What is MTT Assay? - with Principle, Protocol, and Result

There are several assays for the determination of cell viability and cytotoxicity. Some of the assays that use drug exposure, irradiation etc. do not manifest the immediate result; instead, it may take several hours or sometimes even days to acquire results. In such a scenario, MTT, which is a survival assay that delivers instant results based on retention of regenerative capacity or reproductive integrity, are recommended.

Among them, the MTT assay is very popular and has been widely adopted in academic laboratories. The MTT assay stands for tetrazolium salt assay, which is a colourimetric method for accessing cell viability, cell proliferation, and cytotoxicity.

What is an MTT Assay?

The full form of MTT is (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide). It is a tetrazolium salt that is yellow in colour. This assay relies on the conversion of this MTT dye into its insoluble form named Formazan by metabolically viable cells. The result is very evident when the yellow colour MTT dye changes its colour to Violet or purple.

The colour change is only possible when the cells are alive. Mitochondrial oxidoreductase is the enzyme required for this conversion process and comes from the mitochondria of viable cells. If the cells are not alive, enzyme release by the mitochondria automatically stops. And this causes no conversion of MTT into formazan. Hence, you can assess whether the cells are alive or not.

Application of MTT Assay

Used for assessing the viability of cells in culture.
Also, it helps in determining cytotoxicity (loss of viable cells) after treatment of therapeutic agents.
Used in analysing cytostatic activity, i.e., whether the cells are shifting from proliferation to quiescence, to check the aftereffects of any therapeutics drugs.

Principle of MTT Assay

The underlying principle behind this assay is the ability of NADPH-dependent cellular oxidoreductase enzyme secreted by the mitochondria to convert the tetrazolium dye into insoluble formazan crystals. The colour changes from yellow to purple to assure the viability of cells.

The viability of the cell is reflected by the activity of mitochondria. Thereby any increase or decrease in active cell number indicated by the colour change is detected by measuring the concentration of formazan.

The conversion is actually due to the reduction of MTT into insoluble formazan crystals. The colour change from yellow to purple is because of the reduction reaction by the enzyme. The darker the colour of the solution, the higher the number of metabolically viable/active cells.

MTT Assay Protocol

1. Cell Seeding

The MTT assay begins with seeding the cells into 96-well microtiter plates (100 µl/well) from a specific cell line. Later, keep this plate in the incubator at 37^o C for 24 hours.

2. Addition of MTT

The next day, take out the plate and add 20 ul of 5 mg/ml MTT reagent to each well.

Preparation of MTT solution: MTT is a powdered salt. Thus, for preparing the MTT reagent, it is mixed in phosphate buffer saline as per the required concentration.

Note: MTT is a light-sensitive reagent. Thus, the reagent bottle is kept in the dark, and it is preferred to perform the assay in dark settings.

3. Incubation

Incubate the MTT added sample plate for 4 hours at 37^oC.

4. Removal of Medium

Carefully remove the medium along with the MTT.

5. Addition of DMSO

Now, add 200 ul of DMSO to each well of the sample plate.

6. Incubation

Incubate the plate for 2 hours at 37^o.

7. Result Analysis

Note down the reading of the sample plate after measuring the OD at 590 nm using a multi-well spectrophotometer.

MTT Cell Viability Assay

Mitochondrial oxidoreductase is an NADPH-dependent enzyme which remains in the cytosolic compartments of the cells. Thereby, the reduction reaction of MTT relies on the cellular metabolic activity of NADPH flux.

The cells like thymocytes and spherocytes have low metabolic activity rates, due to which the NADPH flux inside these cells is also low. As a result, these cells manifest very little MTT reduction.

In contrast, those cells with high metabolisms, such as rapidly dividing cells, exhibit higher rates of MTT reduction. Thus, one should keep in mind that the assay conditions can get altered by the rate of metabolism and can affect the cell viability count.

MTT Assay Result

To check the viability, we use the concentration of formazan crystals as the indicator. The darker the solution, the more the number of viable cells.

Before analysing the results for an unknown sample, a standard graph is plotted with a known number of cells and their corresponding optical density. The graph contains OD or absorbance at its Y-axis while number of cells at the X-axis. The wavelength for the MTT assay is 560-590 nm. However, 590 nm is preferred.

After plotting the standard graph, the absorbance of the unknown sample is plotted as per the obtained OD. From that point, extrapolate the line to join the X-axis for obtaining the number of cells.

After analysis of the cell number, you can also determine the % viability of alive cells and can exhibit the data in terms of % viability and treatment conditions (control and treated).

MTT Assay for Cytotoxicity

The most general purpose of performing MTT is to estimate the number of viable cells without the need for elaborate cell counting techniques. But this assay is also preferred to check and determine the cytotoxicity of therapeutic drugs and other chemical agents at different concentrations.

Let us understand this with an example.

If suppose you are to check the anti-cancerous activity of a particular drug. For this, you take cells from a cancer cell line and treat these cells with the desired drug.

Take two settings:

Control: Doesn’t contain drug
Treated: Containing drug

Now incubate the cells overnight for drug action.
Perform MTT assay with both settings on the next day.
Take the readings at 590 nm with a spectrophotometer.

If the drug actually possesses anticancer properties, then the reading of control will be more with a darker shade of purple. Whereas the treated one will have a reduced % of cell viability with a lighter intensity.From the above hypothetical example, you might understand how MTT assay can also determine cytotoxicity.

Limitations

It has low sensitivity as it can easily get altered by the metabolic activity of cells.
Chemical interference can hamper the end results.
Development of toxins in samples.
Problems in multiplexing