Comprehensive Overview of In Vitro Cell Viability Assays in Cancer Research

Introduction

Cell viability tests, which are essential to evaluate the percentage of living, metabolically active cells in a population, are essential aspects in biomedical research. These studies are necessary to determine how drugs(or) chemicals affects the health and proliferation of cells.^1-3

Investigators can find out more regarding cytotoxicity, the safety of treatments, and how cells respond physiologically to different stimuli by evaluating the survival of cells.²

Several biological principles are essential for the development of various types of cell viability tests.² The ability of live cells to convert tetrazolium salts or resazurin into fluorescent products that represent mitochondrial or cytosolic metabolic activity is the basis for metabolic activity assays including the MTT, MTS, and resazurin (Alamar Blue) tests.^2,4,5

As an indicator of active metabolic processes in cells, ATP-based assays measure intracellular ATP levels.⁶ Propidium iodide staining and Trypan Blue exclusion are two assays that depend on the membrane integrity where the dead cells with the less intact membrane can take up the dye when compared to the intact cell membranes of the living cells.^2,7,8

Clonogenic or colony formation tests, which measure single cell’s capacity to withstand treatment and develop a colony over time, are commonly used to evaluate the long-term survival and proliferation.²For rapid assessment of living and dead cells, fluorescence-based LIVE/DEAD assays that combine ethidium homodimer-1 and calcein-AM are also often used. When combined together, these various methods provide a well-established foundation for understanding basic cell biology, medication development, toxicity assessment, and cellular responses.⁹

Importance of Cell Viability Assays

Assays for cell viability are crucial in many different biological applications. They are necessary methods in toxicology and drug development, enabling scientists to evaluate the cytotoxic effects of new chemicals on various cell types.^2,10 Viability tests are used in cancer research to assess the effectiveness of chemotherapeutic drugs and evaluate the response of tumour cells are to various treatments.⁷

Measuring the cell longevity, their proliferation, and response to environmental stresses provides aninterpretation of cellular processes and signalling cascades, this is why they are important in cell biology research.^2,11Moreover viability tests are commonly used in stem cell research and regenerative medicine to confirm that cultured cells sustain their ability to proliferateand function properly.¹¹

Table 1: Classification of Cell Viability Assays

Tetrazolium-Based Cell Viability Assays: MTT, XTT, and MTS

Tetrazolium-based assays are frequently used colorimetric methods for measuring cell viability, proliferation, and cytotoxicity. These assays depend on the metabolic reduction of tetrazolium salts into coloured formazan products by metabolically active cells or viable cells, predominantly through mitochondrial dehydrogenase enzyme activity.^{2,4,5,7,12,13}

Principle of Tetrazolium Assay

Tetrazolium salts including MTT, XTT, and MTS are reduced by NAD(P)H-dependent cellular oxidoreductases into formazan products. The extent of formazan formed is directly related to the number of viable cells and the metabolically active cells.^2,14

MTT → insoluble formazan

XTT and MTS → water-soluble formazan

MTT Assay

Principle

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is reduced to insoluble purple formazan crystals, which must be dissolved before absorbance measurement.^2,14

Procedure^12,15,16

• Seed cells (5×10³–1×10⁴ cells/well) in a 96-well plate and incubate for 24 h.
• Treat cells with test compounds and incubate for the desired period. Add MTT solution so that the final solution becomes 0.5mg/ml
• Maintain under stable conditions for 2–4 h at 37°C.
• Remove the medium carefully.
• Add solubilization solution for solubilizing the non-soluble formazan crystals (DMSO or acidified isopropanol).
• Mix to solubilize formazan crystals completely.
• Analyse absorbance at 570 nm (reference: 630–690 nm).^7,16

Advantages

• High sensitivity.¹⁶
• Widely used and standardized.¹⁵

Perceived Limitations:

• Requires the solubilization step to dissolve the non -soluble crystals.¹⁶
• Cytotoxic to cells.

