Flow cytometric analysis of cell suspensions exposed to shock waves in the presence of the radical sensitive dye hydroethidine

Abstract

The occurrence of intracellularly and extracellularly generated free radicals during shock wave exposure on an experimental Siemens lithotripter was tested with the radical sensitive dyes hydroethidine and dichlorofluorescin (DCFH). DCFH, a nonfluorescent compound, is oxidised to dichlorfluorescein (DCF) by hydrogen peroxide in the presence of peroxidase. DCF green fluorescence intensity was used for fluorescence spectrometric measurement of hydrogen peroxide generated during shock-wave treatment of cell-free dye solutions. The fluorescence intensity of ethidium, the oxidised form of hydroethidine, was used for the flow-cytometric measurement of intracellular oxidising reagents present in RT4 tumour cells during shock-wave exposure. Changes in membrane permeability, which influence the intracellular content of ethidium, were controlled by counterstaining the cells with propidium iodide, an indicator for membrane integrity. We observed no increase in intracellular ethidium fluorescence intensity after shock-wave treatment of single cell suspensions and therefore no indication for shock-wave-induced intracellular free radicals.