Table of Contents

1. Introduction
2. MTT Assay
3. Trypan Blue Dye Exclusion Assay
4. Live/Dead Cell Assay
5. ATP Assay
6. XTT Assay
7. WST-8 Assay
8. MTS Assay
9. Comparison Table
10. Conclusion

1. Introduction

Cell viability assays are crucial for evaluating cellular health, proliferation, and response to treatments. This guide provides a comprehensive overview of the most commonly used assays, detailing their procedures, advantages, and limitations to help you choose the best method for your research needs.

2. MTT Assay

Overview

The MTT assay is a colorimetric assay that measures cellular metabolic activity based on the reduction of MTT tetrazolium salt to a purple formazan product by viable cells.

Detailed Procedure

1. Cell Treatment: Incubate cells with MTT reagent for 1-4 hours.
2. Formazan Solubilization: Add a solubilization solution (e.g., DMSO) to dissolve the formazan crystals.
3. Measurement: Measure absorbance at 570 nm using a spectrophotometer.

Advantages

• Well-established and reliable.
• Quantitative measurement.
• Simple procedure.

Limitations

• Requires solubilization step.
• Can be affected by media components.
• Not suitable for non-adherent cells.

3. Trypan Blue Dye Exclusion Assay

Overview

The Trypan Blue Dye Exclusion assay stains dead cells blue, allowing for visualization and counting under a microscope.

Detailed Procedure

1. Cell Staining: Mix cell suspension with Trypan Blue dye.
2. Counting: Load onto a hemocytometer and count the cells under a microscope.

Advantages

• Simple and quick.
• Direct visualization.
• No special equipment needed.

Limitations

• Manual counting can be labor-intensive.
• Semi-quantitative.
• Staining can be subjective.

4. Live/Dead Cell Assay

Overview

Live/Dead Cell assays use fluorescent dyes to differentiate between live and dead cells based on membrane integrity.

Detailed Procedure

1. Dye Addition: Add live/dead dyes to the cell culture.
2. Incubation: Allow cells to incubate with the dyes.
3. Visualization: Examine cells under a fluorescence microscope.

Advantages

• High sensitivity.
• Accurate differentiation between live and dead cells.
• Quantitative.

Limitations

• Requires fluorescence microscopy.
• Can be affected by cell autofluorescence.
• Dyes can be expensive.

5. ATP Assay

Overview

ATP assays measure cellular ATP levels using a bioluminescence reaction involving luciferase and luciferin.

Detailed Procedure

1. Cell Lysis: Lyse cells to release ATP.
2. Reaction Mixture: Mix lysate with luciferase, luciferin, and cofactors.
3. Luminescence Measurement: Measure light emission using a luminometer.

Advantages

• Highly sensitive.
• Direct measure of cellular metabolic activity.
• Minimal sample preparation.

Limitations

• ATP is unstable; requires quick processing.
• Can be affected by interference from other substances.

6. XTT Assay

Overview

The XTT assay uses a colorimetric method to measure cell viability by the reduction of XTT reagent to a colored formazan product.

Detailed Procedure

1. XTT Reagent Preparation: Combine XTT with a co-factor (e.g., PMS).
2. Cell Incubation: Add XTT solution to cells and incubate for 1-4 hours.
3. Formazan Formation: Cells reduce XTT to formazan.
4. Measurement: Measure color intensity using a spectrophotometer at 450-490 nm.

Advantages

• Non-toxic to cells.
• Provides quantitative measurement.
• Simple procedure.

Limitations

• Results can be affected by other substances in the medium.
• Requires a spectrophotometer.

7. WST-8 Assay

Overview

WST-8 is a tetrazolium salt used to measure cell viability by producing a water-soluble orange formazan product upon reduction.

Detailed Procedure

1. Add WST-8: Add WST-8 reagent to cell culture.
2. Incubation: Allow cells to incubate, reducing WST-8 to formazan.
3. Measurement: Measure the color intensity at 450-500 nm using a spectrophotometer.

Advantages

• Highly sensitive.
• Produces water-soluble formazan, no solubilization needed.
• Simple procedure.

Limitations

• May be influenced by other substances.
• Limited to certain cell lines.

8. MTS Assay

Overview

The MTS assay measures cell viability by converting the tetrazolium salt MTS to a purple formazan in the presence of phenazine methosulfate.

Detailed Procedure

1. Cell Treatment: Add MTS reagent to the cells.
2. Incubation: Allow the reaction to proceed, producing purple formazan.
3. Measurement: Measure absorbance at 490–500 nm using a spectrophotometer.

Advantages

• Quantitative measurement.
• Non-toxic to cells.
• Suitable for a wide range of cell types.

Limitations

• Requires a spectrophotometer.
• Similar limitations to MTT regarding solubilization and potential interference.

9. Comparison Table

10. Conclusion

Choosing the right cell viability assay depends on various factors including the type of cells, the need for sensitivity, and the available equipment. Each assay has its own strengths and limitations, so understanding these aspects will help you select the most appropriate method for your research objectives.

Ref:

Ghasemi, M., Turnbull, T., Sebastian, S., & Kempson, I. (2021). The MTT Assay: Utility, Limitations, Pitfalls, and Interpretation in Bulk and Single-Cell Analysis. International Journal of Molecular Sciences, 22(23), 12827.

Madar, I., Sultan, G., Chelliah, R., & Oh, D. (2022). Screening for Anticancer Activity: Trypan Blue Exclusion Assay. In D. Dharumadurai (Ed.), Methods in Actinobacteriology (pp. 59). Humana Press, New York, NY.

Muljadi, M., Lu, T. T., & Cheng, C. M. (2023). Potential Application of the WST-8-mPMS Assay for Rapid Viable Microorganism Detection. Pathogens, 12(2), 343.

 Wang P, Henning SM, Heber D (2010) Limitations of MTT and MTS-Based Assays for Measurement of Antiproliferative Activity of Green Tea Polyphenols. PLoS ONE 5(4): e10202.