XTT Assay

Principle

XTT produces a water-soluble orange formazan dye, eliminating the need for a solubilization step. It requires an electron coupling reagent such as PMS.^2,12,14

Procedure^12,14

• Seed and treat cells in a 96-well plate (5×10³–1×10⁴ cells/well).
• Prepare XTT solution with electron coupling reagent.
• Add XTT mixture directly to wells at the concentration of0.5mg/ml.
• Maintain under stable conditions for 2–4 h at 37°C.
• Measure absorbance at 450–500 nm (reference ~650 nm).

Advantages

• No solubilization step as it forms water soluble crystals.²
• Less toxic to cells.

 PerceivedLimitations

• Requires additional reagent (electron mediator).
• Slightly lower sensitivity than MTT.²

MTS Assay

Principle

MTS is a modified Tetrazolium compound that produces a water-soluble formazan product directly in the culture medium.^2,17

Procedure^{2,12,15,17,18}

• Seed and treat cells in a 96-well plate (5×10³–1×10⁴ cells/well).
• Add MTS reagent (0.5mg/ml) 20 µL per 100 µL medium).
• Maintain under stable conditions for 1–4 h at 37°C.
• Measure absorbance at 490–500 nm.

Advantages

• One-step, simple procedure.
• No solubilization required.¹²
• Suitable for high-throughput screening.

PerceivedLimitations^12,15

• More expensive
• Sensitive to media components and assay conditions.

Dye Exclusion Assays for Cell Viability

Dye exclusion assays are easy and rapid methods used to differentiate viable from non-viable cells based on membrane integrity.^19,20,21 These assays depend on the principle that viable cells possess intact cell membranes that don’t allow the certain dyes to enter in the cell, whereas dead or damaged cells allow dye entry due to the damaged integrity of cell membrane.^2,8,19

Principle

Dye exclusion assays are based on cell membrane permeability.^2,19,20

• Viable cells → intact membrane → exclude dye
• Non-viable cells → less intact membrane → dye can be easily entered

Common dyes used include:

• Trypan Blue
• Eosin
• Propidium Iodide

Recent developments have improved dye exclusion assays by including plate-based and high-throughput formats, decreasing subjectivity and increasing reproducibility.

Trypan Blue Exclusion Assay

Principle

Trypan Blue cannot be taken up by the viable cells due to its intact membrane but the dead cells have lost their membrane integrity and can take up the dye. Apart from this as the cell membrane of live cells is negatively charged and dye is also negatively charged which causes the repulsion and dye cannot enter the cell but different in the case of the dead cells as the damaged cell has the decreased negative charge and cells can take up the dye easily.^2,22,23

Procedure

• Prepare a single-cell suspension (1×10³).
• Mix cells with 0.4% Trypan Blue (1:1 ratio).^24-27
• Maintain under stable conditions for 2–5 minutes at room temperature.
• Place the sample onto a hemacytometer.
• Count unstained (viable) and stained (dead) cells.
• Calculate percentage viability.

Automated cell counters, such as the countess™, can streamline this process by acquiring    images and analysing cell count and viability automatically.²⁵

Advantages

• Simple and cost-effective.
• Rapid assessment.

PerceivedLimitations:

• Need operator.
• Low throughput.
• Cannot detect early apoptosis.

Eosin Dye Exclusion Assay

Principle

Eosin dye that enters the dead cells with less membrane integrity same as the trypan blue but staining non-viable cells red or pink.^{2,19,21,27,28,41}

Procedure

• Mix cell suspension with eosin dye
• Maintain under stable conditions for 2–5 minutes at room temperature.
• Load on haemocytometer.
• Count stained and unstained cells that represents the live and dead cells

Advantages

• Simple and inexpensive

Fluorescence-Based Cell Viability Assays

Fluorescence-based assays are highly responsive methods for measuring cell viability, cytotoxicity, and metabolic activity. They depend on fluorescent dyes or probes that either enter the viable cells ideally stain dead or apoptotic cells due to their decreased cell membrane integrity. These assays are widely used in high-throughput screening, flow cytometry, and imaging studies.^2,9,15,21,29

