Cell Growth Determination Kit, MTT Based From Sigma

The Cell Growth Determination Kit from Sigma is a colorimetric method for determining the number of viable cells in culture and is useful in the measurement of cell growth in response to mitogens, antigenic stimuli, growth factors and other growth promoting reagents; it is also useful in cytotoxicity studies and the derivation of cell growth curves. The kit comes with enough reagents to perform 500 assays in 96-well format. However, the reagents are supplied in such a way that you can perform assays in any well format. There are only two reagents supplied in the kit. The first reagent is the MTT solution which comes as 5 mg/ml MTT in RPMI-1640 without phenol red; it is supplied in 1 ml aliquots (5 total). The second reagent is the MTT solvent: 50 mls of 0.1 N HCl in anhydrous isopropanol. It should be noted that the MTT solution may be harmful if swallowed, inhaled or absorbed through skin. MTT may alter genetic material. MTT solvent is flammable and corrosive.

The assay is based upon the use of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) which is reduced to form formazan crystals (not soluble in aqueous solutions) on the outside of the cell (reduction is based upon the activity of mitochondria in the cells). The MTT solution is added to the cells (10% of total volume) and after a period of 2-4 hours, the media is removed and replaced with the acidified isopropanol (which dissolves the crystals) and then simply read at 570 nm. An increase in absorption corresponds to an increase in cell number (or cell growth) whereas a decrease indicates senescence (or lack of growth). This kit does not indicate apoptosis and as such a separate kit must be used to determine apoptosis.

The biggest problem I have noticed with this kit is that the crystals tend not to stick and can actually be washed away when pipetting the media off the cells before adding the acidified isopropanol. As a result, I have seen significant variation between replicate samples. Sigma does offer the suggestion that instead of removing the media, the solvent can be added directly to the media (use solvent in direct proportion to the volume of media in the wells). However, I have had problems with the crystals completely dissolving.

I don’t like doing the multiple pipetting steps, nor do I like constantly having to check to make sure all crystals are dissolved. As such, I have actually switched kits to a kit from Promega (CellTiter 96 AQueous One Solution Cell Proliferation Assay ) which is based similarly to this one except that the formazen is soluble in media and as a result, there is no need to do extra pipetting steps which can lead to errors if close attention to detail is not employed.

Some other tips that I have found useful for this kit as well as the Promega kit are that you should use phenol red free media to reduce background and you should use a trustworthy microplate reader that it is calibrated/up to date. I have also found it very useful to subtract background at 630 nm as this corrects for background caused by deficiencies in the plastic or by fingerprints.