A new approach for measurement of cytotoxicity using colorimetric assay

Using in vitro established tumour cell lines attempts were made to assess the suitability of tetrazolium salt reduction (MTT) assay to replace the conventional radioactive base techniques for measuring cell proliferation and cell killing. The optimum conditions for MTT loading time, concentration of MTT and the time for colour development were found to be 4 h, 5 mg/ml and 30 min respectively, conditions which were used for subsequent experiments. Analysis of the correlation between increasing cell numbers and optical densities (OD) showed a direct relationship with correlation of coefficient values of r > 0.98 and 10,000 cells/well was found to provide an accurate ODs for a wide variety of cell types. The accuracy of replicate readings of the assay was investigated by setting a wide range of cell numbers and the variation among seven replicates was calculated and found to be less that 6% of the mean values. The reproducibility of the assay for two cell lines was tested using the lines on four different occasions. The ODs for Jar and Fen cell lines were 0.80 +/- 0.01, 0.82 +/- 0.02, 0.90 +/- 0.02, 0.79 +/- 0.05 and 0.56 +/- 0.01, 0.58 +/- 0.03, 0.60 +/- 0.02 and 0.61 +/- 0.02 respectively giving maximum variation of less than 11% of mean on repeated testings. Comparison between the results of MTT with 3H-Tdr or 51Cr release assays showed a high degree of correlation over a wide range of cell numbers and cell types. The r values between the results of MTT with 3H-Tdr (for cell number ranging from 1.8 to 60 x 10(3)/well) or 51Cr release assays (for E/T ratios of between 5:1 and 40:1) were 0.89 (p = 0.001) and 0.96 (p < 0.03) respectively. These results demonstrate that it is possible to use the MTT assay interchangeably with radioactive base techniques to measure cell proliferation and cytotoxicity. The ease of its execution, safety and its suitability for analysing as few as 3000 cells makes this method a serious contender for replacing the conventional radioactive techniques.