Principle of Fluorescence-Based Assays

The major principle of fluorescent based assays depends on:

• Membrane integrity (e.g., Propidium Iodide, Calcein-AM).^31,32
• Metabolic activity (e.g., Calcein-AM, Resazurin).^33,34,41
• Apoptotic changes (e.g., Annexin V-FITC, JC-1)

Viable cells have the enzyme esterases that are able to convert the non-fluorescent dye to the fluorescent dyes where the dead cells are unable to stain.^19,30 

Common Fluorescent Assays

Calcein-AM Assay

Principle

Calcein-AM is a non-fluorescent, that has the ability to cross the viable cells that gets converted by intracellular esterases into green-fluorescent calcein in viable cells.Whereas the dead cells don’t have any effect by this calcein.^2,31-33,35

Procedure

• Seed cells (1×10³) in a microplate or chamber slide.
• After the 24hrs of treatment remove the media wash with PBS and then Add Calcein-AM (1–5 µM) and incubate 30–60 min at 37°C.^35-38
• Wash cells with PBS to remove excess dye.
• Measure fluorescence (Ex: 490 nm, Em: 520 nm) using a plate reader or fluorescence microscope.

Advantages

• High sensitivity
• Compatible with live-cell imaging
• Non-toxic for short-term studies

Resazurin / Alamar Blue Assay

Principle

Resazurin is also a non-fluorescent dye that has the ability to enter the viable cells and gets reduced into resorufin, a fluorescent product (Ex: 560 nm, Em: 590 nm) and it cannot enter the dead cells.^2,33

Procedure^37,39,40

• Seed cells(1×10³) in a microplate or chamber slide
• After the 24hrs of treatment remove the media wash with PBS and then Add resazurin solution (10% v/v) and incubate 1–4 hrs at 37°C.^33,39,40
• Wash cells with PBS to remove excess dye.
• Measure fluorescence using a plate reader.

Advantages

• Non-toxic; cells can be used for further assays.^39,40
• High-throughput compatible.

Propidium Iodide (PI) and SYTOX Dyes

Principle

Membrane-impermeable fluorescent dyes enter only dead cells, as the membrane because of the less membrane integrity binding DNA/nucleic acids and emitting red fluorescence (Ex: 535 nm, Em: 617 nm).^2,33,41,42

Procedure

• Seed cells(1×10³) in a microplate or chamber slide (5×10³).
• After the 24hrs of treatment remove the media wash with PBS Add PI or SYTOX dye to the cell suspension.
• Incubate 5–15 min at room temperature in the dark.
• Analyse by fluorescence microscopy or flow cytometry.

Advantages

• Accurate identification of live/dead cells
• Can use along with other dyes like calcein

Table 2: Comparative summary of cell viability assays

 Conclusion

Cell viability assays are important for determining cellular health, proliferation, and cytotoxicity. In this review, three major aspects tetrazolium-based assays, dye exclusion assays, and fluorescent-based assaywere explained,each having various advantages and limitations.

Tetrazolium-based assays (such as MTT, XTT, and WST) facilitatea quantitative evaluation of metabolic activity, acting as an indirect measure of viable cells. These assays are sensitive, relatively simple, and suitable for high-throughput screening, but they depend on cellular metabolism, which may not always directlyrelates with cell number.

Dye exclusion assays, such as trypan blue staining, have straightforward and cost-effective method to differentiate live and dead cells based on membrane integrity. However, they are less sensitive, and not ideal for large-scale or automated analysis.

Fluorescent-based assays, including those using dyes like calcein-AM and propidium iodide, allow for more Accurate and multiparametric analysis of cell viability. These assays can differentiate between live, dead, and even apoptotic cells with high sensitivity and are well-established for advanced imaging and flow cytometry applications, though they need specialized equipment and can be more expensive.

