High-Throughput Assessment of Mitochondrial Fluorescence Labeling at Single-Particle Level

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.