Determining cell numbers is a basic procedure used widely in laboratories for testing the effects of growth stimulating factors, cytotoxic agents and drugs on cell growth. There are several assays and kits available for determining cell numbers. Of these, we have used the Vybrant® MTT Cell Proliferation Assay Kit from Molecular Probes (Invitrogen) for the past 3 years.
The basic principle of the assay involves the conversion of the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) to formazan crystals by viable cells in culture. The formazan crystals are solubilized by the addition of SDS-HCL and the resultant color is measured in a spectrophotometer at 570nm. The absorbance correlates directly with cell number.
The Vybrant MTT Cell Proliferation Assay Kit comes with 10 vials of MTT reagent, each containing 5g of MTT, and 10 vials of SDS, each containing 1g of SDS. Basic reagents such as HCl, DMSO and PBS are to be provided by the user. The MTT reagent needs to be re-constituted from solid state by dissolving 5g in 1ml of PBS. We have encountered a problem with dissolving and occasionally had to filter the solution to get rid of the undissolved reagent. This may lead to compromising the exact concentration of MTT in solution. However, this did not seem to affect our final result. We were able to verify this by seeding a linear range of known numbers of cells in 96 well plates and testing the linearity. Additionally, the reagent is stable for only a month after reconstitution and therefore at times there is wastage of reagent. Further, SDS solution in 0.01M HCl needs to be prepared freshly each time.
The procedure for the assay itself is reasonably simple. The assay can be used to determine cell growth in both adherent and non-adherent cell cultures. It is best to use media without phenol red to minimize interference. The cells are grown in 96 well plates to which 10ul of MTT reagent is added. It is important to have a negative control with just the media and no cells to eliminate background color. The cells are incubated with MTT agent for 4h and 100ul of SDS-HCl is added to each well. The cells are then incubated overnight at 37°C in a humidified chamber. The samples are then mixed and absorbance read at 570nm.
The protocol suggests adding DMSO to the cell cultures after MTT incubation to shorten the time of the assay. After 10 min incubation with DMSO, the absorbance can be read at 540nm. Although theoretically this procedure appears feasible, we have not been able to get consistent results with this procedure. We have used MTT assay for primary cell cultures such as neural stem and progenitor cells as well as secondary cancer and tumor cell lines. We have been very successful in getting accurate and consistent results by using the overnight incubation method.
In summary, the Vybrant MTT Cell Proliferation Assay Kit from molecular probes is a very convenient tool for determining the cell numbers in a variety of cell cultures.