摘要
目的探讨Hoechst 33342与DAPI两种荧光染料标记细胞核后对细胞内活性氧水平的影响。方法静息培养的血管平滑肌细胞加入晚期糖基化终末产物作用10 min,加入标记活性氧的荧光探针H2DCFDA,再分别加入Hoechst 33342和DAPI不同荧光染料进行核标记。荧光显微镜下观察细胞核被标记的数目与细胞内活性氧的荧光水平。结果 Hoechst 33342染料标记5 min后即可见细胞核被标记上,随着时间的延长被标记的核数目并不发生改变;而与之明显不同的是,DAPI染料标记5 min时,只有几个细胞核被标记上,但随着时间的延长被标记的核数目越来越多。Hoechst 33342标记后细胞内活性氧的荧光强度并不随时间的延长发生变化,而DAPI标记后细胞内活性氧绿色荧光的细胞数就越少,DAPI标记的细胞核数与显示活性氧绿色荧光的细胞数呈反比。这些结果提示,DAPI染料在标记细胞核时破坏了活性氧在细胞内的储存,干扰了实验结果。结论检测细胞内活性氧时,应使用Hoechst 33342核标记染料而不能用DAPI。
Aim To investigate the influence on the level of intracellular reactive oxygen species （ROS） by stai- ning 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 in tensity 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 dur ing the detection of intracellular ROS.
出处
《中国动脉硬化杂志》
CAS
CSCD
北大核心
2014年第1期75-78,共4页
Chinese Journal of Arteriosclerosis
基金
国家自然科学基金(30871023和81070124)
广东省自然科学基金(S2012010009199)
高等学校博士学科点专项科研基金(20090171110049)
