although inclusion can speed up signal generation.

Resazurin assays are relatively inexpensive and can be used in combination with other methods to achieve a greater understanding of the cytotoxicity mechanisms. Despite these advantages, care must be taken to avoid fluorescent interference from other compounds.

As with the other dye reduction assays, extended incubation times are not recommended and should be optimized to balance sensitivity and toxicity.

Figure 4. Reduction of resazurin to resorufin in viable cells.

Advantages:

• Relatively inexpensive
• Increased sensitivity over tetrazolium assays

Disadvantages:

• Risk of fluorescence interference from other compounds

Figure 5. Jurkat cells treated with idarubicin (left) or staurosporin (right) were analyzed with Resazurin assay (ab129732).

Mitochondrial membrane potential-dependent dyes

Membrane potential is closely linked to a cell’s ability to generate ATP and therefore can be used as an indicator of cell health. There are several dyes available that accumulate in mitochondria due to the mitochondrial membrane potential and you can use these to identify viable cells.

A loss of membrane potential and loss of staining is used to assay for apoptosis.

Advantages:

• TMRE is reversible and suitable for live cell analysis
• JC-1 and JC-10 are ideal for comparative measurements

Disadvantages:

• Some assays not suitable for fixed samples
• Controls and technical details need careful attention to ensure performance⁶

Figure 6. Cell staining with TMRE kit (ab113852). A: (left): Healthy HeLa cells. B (right): Healthy Jurkat cells.

Esterase cleavage

Calcein and similar hydrophobic dyes diffuse into cells and are cleaved by intracellular esterases in live cells, providing a measurement of enzymatic activity which is a proxy for metabolic health and membrane integrity. The hydrophilic fluorescent product is retained within the cell.

Advantages:

• Non-toxic

Disadvantages:

• Potential fluorescent interference from other compounds

ATP assays

Most assays use a cell membrane permeabilization agent to release ATP; light is produced using ATP-dependent luciferase. Other ATP assays use the ATP-dependent phosphorylation of glycerol (or other substrates).

Advantages:

• Sensitive
• Fast

Disadvantages:

• Requires cell lysis

Cytotoxicity assays

Cytotoxicity is a measure of how toxic a substance is to a cell. A cell may either cease to proliferate or die due to apoptosis or necrosis in response to a toxic substance.

Cytotoxicity assays are commonly used to screen for any cytotoxic effects of a compound in drug screening. To determine the toxicity of a chemical agent, the assays detect markers of severe cellular damage or cell death, most commonly, damage to the cell membrane.

Methods that assess damage to the cell membrane include:

• Measurement of the activity of enzymes that leak into the extracellular medium
• Amine-reactive dyes bind weakly to the surface of live cells and create a brighter staining in dead cells
• Membrane impermeable dyes that enter and stain cells upon membrane damage. Membrane
• impermeable dyes. Often used with dyes that stain live cells, in combined live:dead cell assays.
• Membrane impermeable dyes that enter and stain cells upon membrane damage. Often used with dyes that stain live cells in combined live: dead cell assays.

In addition to causing cell membrane damage, cytotoxic agents can also affect cells by stopping protein synthesis, irreversibly binding to receptors, or causing other losses of structure or function⁷.

Alternatives to methods that assess damage to the cell membrane include the SRB assay, which uses the level of binding of the fluorescent Sulforhodamine B dye as a proxy for the number of live cells. The Crystal violet assay works similarly. Both methods should be used with adherent cell cultures as they rely on the detachment of adherent cells from cell culture plates during cell death.

See below to learn more about these assay methods, or review our most popular cytotoxicity assay kits, including the LDH assay, DRAQ7®, and our combined dye live: dead cell assay.

Enzyme leakage

These assays measure the activity of enzymes that leak into the extracellular medium on cell membrane damage. The most popular assay is for lactate dehydrogenase (LDH).

Advantages:

• Fast
• Reliable
• Sensitive
• Non-toxic to healthy cells
• Can be automated for high throughput applications

Disadvantages:

• Susceptible to interference from other compounds including serum (LDH only)

Figure 8. LDH assay used with staurosporine or cycloheximide HeLa cells, untreated cells, LDH positive control, and lysed cells.

