OBJECTIVE: The color density of the methyl tetrazolium (MTT) test is proportional to mitochondrial enzyme activity thus reflecting cellular viability. The aim was to evaluate the MTT test as a viability assay for vein homograft studies. MATERIALS AND METHODS: Fresh intact vein samples were harvested during multi-organ procurement. The reliability of the MTT assay was tested by a fluorescent dye combination (1 microg/ml propidium iodide PI and 4 microM/ml SYTO-16 stains). The enzyme kinetics of the reaction was also investigated. The optimal reagent concentration, biopsy size and incubation period were established. RESULTS: There was a linear relationship between the vein homograft's weight and the pigment production activity. A nonspecific reaction (8.6%) was observed in negative controls. The MTT cleavage up to 0.1% (w/v) follows the Michaelis kinetics. The Michaelis constant (2,805 +/- 130 microM), the maximal velocity (196 +/- 2.2 x 10(-5 )microM s(-1)) and the velocity constant (6.98 +/- 0.2 x 10(-7) s(-1)) was calculated. The viability assessed by fluorescent dyes simultaneously visualized the live/dead cell ratio, which can be calculated by image analysis software. CONCLUSION: The use of MTT in colorimetric assays offers high sensitivity. The assay is simple, inexpensive, and reproducible in vein homograft studies. 2004 S. Karger AG, Basel.
OBJECTIVE: The color density of the methyl tetrazolium (MTT) test is proportional to mitochondrial enzyme activity thus reflecting cellular viability. The aim was to evaluate the MTT test as a viability assay for vein homograft studies. MATERIALS AND METHODS: Fresh intact vein samples were harvested during multi-organ procurement. The reliability of the MTT assay was tested by a fluorescent dye combination (1 microg/ml propidium iodide PI and 4 microM/ml SYTO-16 stains). The enzyme kinetics of the reaction was also investigated. The optimal reagent concentration, biopsy size and incubation period were established. RESULTS: There was a linear relationship between the vein homograft's weight and the pigment production activity. A nonspecific reaction (8.6%) was observed in negative controls. The MTT cleavage up to 0.1% (w/v) follows the Michaelis kinetics. The Michaelis constant (2,805 +/- 130 microM), the maximal velocity (196 +/- 2.2 x 10(-5 )microM s(-1)) and the velocity constant (6.98 +/- 0.2 x 10(-7) s(-1)) was calculated. The viability assessed by fluorescent dyes simultaneously visualized the live/dead cell ratio, which can be calculated by image analysis software. CONCLUSION: The use of MTT in colorimetric assays offers high sensitivity. The assay is simple, inexpensive, and reproducible in vein homograft studies. 2004 S. Karger AG, Basel.