A rapid and accurate 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetric assay for quantification of bacteriocins with nisin as an example

The objective of this study is to propose a more accurate and faster MTT 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetric assay (MCA) for quantitative measurement of polypeptide bacteriocins in solutions with nisin as an example. After an initial incubation of nisin and indicator bacterium Micrococcus luteus NCIB 8166 in tubes, MTT was added for another incubation period. After that, nisin was quantified by estimating the number of viable bacteria based on measuring the amount of purple formazan produced by cleavage of yellow tetrazolium salt MTT. Then MCA was compared to a standard agar diffusion assay (ADA). The results suggested a high correlation coefficient (r2=0.975±0.004) between optical density (OD) and the inhibitory effect of nisin on a bacterial strain Micrococcus luteus NCIB 8166 at a range of 0.125～32 IU/ml. The MCA described in this study was very quick. Quantification of nisin took only 7～8 h and the detection limit was at the level of 0.125 IU/ml when compared to 12 IU/ml and 24～28 h for ADA. The MCA provides an accurate and rapid method for quantifi-cation of nisin in solutions and is expected to be used for quantification of other antimicrobial substances.

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

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 100μg/ml,incubation time with HSS 36 h.Additionally,several experiments demonstrated the optimal combination of other conditions was 50μg 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.

The feasibility of antitumor drugs chemosensitivity testing by flow cytometry

Objective: To investigate the feasibility of chemosensitivity testing of antitumor drugs by flow cytometry in clinical applications so as to provide experimental and theoretical basis for the establishment of a novel antitumor drugs sensitivity testing and the screening of particular antitumor drugs. Methods: Detect the apoptosis rate of 12 cases of Molt-4 cell line, 57 cases of fresh clinical gastrointestinal tumor cells by Sub-G1 and Annexin V assay of flow cytometry under the effects of antitumor drugs at different times and the outcomes were compared with the ones of the MTT (3-(4,5-dimethylthiazolyl-2) -2,5-diphenyltetrazolium bromide) assay. Results: The lethality of drugs on Molt-4 cell, clinical gastrointestinal tumor cells had a positive correlation with the acting time of antidrugs by employing Annexin V, Sub-G1 and MTT assay. Drug-incurring maximum lethality of Annexin V assay was higher than MTT colorimetric assay, that of Sub-G1 was lower than MTT assay, the virtual times of Annexin V and Sub-G1 assay were obviously earlier than that of MTT colorimetric assay. Conclusion: Annexin V and Sub-G1 assay of flow cytometry can be taken as potent protocols testing anti-tumor drug chemosensitivity. Annexin V assay is featured by more sensitive, concise, reliable compared with the classical chemosensitivity testing assay of MTT colorimetric assay and it possesses clinical applied value.