Detection of ROS Induced by Light Irradiation in Endothelial Cell During Light Irradiation Applying H_2DCF DA∶A Preliminary Study

Objective To detect the reactive oxygen species generated during light irradiation in endothelial cells(ECV 304) with fluorescent microscope and H2DCF DA, which is widely used as fluorescent probe in biomedical study Methods ECV 304 cells were subcultured for 24 hours, and H2DCF DA was added into it for 30 minutes with the final concentration of 10μmol/L The procedure of light irradiation was carried out during collecting the fluorescent image of DCF The wavelength range of the light irradiation was 460 490nm and the power density was about 100 mW/cm2 The sequential images were obtained by using fluorescent microscope The mean fluorescence intensity of DCF in cells and its variety with time were calculated by image analyzing and processing technique In another group the fluorescence images of MitoFluor Red 589 were collected, and the distributed site of DCF was determined by comparing the fluorescence images of MitoFluor Red 589 and DCF of the same cell Results The fluorescence intensity of DCF in ECV 304 cell increased rapidly during the early stage of light irradiation, and the increase of it became weak with the prolongation of irradiation time Moreover, the fluorescence in ECV 304 cell distributed mainly in mitochond ria The values of I1/I2 in mitochondria region,nucleus region and non mitochondria cytosol region were as follows: 1 9490±0 2209,1 1128±0 2256,0 9413±0 0853 Conclusions It is simple and reliable to detect the intracellular reactive oxygen species applying fluorescence technique and image analyzing and processing technique The light irradiation induced reactive oxygen species distributed mainly in mitochondria, which may be associated with its abundant endogenesis photosensitizer But we can not absolutely exclude the possibility that the property of this probe itself induces the above mentioned phenomena