List of Components

Overview

Assay Principle

Determination of cell growth rates is widely used in the testing of drug action, cytotoxic agents and screening other biologically active compounds. Several methods can be used for such determinations, but indirect approaches using fluorescent or chromogenic indicators provide the most rapid and large scale assays. The non-radioactive, colorimetric assay system using MTT was first described by Mosmann. The assay is designed for the spectrophotometric quantification of cell growth and viability without the use of radioactive isotopes. The MTT assay involves the conversion of the water soluble MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to an insoluble purple formazan crystals. The formazan crystals formed are then solubilized, and the concentration of resulting colored solution determined by optical density at 570 nm. The result is a sensitive assay with excellent linearity up to approximately 10⁶ cells per well.

Important Product Information

• The culture conditions used to grow the cells can affect the results and must be taken into consideration when analyzing the data. The age of the cultures, number of passages and details of the growth medium can all be important factors.

• For Formazan solubilization solution, frozen block or precipitates may form during shipment or storage, in which case the container should be warmed to +37°C and thoroughly mixed.

• After thawing, the MTT labeling reagent may be stored protected from light at 4°C for up to 4 weeks, in which case a sterile filtration of the reagent is recommended.

• The presence of phenol red in the final assay samples can seriously affect results. We recommend that the cells be cultured in medium free of phenol red, if possible.

• MTT is oncogenic. Please wear gloves to operate.

Additional Materials Required

• 10μL, 100-200μL and multi-channel pipettes

• 96 well plate

• CO₂ incubator

• DMSO

• PBS

• Plate reader capable of reading absorbance at 570nm

Assay Protocol

1. Collect cells at logarithmic phase, count the cells.
Note: Add 2000 cells per well for cell proliferation assay and 5000 cells per well for cell cytotoxicity assay. Cells number of each well depends on cell density and growth rate.

2. For adherent cells, remove the medium and replace it with 100 µL of fresh culture medium. For non-adherent cells, centrifuge the 96 well microplate, pellet the cells, carefully remove as much medium as possible and replace it with 100 µL of fresh medium.

3. Add 10 μL of the MTT labeling reagent to each well. Include a negative control of 10μL of the MTT labeling reagent added to 100 µL of medium alone.

4. Incubate the microplate at 37°C for 4 hours in a humidified chamber (5% CO₂ ). At high cell densities (>100,000 cells per well) the incubation time can be shortened to 2 hours.

5. Add 100 μL of Formazan solubilization solution to each well and mix thoroughly.

6. Incubate the microplate at 37°C for 4-18 hours in a humidified chamber (5% CO₂ ).

7.Check for complete solubilization of the purple formazan crystals and measure the spectropho- tometrical absorbance of the samples using a microplate reader at 570 nm.
Note: The wavelength between 560 and 600 nm can be chosen if 570nm is unavailable.

Quick Protocol Option

To shorten the time of the assay it is possible to use DMSO (not provided) as a solubilizing agent to dissolve the formazan.

1. After labeling the cells with MTT, as described above, remove all but 25 µL of medium from the wells. For non-adherent cells it may be necessary to first centrifuge the plates to sediment the cells.

2. Add 50 µL of DMSO to each well and mix thoroughly with the pipette.

3. Incubate at 37°C for 10 minutes.

4. Check for complete solubilization of the purple formazan crystals and measure the spectropho- tometrical absorbance of the samples using a microplate reader at 540 nm.

MTT Cell Proliferation and Cytotoxicity Assay Kit Images

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Quantitation of U937 cells using the MTT Cell Proliferation Assay Kit. Cells in the parent culture were counted in a hemacytometer and then diluted to the indicated cell numbers in 100 µL volumes, delivered to the wells of a microplate and incubated for 4 hours to allow time for adsorption before being assayed. Absorbance measurements at 570 nm were made using a microplate reader. Each data point represents the mean value of samples in triplicate.