Overall, no single assay is universally; the selection depends on the experimental objectives, required sensitivity, available resources, and throughput needs. Combining multiple assay types can provide a more comprehensive and reliable assessment of cell viability. 

Acknowledgement

We express our sincere gratitude to the management of school of pharmaceutical sciences and technologies, JNTU Kakinada for the constant support and encouragement throughout the development of this manuscript.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of Interest

The authors do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

Informed Consent Statement

This study did not involve human participants, and therefore, informed consent was not required.

Clinical Trial Registration

This research does not involve any clinical trials.

Permission to reproduce material from other sources

Not Applicable.

Author Contributions

• Korimelli Sridevi: Supervision, Designed the overall structure, Critical review, and Validation
• Himaja Kommineni: Conceptualization, Design of the study, Data collection and analysis, Prepared the original manuscript draft
• Gunadeep Naidu Vennela: Literature review and Data verification 

References

1. Khalef L, Lydia R, Filicia K, Moussa B. Cell viability and cytotoxicity assays: biochemical elements and cellular compartments. Cell Biochem Funct. 2024;42:e4007. doi:10.1002/cbf.4007
   CrossRef
2. Sukumaran A, Sweety VK, Vikas B, Joseph B. Cytotoxicity and cell viability assessment of biomaterials. In: Cytotoxicity—Understanding Cellular Damage and Response. IntechOpen; 2023. doi:10.5772/intechopen.111822
   CrossRef
3. Kamiloglu S, Sari G, Ozdal T, Capanoglu E. Guidelines for cell viability assays. Food Front. 2020;1:332-349. doi:10.1002/fft2.44
   CrossRef
4. Stockert JC, Horobin RW, Colombo LL, Blázquez-Castro A. Tetrazolium salts and formazan products in cell biology: viability assessment, fluorescence imaging, and labeling perspectives. Acta Histochem. 2018;120(3):159-167. doi:10.1016/j.acthis.2018.02.005
   CrossRef
5. Präbst K, Engelhardt H, Ringgeler S, Hübner H. Basic colorimetric proliferation assays: MTT, WST, and resazurin. In: Gilbert D, Friedrich O, eds. Cell Viability Assays. Methods Mol Biol. Vol 1601. Humana Press; 2017. doi:10.1007/978-1-4939-6960-9_1
   CrossRef
6. Xu N, Wang Y, Xu X. Cell growth measurement. IntechOpen; 2020. doi:10.5772/intechopen.86835
   CrossRef
7. Singhal M, Shaha S, Katsikogianni M. Comparative analysis of cytotoxicity assays, from traditional to modern approaches. In: Erkekoğlu P, ed. Cytotoxicity—A Crucial Toxicity Test for In Vitro Experiments. IntechOpen; 2024. doi:10.5772/intechopen.1006842
   CrossRef
8. Forgie BN, Prakash R, Goyeneche AA, Telleria CM. Vitality, viability, long-term clonogenic survival, cytotoxicity, cytostasis and lethality: what do they mean when testing new investigational oncology drugs? Discov Oncol. 2024;15(1):5. doi:10.1007/s12672-023-00857-2
   CrossRef
9. Sazonova EV, Chesnokov MS, Zhivotovsky B, et al. Drug toxicity assessment: cell proliferation versus cell death. Cell Death Discov. 2022;8:417. doi:10.1038/s41420-022-01207-x
   CrossRef
10. Çetin Y. Assessing the toxic potential of new entities: the role of cytotoxicity assays. IntechOpen; 2025. doi:10.5772/intechopen.1008309
    CrossRef
