Measurement of peroxidase activity in single neutrophils by combining catalyzed-enzyme reaction and epi-fluorescence microscopy

An epi-fluorescence microscopy for determination of peroxidase in individual neutrophils was developed by a combination of enzyme-catalyzed reaction and fluorescence detection. In this method, an individual cell was transferred into a microliter-volume vessel and lysed by freeze-thawing and ultrasonication. The peroxidases-catalyzed reaction was initiated by adding the buffer solution containing nonfluorescent substrates 10-acetyl-3,7-dihydroxyphenoxazine and H2O2 to the vessel. Peroxidase activity could be determined via measuring the fluorescent signal of the product resorufin of enzyme-catalyzed reaction. When the slope of kinetic curve of the enzyme-catalyzed reaction was used to quantify peroxidases in single cells, the effect of the time difference between each measurement and the interference from other intracellular compounds that could emit fluorescence can be eliminated.