Catalog No. 30-1010K

Features
Background
FAQ
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Product Instruction Manual

The ATCC MTT Cell Proliferation Assay offers a quantitative, convenient method for evaluating a cell population's response to external factors, whether it be an increase in cell growth, no effect, or a decrease in growth due to necrosis or apoptosis.

Features

• Proven technology. The utility of the MTT method has been documented in the literature for many different applications.
• Accurate measurements. The spectrophotometric procedure can detect slight changes in cell metabolism, making it much more sensitive than trypan blue staining.
• Safer reagents. There's no need to store or manipulate radioactive substances.
• Easy to use. The procedure is relatively simple and uses equipment already available in most labs.
• Rapid processing. Assays are run in a 96-well plate and read with a microtitre plate reader, allowing high-throughput handling of samples.
• Convenient storage. The kit is stable for 18 months when stored under refrigeration in the dark.

Background

The measurement of cell viability and growth is a valuable tool in a wide range of research areas. Several approaches have been used in the past. Trypan blue staining is a simple way to evaluate cell membrane integrity (and thus assume cell proliferation or death) but the method is not sensitive and cannot be adapted for high-throughput screening. Measuring the uptake of radioactive substances, usually tritium-labeled thymidine, is accurate but it is also time-consuming and involves handling of radioactive substances.

The reduction of tetrazolium salts is now recognized as a safe, accurate alternative to radiometric testing. The yellow tetrazolium salt (MTT) is reduced in metabolically active cells to form insoluble purple formazan crystals, which are solubilized by the addition of a detergent. The color can then be quantified by spectrophotometric means. For each cell type a linear relationship between cell number and absorbance is established, enabling accurate, straightforward quantification of changes in proliferation. Among the applications for the method are drug sensitivity, cytotoxicity, response to growth factors, and cell activation.

References
van de Loosdrecht, A.A., et al. J. Immunol. Methods 174: 311-320, 1994.
Ohno, M., and T. Abe. J. Immunol. Methods 145:199-203, 1991.
Ferrari, M., et al. J. Immunol. Methods 131: 165-172, 1990.
Alley, M.C., et al. Cancer Res. 48: 589-601, 1988.
Carmichael, J., et al. Cancer Res. 47:936-42, 1987.
Gerlier, D., and N. Thomasset. J. Immunol. Methods 94: 57-63, 1986.
Mosmann, T. J. Immunol. Methods 65: 55-63, 1983.

1. How does the MTT Cell Proliferation Assay work?

   The MTT Cell Proliferation Assay is a colorimetric assay system which measures the reduction of a tetrazolium component (MTT) into an insoluble formazan product by the mitochondria of viable cells. After incubation of the cells with the MTT reagent for approximately 2 to 4 hours, a detergent solution is added to lyse the cells and solubilize the colored crystals. The samples are read using an ELISA plate reader at a wavelength of 570 nm. The amount of color produced is directly proportional to the number of viable cells.

    
2. What does the MTT system do that trypan blue cannot?

   The MTT system is a quantitative, more sensitive test. Because there is a linear relationship between cell activity and absorbance, the growth or death rate of cells can be measured; the trypan blue test is qualitative and indicates only if a cell is alive. The MTT assay can also be adapted to high-throughput screening, whereas trypan blue tests must be read individually.

    
3. How long does it take to develop purple formazan precipitate?

   We recommend 2 to 4 hours as a starting point. This will vary somewhat among different cell types; there may be some cells that require up to 24 hours. The purple formazan has to be visible inside the cells before the detergent reagent can be added.

    
4. How many cells are required to obtain an efficient reading with the MTT Cell Proliferation Assay?

   For most tumor cells, hybridomas, and fibroblast cell lines, we recommend 5,000 cells per well to perform proliferation assays, although as few as 1,000 cells per well have been used successfully. The known exceptions are blood lymphocytes, which require approximately tenfold more cells (25,000-250,000 cells/well) to obtain a sufficient absorbance reading.

    
5. Are there cell types which will not work with the MTT Cell Proliferation Assay?

   Cells with functional mitochondria are needed to convert the tetrazolium dye into its reduced form. Most eukaryotic cells in culture, including mammalian (suspension and adhesion), plant, and yeast cell types, reduce the dye sufficiently to perform accurate assays.

    
6. How do MTT Cell Proliferation Assay results compare to [3H]thymidine incorporation assays?

   Because the MTT Cell Proliferation Assay requires less cell manipulation than [3H]thymidine incorporation assays (no cell harvesting or medium changes are necessary), the possibility of error is reduced and the standard deviation values are lower. Comparisons between [3H]thymidine incorporation and MTT assays have demonstrated less than 5% difference for determination of growth factor response.

    
7. Can the MTT Cell Proliferation Assay directly replace [3H]thymidine incorporation assays?

   Yes, the addition of dye solution can be substituted at the point in the assay when radioactive thymidine is added.

    
8. Can the MTT Cell Proliferation Assay be used for cytotoxicity studies?

   Yes, MTT Cell Proliferation Assay can be used to study cell death mediated by a cytotoxic agent.

    
9. How do I store the reagents?

   The MTT Reagent must be kept at 4C in the dark. The Detergent Reagent can be stored at either 4C or ambient temperature. If the detergent reagent is kept at 4C, warm the bottle for 5 minutes at 37C and gently mix by inverting before use (avoid creating bubbles).

    

You can order the MTT Cell Proliferation Assay from our online catalog if you have an ATCC account. Or call 800-638-6597 in the United States, Canada, and Puerto Rico or 703-365-2700 elsewhere. Customers in Europe, Australia, Hong Kong, India, Japan, Korea, New Zealand, Singapore and Taiwan, R.O.C. must order from our official distributors.

This product is intended for research purposes only. It is not intended for use in humans.

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Frequently Asked Questions