Digital Image Analysis of Reactive oxygen species and Ca2+ in mouse 3T3 fibroblasts

Recently, analysis of digital images with confocal microscope has become a routine technique and indispensable tool for cell biological studies and molecular investigations. Because the light emitted from the point out-of-focus is blocked by the pinhole and can not reach the detector, thus only an image of the fluorescence from the focal plane is imaged. In present studies, we use the probes 2′, 7′-dichlorof luorescein diacetate (H2DCF-DA) and Fluo-3 AM to research reactive oxygen species (ROS) and Ca2+ in mouse 3T3 fibroblasts, respectively. Our results indicate that the distribution of ROS and Ca2+ were clearly seen in mouse 3T3 fibroblasts. Moreover, we acquired and quantified the fluorescence intensity of ROS and Ca2+ with Leica Confocal Software. It was found that the quantified fluorescence intensity of ROS and Ca2+ was 123.30.26±8.99 and 125.13±12.16, respectively. Taken together, our results indicate that it is a good method to research the distribution and fluorescence intensity of ROS and Ca2+ in cultured cells with confocal microscope.