Verification and Improvement of the MTT Method for in vitro HSS Bioactivity Activity Determination

Abstract

To optimize the experimental conditions of MTT colorimetric assay for HSS bioactivity in vitro, we studied the optimal combination of the major conditions of the MTT assay by orthogonal test and other experiments, and compared HSS bioactivity in vitro measured by the improved MTT protocol and published MTT assay at serial protein doses. Results showed that the absorbance value (A value) of the MTT assay directly correlated with the number of human hepatoma cell lines SMMC7721. The result of orthogonal test was the number of 5×104 SMMC7721 cells/ml, culture period 6 h before adding HSS, concentration of HSS 100 ?g/ml, incubation time with HSS 36 h. Additionally, several experiments demonstrated the optimal combination of other conditions was 50 ?g MTT, incubation time for MTT 6 h, DMSO was used to dissolve the MTT formazan crystals and measured with ELISA scanner at 570 nm. The result of determining HSS bio-activity in vitro by optimized MTT protocol showed that sHSS bio-activity increased with the growth of protein dose, but decreased when it beyond a certain dose. The optimized MTT protocol was a sensitive, convenient and stable quantitative method to evaluate HSS bio-activity.