Step-by-Step Guide to Performing a Cell Viability Assay

Importance of a Cell Viability Assay 

Cell viability is the number of healthy cells in a given cell population. Most importantly, it indicates the cell health and the response of cells to external stimuli and cytotoxic chemicals. Cell viability assay helps to understand the mechanism underlying cell survival, proliferation, and death. In drug development, cell viability assays help to assess the in vitro drug cytotoxicity. Regardless of the method employed, cell viability is the ratio of viable and dead cells within a cell population.

Choosing the Assay

Different cell viability assays include Luminescent cell viability assay, MTT assay, Luminescent ATP assay, and Caspase assay, among others. Cell viability assay, cell proliferation assay, or cell cytotoxicity assay use different markers that indicate the presence of healthy and living cells. The MTT assay, also known as the 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide assay, is highly sensitive and reliable compared to other cell-based functional assays. MTT assays help to evaluate cellular metabolic activity, which consequently helps to assess cell viability, cell proliferation, and cytotoxicity. The MTT assay is considered a gold standard cell-based screening assay in drug screening.

Step-by-Step Guide

This section offers a step-by-step guide to the MTT assay, as detailed below.

The first step involves the preparation of an MTT solution using the phosphate-buffered saline (PBS) solution at a recommended strength of 5 mg/mL. The MTT solution is commercially available in optimized MTT assay kits but can also be prepared fresh in an assay lab.

The next step involves plating cells at 103 to 105 cells/well in a culture medium and incubating the microtiter plate for 6 to 24 hours at 37 °C and 5% CO2. 

After adding 10 µL MTT reagent solution to each well, the microtiter plate is incubated in a humidified environment at 37 °C and 5% CO2 for 2 to 4 hours. The formation of a purple-colored precipitate due to formazan crystals occurs.

After adding 100 µL of the solubilization or detergent solution provided in the commercially available MTT kit to each well, the microtiter plate is incubated for overnight.

After ensuring the complete solubilization of the purple precipitate, a microtiter plate reader is used to measure the absorbance of the sample between 550 and 600nm based on the wavelength filters available. Record the measurements in duplicates or triplicates and report the average value.

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The elimination of background absorbance will need the measurement of blank wells containing only the culture medium and the subtraction of the absorbance value of the blank from the sample measurements.

A standard curve of absorbance (within the range of 0.75 to 1.25) versus known cell concentrations may be established for cell counting. For this purpose, plate serial dilutions of cells in the culture medium can be plated out into the microtiter plate wells in triplicates. The average absorbance value corresponding to each known cell concentration is plotted against the number of cells/mL to obtain a standard linear plot. From the standard plot, determine the cell counts or cell viability of a sample treated with a cytotoxic agent or any chemical from its corresponding absorbance value.