The Optimal Condition of Performing MTT Assay for the Determination of Radiation Sensitivity

Hong, Semie; Kim, Il Han

Abstract

PURPOSE: The measurement of radiation survival using a clonogenic assay, the established standard, can be difficult and time consuming. In this study, We have used the MTT assay, based on the reduction of a tetrazolium salt to a purple formazan precipitate by living cells, as a substitution for clonogenic assay and have examined the optimal condition for performing this assay in determination of radiation sensitivity.
MATERIALS AND METHODS
Four human cancer cell lines-PCI-1, SNU-1066, NCI-H630 and RKO cells have been used. For each cell line, a clonogenic assay and a MTT assay using Premix WST-1 solution, which is one of the tetrazolium salts and does not require washing or solubilization of the precipitate were carried out after irradiation of 0, 2, 4, 6, 8, 10 Gy. For clonogenic assay, cells in 25 cm2 flasks were irradiated after overnight incubation and the resultant colonies containing more than 50 cells were scored after culturing the cells for 10~14 days. For MTT assay, the relationship between absorbance and cell number, optimal seeding cell number, and optimal timing of assay was determined. Then, MTT assay was performed when the irradiated cells had regained exponential growth or when the non-irradiated cells had undergone four or more doubling times.
RESULTS
There was minimal variation in the values gained from these two methods with the standard deviation generally less than 5%, and there were no statistically significant differences between two methods according to t-test in low radiation dose (below 6 Gy). The regression analyses showed high linear correlation with the R2 value of 0.975~0.992 between data from the two different methods. The optimal cell numbers for MTT assay were found to be dependent on plating efficiency of used cell line. Less than 300 cells/well were appropriate for cells with high plating efficiency (more than 30%). For cells with low plating efficiency (less than 30%), 500 cells/well or more were appropriate for assay. The optimal time for MTT assay was after 6 doubling times for the results compatible with those of clonogenic assay, at least after 4 doubling times was required for valid results. In consideration of practical limits of assay (12 days, in this study) cells with doubling time more than 3 days were inappropriate for application.
CONCLUSION
In conclusion, it is found that MTT assay can successfully replace clonogenic assay of tested cancer cell lines after irradiation only if MTT assay was undertaken with optimal assay conditions that included plating efficiency of each cell line and doubling time at least.