11. Niu J, Li M, Wang Y. Cell proliferation and cytotoxicity assays: the fundamentals for drug discovery. Int J Drug Discov Pharmacol. 2024;3(3):100013. doi:10.53941/ijddp.2024.100013
    CrossRef
12. Karatop EU, Cimenci CE, Aksu AM. Colorimetric cytotoxicity assays. IntechOpen; 2022. doi:10.5772/intechopen.105772
    CrossRef
13. Sylvester PW. Optimization of the tetrazolium dye (MTT) colorimetric assay for cellular growth and viability. In: Satyanarayanajois S, ed. Drug Design and Discovery. Methods Mol Biol. Vol 716. Humana Press; 2011. doi:10.1007/978-1-61779-012-6_9
    CrossRef
14. Schanne G, Demignot S, Policar C, Delsuc N. Cellular evaluation of superoxide dismutase mimics as catalytic drugs: challenges and opportunities. Coord Chem Rev. 2024;514:215906. doi:10.1016/j.ccr.2024.215906
    CrossRef
15. Arunachalam K, Sreeja PS. MTT assay protocol. In: Advanced Cell and Molecular Techniques. Springer; 2025. doi:10.1007/978-1-0716-4518-5_45
    CrossRef
16. Mazlumoğlu BŞ. In vitro cytotoxicity test methods: MTT and neutral red uptake. Pharmata. 2023;3(2):50-53. doi:10.5152/Pharmata.2023.128796
    CrossRef
17. Kıvrak İ, Kıvrak Ş. In vitro assessment of antioxidant capacity and potential to induce autophagy/mitophagy. IntechOpen; 2023. doi:10.5772/intechopen.112278
    CrossRef
18. Petosa AR, Nowierski M, Yargeau V, et al. Biological activity of wastewater assessed using in vitro cell-based assays. Res Sq. Preprint. 2021. doi:10.21203/rs.3.rs-513231/v1
    CrossRef
19. Naveen KV, Tyagi A, Ibrahium OMH, et al. From dye exclusion to high-throughput screening: a review of cell viability assays and their applications. Biotechnol Adv. 2026:108764. doi:10.1016/j.biotechadv.2025.108764
    CrossRef
20. In vitro cytotoxicity and cell viability assays: principles, advantages, and disadvantages. In: Genotoxicity—A Predictable Risk to Our Actual World. IntechOpen; 2018. doi:10.5772/intechopen.71923
    CrossRef
21. Riss TL, Niles AL, Moravec RA, Karassina N, Vidugiriene J. Cytotoxicity assays: in vitro methods to measure dead cells. In: Markossian S, et al., eds. Assay Guidance Manual. Eli Lilly & Company; 2019.
22. Ude A, Afi-Leslie K, Okeke K, Ogbodo E. Trypan blue exclusion assay, neutral red, acridine orange and propidium iodide. In: Sukumaran A, Mansour MA, eds. Cytotoxicity: Understanding Cellular Damage and Response. IntechOpen; 2022. doi:10.5772/intechopen.105699
    CrossRef
23. Kerschbaum HH, Tasa BA, Schürz M, Oberascher K, Bresgen N. Trypan blue—adapting a dye used for labelling dead cells to visualize pinocytosis in viable cells. Cell Physiol Biochem. 2021;55(S1):171-184. doi:10.33594/000000380
    CrossRef
24. Yadav AR, Mohite SK, Magdum CS. Synthesis, characterization and biological evaluation of some novel 1,3,4-oxadiazole derivatives as potential anticancer agents. Int J Sci Res Sci Technol. 2020;7(2). doi:10.32628/IJSRST207234
    CrossRef
25. di Vito R, Levorato S, Fatigoni C, et al. In vitro toxicological assessment of PhSeZnCl in human liver cells. Toxicol Res. 2023;39(1):105-114. doi:10.1007/s43188-022-00148-y
    CrossRef
26. Kuijpers L, van Veen E, van der Pol LA, Dekker NH. Automated cell counting for trypan blue-stained cell cultures using machine learning. PLoS One. 2023;18(11):e0291625. doi:10.1371/journal.pone.0291625
    CrossRef
