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Comparative Analysis of Cell Viability Assay Methods: Choosing the Right One for Your Research

In-vitro cell viability assessment is now a routine approach to address several biological questions in biomedical research. Cell viability assays can screen compounds and determine whether these molecules can affect cell proliferation or exhibit cytotoxicity. Depending on the cell population and their number, selecting a proper assay method can help acquire the desired results. 

Multiple cell viability and cell proliferation assays are available to study cellular health. These assays can be cell-based screening assays or cell-based functional assays. Besides, assay development and HPLC testing services are focusing on offering solutions for cell viability assays. 

Choosing a cell viability assay from different alternatives can be challenging. Determining the best cell viability method that can suit individual research needs requires an in-depth understanding of each assay type and its associated marker, the measurement that correlates with cell viability, and the limitations of individual assay chemistries. Additionally, researchers also have the option of multiplexing compatible assays to generate more data and have a statistical edge. The current article compares several cell viability assay methods to help researchers choose the right one for each research. 

Choosing an ideal cell viability assay method

MTT tetrazolium assay

The MTT tetrazolium assay is the first cell viability assay suitable for high throughput screening. The enzyme NADPH-dependent cellular oxidation in viable cells can reduce the dye MTT into its insoluble component having a purple color. This insoluble component, formazan, can be measured at 570 NM. This assay can measure viable cells, cytotoxicity, and cytostatic activity of toxic materials or medicinal agents. 

MTS tetrazolium assay

MTS, a new tetrazolium salt, is readily reduced by a viable cell population to produce formazan products. These products are soluble in cell culture medium. This assay protocol requires adding the tetrazolium reagent to the culture medium and reading the absorption of the generated product. The primary benefit of this method is that it does not require a liquid handling step, which saves time and reduces errors such as cell loss. 

CCK 8 (WST-8) assay

WST-8 is a second-generation tetrazolium salt with better solubility than MTS. Dehydrogenases in cells reduce WST-8 into an orange-colored product that is soluble in tissue media. The generated product is directly proportional to viable cell count. 

Sulforhodamine B colorimetric assay

The Sulforhodamine B (SRB) assay is one of the earliest techniques for in vitro cytotoxicity testing. This assay is based on the SRB’s ability to bind to cellular protein components. SRB contains two sulfonic groups that can bind to the amino acid residues of a cell in mildly acidic conditions while dissociating in basic conditions. Researchers use the dye amount obtained from stained cells as a proxy for cellular mass. This amount of dye is directly proportional to the cellular mass. 


Today, multiple alternatives are available to plan and design cellular health experiments. Choosing a cell viability assay can be challenging. However, adequate research and information about cell-based assays and the intended cell population may prove beneficial in identifying the right cell viability assay method.



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