Membrane impermeable dyes (dye exclusion assays)

These assays use membrane-impermeable fluorescent dyes (mostly DNA stains) that stain cells with damaged cell membranes. They offer a simple and widely used method for determining the membrane integrity of cells in suspension⁷.

The once commonly used propidium iodide has largely been replaced by DRAQ7™ and 7-AAD for cell viability assays due to its broad emission spectra and tendency to bind to live cells.

Advantages:

• Simple
• Rapid
• Can be used for low numbers of cells

Disadvantages:

• Trypan blue requires cell counting and is therefore susceptible to error

Figure 9. Jurkat cells treated with staurosporine to induce cell death show DRAQ7™ staining (top half of the plot).

Amine-reactive dyes for live:dead cell assays

Amine-reactive dyes weakly stain viable cells by binding to cell surface amines and strongly stain membrane-compromised cells by reacting with intracellular amines. Dead and live cells can be differentiated by fluorescence level.

Advantages:

• Accurate
• Clear distinction between dead and live cells
• Fixation compatible – allowing for post-fixation analysis as required for immunophenotyping experiments⁸

Disadvantages:

• Potential background fluorescence
• Requires a flow cytometer

Combined dye live:dead cell assays

Multiple dyes can be combined in a single live:dead cell assay to differentiate between dead and live cells. Kits allow for rapid quantitation of cell viability and are often suitable for proliferating and non-proliferating cells.

Advantages:

• Rapid
• More robust and accurate than other viability/cytotoxicity assays

Disadvantages:

• Potential background fluorescence

Figure 10. Left: Live: dead cell assay (ab115347) used with live (upper left) and dead (lower right) cells. Right: Etoposide-treated cells stained with ab115347. Live cells stain green and dead cells are red.

Cell proliferation and cell cycle assays

Proliferation is the process by which cells increase in number, usually through the phases of the cell cycle (mitosis). In addition to being key to development and size attainment in organisms, dysfunction of proliferative mechanisms can lead to diseases such as cancer⁹.

Cell proliferation assays measure the number of cells in a population that are actively dividing⁴, whereas viability assays measure all live cells, which can also be quiescent or senescent. Proliferation assays can additionally provide an indication of cell viability, as only viable cells can proliferate. The number of dividing cells can be measured through analyzing DNA levels, DNA synthesis, metabolic activity, or proliferation-specific proteins.

Proliferation can be used as an indication of prognosis in cancer, with a high rate of proliferation associated with aggressive cancers. For this reason, proliferation assays are commonly used in tissue analysis, using well-known markers such as Ki67, proliferating cell nuclear antigen (PCNA), and minichromosome maintenance (MCM)¹⁰ – see protein markers of proliferation below.

Methods include:

• DNA staining dyes for cell cycle analysis
• Dye dilution assays
• Incorporation of nucleoside analogs during DNA synthesis

Our most popular assay kits include: EdU, propidium iodide, and CFSE.

Cell proliferation can also be measured using metabolic assays – see the section in this guide on metabolic assays above.

Using DNA content to measure proliferation (cell cycle assays)

DNA-staining dyes are commonly used in flow cytometry to measure the DNA content in cell populations and assay for cell cycle state. These dyes emit fluorescence once bound to DNA. They bind proportionately to the amount of DNA present in the cell; therefore, cells undergoing DNA replication and preparing to divide (phases S and G2) will take up proportionally more dye and fluoresce more brightly. Propidium iodide is the most commonly used dye.

Advantages:

• Widely used
• Straight forward

Disadvantages:

• Some dyes require cells to be fixed or permeabilized (propidium iodide, DAPI), which is often incompatible with fluorescent proteins and some surface markers.

Figure 11. Propidium iodide flow cytometry kit (ab139418) used with thymidine (B) and nocodazole (C) treated HeLa cells. Peaks show 2N and 4N DNA content.

