Comparison of the Influence of Hoechst 33342 and DAPI on the Level of Intracellular Reactive Oxygen Species

Aim To investigate the influence on the level of intracellular reactive oxygen species( ROS) by staining the cell nuclei using two fluorescent dyes —Hoechst 33342 and DAPI,respectively. Methods Vascular smooth muscle cells( VSMC) were stimulated with advanced glycation end products( AGE) for 10 minutes and then incubated with 2',7'-dichlorofluorescin diacetate( H2DCFDA). After that,cell nuclei were stained with Hoechst 33342 and DAPI respectively. And through the analysis of the number of labeled nuclei and the level of intracellular ROS by fluorescence microscopy,the fluorescence intensity of intracellular ROS were detected by staining with two different fluorescent dyes. Results After staining with Hoechst 33342 for 5 min,cell nuclei were labeled immediately and the number of them did not change with the increase of staining time. However,there were only a few cell nuclei could be labeled when the cells were stained with DAPI for 5 min,with the increase of staining time,more and more cell nuclei could be labeled. Surprisingly,the fluorescence intensity of Hoechst 33342 group showed no significant differences staining at 5,10 and 20 min. However,with the increase of staining time,the more the cell nuclei were labeled with DAPI,the less the fluorescence intensity of ROS was. These results suggest that DAPI damages the storage of intracellular ROS during the nuclei staining, thus disturbing the experimental results. Conclusion Cell nuclei should be stained with Hoechst 33342 not DAPI during the detection of intracellular ROS.