MTT Assay

Introduction:
Cell-based assays are often used for screening collections of compounds to determine if the test molecules have effects on cell proliferation or show direct cytotoxic effects that eventually lead to cell death. Cell-based assays also are widely used for measuring receptor binding and a variety of signal transduction events that may involve the expression of genetic reporters, trafficking of cellular components, or monitoring organelle function.

MTT Tetrazolium Assay Concept

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay was the first homogeneous cell viability assay developed for a 96-well format that was suitable for high throughput screening (HTS) (2).
The MTT substrate is prepared in a physiologically balanced solution, added to cells in culture, usually at a final concentration of 0.2 - 0.5mg/ml, and incubated for 1 to 4 hours. The quantity of formazan (presumably directly proportional to the number of viable cells) is measured by recording changes in absorbance at 570 nm using a plate reading spectrophotometer. A reference wavelength of 630 nm is sometimes used, but not necessary for most assay conditions.

Viable cells with active metabolism convert MTT into a purple colored formazan product with an absorbance maximum near 570 nm . When cells die, they lose the ability to convert MTT into formazan, thus color formation serves as a useful and convenient marker of only the viable cells.

Reagent Preparation

MTT Solution

1.

Dissolve MTT in Dulbecco’s Phosphate Buffered Saline, pH=7.4 (DPBS) to 5 mg/ml.

2.

Filter-sterilize the MTT solution through a 0.2 µM filter into a sterile, light protected container.

3.

Store the MTT solution, protected from light, at 4°C for frequent use or at -20°C for long term storage.

Solubilization Solution

1.

Choose appropriate solvent resistant container and work in a ventilated fume hood.

2.

Prepare 40% (vol/vol) dimethylformamide (DMF) in 2% (vol/vol) glacial acetic acid.

3.

Add 16% (wt/vol) sodium dodecyl sulfate (SDS) and dissolve.

4.

Adjust to pH = 4.7

5.

Store at room temperature to avoid precipitation of SDS. If a precipitate forms, warm to 37°C and mix to solubilize SDS.

MTT Assay Protocol

1.

Prepare cells and test compounds in 96-well plates containing a final volume of 100 µl/well.

2.

Incubate for desired period of exposure.

3.

Add 10 µl MTT Solution per well to achieve a final concentration of 0.45 mg/ml.

4.

Incubate 1 to 4 hours at 37°C.

5.

Add 100 µl Solubilization solution to each well to dissolve formazan crystals.

6.

Mix to ensure complete solubilization.

7.

Record absorbance at 570 nm.

References
1-http://www.ncbi.nlm.nih.gov/books/NBK144065/#mttassays.REF.1 
2-Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Meth. 1983;65:55–63.