Incorporation of nucleoside analogs during DNA synthesis

The most reliable and accurate method of assessing cell proliferation is a measurement of DNA-synthesis. This relies on incubating live cells with compounds capable of being incorporated into newly synthesized DNA. These compounds can then be detected with a reporter.

Thymidine analogs are the compound of choice for incorporation into DNA, substituting thymidine during DNA replication. This labels proliferating and daughter cells. However, it is important to be aware that these thymidine analogs can lead to mutations and DNA damage in some instances, affecting the cycle^11,12.

Bromodeoxyuridine (BrdU) and ethynyldeoxyuridine (EdU) assays measure the incorporation of BrdU or EdU into newly synthesized DNA during DNA replication. Unlike BrdU, which is detected using antibodies, EdU can be easily directly labeled with a fluorescent dye or biotin for colorimetric or fluorometric detection via streptavidin-HRP. Unlike the harsher BrdU protocol, Edu staining is consistent with further antibody staining.

This method is suitable for immunohistochemistry (IHC), immunocytochemistry (ICC), ELISAs, flow cytometry, and some multiplex assays. Bromo-deoxyuridine (BrdU) can be detected with immuno-detection, whilst chemical detection is used for ethinyl-deoxyuridine (EdU)¹³.

For more information on these assays, review our cell proliferation guide .

Advantages:

• Accurate and reliable
• High- and low-throughput options

Disadvantages:

• Protocol can be lengthy and complex
• DNA denaturation prohibits subsequent co-staining experiments (BrdU only)

Figure 12. EdU staining of proliferating HeLa cells. DNA (blue) was stained with Hoechst 33342 (ab145597). Green cells are EdU + Hoechst-positive.

Dye dilution assays

The dyes in dye dilution assays (or fluorescent dye proliferation assays) are retained within cells over multiple generations. Daughter cells receive half of the dye of parent cells, and assays are analyzed on a flow cytometer. Carboxyfluorescein succinimidyl ester (CFSE) is the longest-established dye.

Advantages:

• Fluorescence is retained after formaldehyde and alcohol fixation
• Enables multicolor analysis

Disadvantages:

• CFSE is cytotoxic at higher concentrations (non-toxic to cells at appropriate concentrations)

Figure 13. Flow cytometry analysis of CFSE (ab113853) dilution assay.

Protein markers of proliferation

Another method to study cell proliferation is by looking at specific proteins that are expressed in proliferating cells but absent from non-proliferating cells. This requires the use of specific primary antibodies against the antigens expressed during proliferation.

These antigens are typically expressed in the perinuclear or nuclear interior regions across all cell cycle phases except G0, making them excellent cellular markers for proliferation. Ki67 is a very popular proliferation marker and is routinely used in pathology labs due to its diagnostic and prognostic power in cancer. PCNA is another common marker, yet multiple studies have shown that Ki67 is more sensitive and specific when evaluating cell proliferation in tumors from various origins^{14, 15, 16, 17}. A marker growing in prominence is MCM-2, and recent work suggests this may be a better choice for informing cancer prognoses than Ki67 and PCNA^{18, 19}.

However, much of the data is inconclusive regarding a ‘best’ marker of proliferation, especially in a clinical context.

These immunoassays are excellent for fixed tissue samples and analysis by IHC.

Advantages:

• Accurate and reliable
• Large body of supporting data
• Clinical diagnostic and prognostic value in some cases

Disadvantages:

• Limited high-throughput options
• Scoring of results can be subjective
• Conflicting data around the ‘best’ marker of cell proliferation in a clinical setting

Figure 14. Immunohistochemical analysis of frozen sections from adult zebrafish intestine, labeled with an anti-PCNA antibody [PC10] (ab29).

Figure 15. Immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded sections from mouse spleen, labeled using an anti-Ki67 antibody (ab15580).

Figure 16. Immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded sections from human small cell lung cancer tissue, labeled with an anti-MCM2 antibody (ab4461).

Clonogenicity assays

Although little used for high throughput, the classical method of assaying cell proliferation is to use a clonogenic/clonogenicity assay. In this assay, cells are plated out at a low density and then the number of colonies formed is counted.

