线粒体荧光标记的单颗粒水平高通量评估方法

摘要

Mitochondria play a central role in the regulation of energy metabolism and signal transduction in eukaryotic cells. Although many fluorescent labeling strategies have been developed for mitochondrial studies, the methods that enable labeling efficiency assessment at the single-mitochondrion level are still lacking. By employing the unique advantages of high sensitivity flow cytometry ( HSFCM ) in the sensitive, rapid, and quantitative multiparameter analysis of individual mitochondria, here we examined the performance of several different mitochondrial labeling strategies from the perspectives of brightness, labeling ratio, and stability. Mitochondria isolated from HeLa cells transfected with pAcGFP1-Mito plasmid upon transient or stable transfections, and mitochondria directly labeled with MitoTracker Green or SYTO 62 were analyzed by a laboratory-built three-channel HSFCM. Upon the quantitative measurement of fluorescence brightness, it was found that the fluorescence intensity of green fluorescent protein ( GFP ) in mitochondria isolated from cells with stable transfection was about 17. 7-fold higher than the transient transfection ones, and was approximately two orders of magnitude brighter than mitochondria labeled with MitoTracker Green. On the other hand, the fluorescence signal of SYTO 62 labeling decreased upon washing, indicating its rapid dissociation rate. The strong fluorescence intensity and good labeling stability make stable transfection an efficient method to label mitochondria. The experimental results demonstrates that HSFCM provides a powerful analytical tool to assess the performance of mitochondrial fluorescence labeling via high throughput single mitochondria analysis.%线粒体是真核细胞能量代谢和信号转导的调控中枢,虽然各种灵敏、特异的线粒体荧光标记技术已经被广泛应用于线粒体研究,但仍缺乏单线粒体水平的荧光染色性能评估方法。基于超高灵敏流式检测技术( High sensitivity flow cytometry, HSFCM)能对单个线粒体进行高灵敏、高通量、多参数定量分析的独特优势,本研究发展了一种单线粒体水平的荧光标记高通量评估方法。将携带靶向线粒体绿色荧光蛋白基因的pAcGFP1-Mito质粒转染至人宫颈癌HeLa细胞中,用G418筛选出稳定转染细胞,分别从瞬时转染和稳定转染的细胞中提取线粒体。此外,从未转染质粒的正常HeLa细胞中提取线粒体,分别进行MitoTracker Green标记以及SYTO 62线粒体DNA染色,应用实验室自行研制的超高灵敏流式检测装置在单线粒体水平对这4种线粒体标记方法的荧光亮度、标记效率和稳定性进行评估。实验结果表明,稳定转染细胞中单个线粒体的绿色荧光蛋白(GFP)荧光亮度为瞬时转染的17.7倍,比MitoTracker Green标记的线粒体亮度高约两个数量级,且标记稳定性好。本研究为线粒体标记方法的选择提供了一种先进的分析方法。