27. Sasikala S, Jenifer MM, Velavan K, et al. Predicting the relationship between pesticide genotoxicity and breast cancer risk in South Indian women in vitro and in vivo. Sci Rep. 2023;13:35552. doi:10.1038/s41598-023-35552-3
    CrossRef
28. Jeevanandam J, Barhoum A, Chan YS, et al. Toxicity evaluation and biocompatibility of nanostructured biomaterials. In: Applications of Nanobiotechnology. IntechOpen; 2023. doi:10.5772/intechopen.109078
    CrossRef
29. Sabirova S, Sharapova G, Budyukova A, et al. Comprehensive analysis of cellular metrics: from proliferation to mitochondrial membrane potential and cell death in a single sample. Cell Death Discov. 2025;11:119. doi:10.1038/s41420-025-02391-2
    CrossRef
30. Roger LM, Adarkwa Darko Y, Bernas T, et al. Evaluation of fluorescence-based viability stains in cells dissociated from coral Pocillopora damicornis.Sci Rep. 2022;12:15297. doi:10.1038/s41598-022-19586-7
    CrossRef
31. Azqueta A, Stopper H, Zegura B, et al. Do cytotoxicity and cell death cause false positive results in the in vitro comet assay? Mutat Res Genet Toxicol Environ Mutagen. 2022;881:503520. doi:10.1016/j.mrgentox.2022.503520
    CrossRef
32. Tolosa L, Martínez-Sena T, Schimming JP, et al. In vitro assessment of toxicity of volatile, oxidisable compounds. Arch Toxicol. 2021;95(6):2109-2121. doi:10.1007/s00204-021-03036-w
    CrossRef
33. Rodríguez Rego JM, Mendoza-Cerezo L, Iñesta Vaquera FA, et al. From medical imaging to bioprinted tissues: importance of workflow optimisation. Expert Rev Mol Med. 2025;27:e33. doi:10.1017/erm.2025.10018
    CrossRef
34. Grigoryeva NY. Studying cyanobacteria by fluorescence methods: a review. IntechOpen; 2020. doi:10.5772/intechopen.93543
    CrossRef
35. Skvortsov DA, Kalinina MA, Zhirkina IV, et al. From toxicity to selectivity: coculture of tumor and non-tumor lung cells. Front Pharmacol. 2021;12:713103. doi:10.3389/fphar.2021.713103
    CrossRef
36. Wilson-Robles H, Miller T, Sima C, et al. Evaluation of two novel therapeutics against osteosarcoma. Res Sq. Preprint. 2020. doi:10.21203/rs.3.rs-51104/v1
    CrossRef
37. Coelho CC, Grenho L, Gomes PS, et al. Nano-hydroxyapatite in oral care cosmetics: characterization and cytotoxicity assessment. Sci Rep. 2019;9:11050. doi:10.1038/s41598-019-47491-z
    CrossRef
38. Seo Y, Park C, Son J, et al. Carbon nanotubes modified with silver nanoparticles: antibacterial and cytotoxic properties. J Vis Exp. 2018;(135):e57384. doi:10.3791/57384
    CrossRef
39. Petiti J, Revel L, Divieto C. Standard operating procedure to optimize resazurin-based viability assays. Biosensors. 2024;14(4):156. doi:10.3390/bios14040156
    CrossRef
40. Vieira-da-Silva B, Castanho MARB. Resazurin reduction-based assays revisited. Molecules. 2023;28(5):2283. doi:10.3390/molecules28052283
    CrossRef
41. Costigan A, Hollville E, Martin SJ. Discriminating between apoptosis, necrosis, necroptosis, and ferroptosis. Curr Protoc. 2023;3: e951. doi:10.1002/cpz1.9
    CrossRef
42. Abdel-Malek AR, Moustafa AY, Salem SH. Antimicrobial and cytotoxic activities of flavonoid and phenolics extracted from Sepia pharaonis ink (Mollusca: Cephalopoda). BMC Biotechnol. 2024;24:54. doi:10.1186/s12896-024-00880-3
    CrossRef

Abbreviations

MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium

XTS: 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide

PI: Propidium iodide

PBS: Phosphate buffered saline