Advantages:

• Low risk of interference²⁰

Disadvantages:

• Susceptible to error due to manual counting
• Time-consuming
• Limited to adherent cells

Senescence assays

Senescence is thought to be a tumor suppressive mechanism and an underlying cause of aging. Senescence represents an arrested metabolic state in which the cells remain viable, but not actively dividing.

The most common marker of senescent cells is the overexpression and accumulation of the endogenous lysosomal beta-galactosidase (SA-beta-gal). Beta-gal activity is detected using a colorimetric or fluorometric substrate.

Advantages:

• Suitable for cells or tissues
• Simple
• Reliable

Disadvantages:

• Requires fixation of cells (colorimetric assay only)
• Certain cells (osteoclasts and macrophages) have higher levels of β-gal activity, potentially leading to false positives²¹

Scoring proliferating cells

Scoring the extent of proliferation is especially important in a clinical setting. The percentage of Ki67-positive cells, for example, can be used to score the severity and course of cancer. There are several scoring techniques available for use with the proliferation proteins methods, each with their own strengths and limitations.

See our complete proliferation guide for more information on identifying and scoring proliferating cells.

References

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3. Cobb, L. Cell Proliferation Assays and Cell Viability Assays Materials and Methods 3 ,2799 (2013)
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8. Matson, J.,, Cook, J. Cell cycle proliferation decisions: the impact of single cell analyses The FEBS Journal 284 ,362-375 (2016)
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10. Breunig, J. J.,, Arellano, J. I.,, Macklis, J. D.,, Rakic, P. Everything that Glitters Isn’t Gold: A Critical Review of Postnatal Neural Precursor Analyses Cell Stem Cell 1 ,612–627 (2007)
11. Anda, S.,, Boye, E.,, Grallert, B. Cell-cycle analyses using thymidine analogues in fission yeast PLoS One 9 ,1–9 (2014)
12. Tuttle, A.H.,, Rankin, M.M.,, Teta, M.,, et al Immunofluorescent detection of two thymidine analogues (CldU and IdU) in primary tissue J Vis Exp 46 ,2166 (2010)
13. Oka, S.,, Uramoto, H.,, Shimokawa, H.,, Iwanami, T.,, Tanaka, F. The expression of Ki-67, but not proliferating cell nuclear antigen, predicts poor disease free survival in patients with adenocarcinoma of the lung Anticancer Res 31 ,4277–4282 (2011)
14. Mateoiu, C.,, Pirici, A.,, Bogdan, F. L. mmunohistochemical nuclear staining for p53, PCNA, ki-67 and bcl-2 in different histologic variants of basal cell carcinoma. Rom. J. Morphol. Embryol 52 ,315–319 (2011)
15. Salehinejad, J.,, et al Immunohistochemical detection of p53 and PCNA in ameloblastoma and adenomatoid odontogenic tumor Oral Sci 53 ,213–217 (2011)
16. Bologna-Molina, R.,, Mosqueda-Taylor, A.,, Molina-Frechero, N.,, Mori-Estevez, A. D.,, Sánchez-Acuña, G. Comparison of the value of PCNA and Ki-67 as markers of cell proliferation in ameloblastic tumors Med. Oral Patol. Oral Cir. Bucal 18 , (2013)
17. Carreón-Burciaga, R. G.,, González-González, R.,, Molina-Frechero, N.,, Bologna-Molina, R. Immunoexpression of Ki-67, MCM2, and MCM3 in Ameloblastoma and Ameloblastic Carcinoma and Their Correlations with Clinical and Histopathological Patterns Dis. Markers , (2015)
18. Joshi, S.,, et al Digital imaging in the immunohistochemical evaluation of the proliferation markers Ki67, MCM2 and Geminin, in early breast cancer, and their putative prognostic value BMC Cancer 15 ,546 (2015)
19. Gutiérrez, L.,, et al Nanotechnology in Drug Discovery and Developmen Comprehensive Medicinal Chemistry III ,264-295 (2017)
20. Noren Hooten, N.,, Evans, M. K. Techniques to induce and quantify cellular senescence J. Vis. Exp. 123 (e55533), (2017)