By NanoFCM
The Flow NanoAnalyzer is applied for the high-throughput multiparameter analysis of individual mitochondria. By simultaneous labeling of mitochondrial cardiolipin (inner membrane) and mtDNA (matrix) with NAO and SYTO 62, respectively, the Flow NanoAnalyzer demonstrates the assessment of the purity and structural integrity of individual mitochondria. By simultaneous labeling of porin and cytochrome C proteins, the functional integrity of individual mitochondria can be characterized. The Flow NanoAnalyzer can characterize the functional integrity of individual mitochondria via simultaneous detection of side scatter, porin (outer membrane) and cytochrome C (intermembrane space) proteins. Employing an immunofluorescent labelling strategy, the copy number and distribution of proteins in single mitochondria are accessed via the Flow NanoAnalyzer. A series of fluorescent nanospheres with fluorescence intensity calibrated by the unit of molecules of equivalent soluble fluorochrome (MESF)-AF488 were used to construct a calibration curve. The fluorescence intensity of a single mitochondrion could then be converted to the number of proteins per mitochondrion. This result is achieved by treating isolated mitochondria with potential anticancer compounds, followed by simultaneously measuring the side scatter and mitochondrial membrane potential (ΔΨm, labeled with DiOC6(3)) fluorescence of individual mitochondria using the Flow NanoAnalyzer. The feasibility of this method was tested with eight widely used anticarcinogens. A potent mitochondrial membrane uncoupling agent, CCCP, was used as the positive control. By labeling with DiOC6(3), the Flow NanoAnalyzer can monitor the change of mitochondrial membrane potential and quickly and accurately detect whether the drug can act directly on mitochondria and its effect. Isolated mitochondria expressing green fluorescent protein (GFP-mito) or Discosoma red fluorescent protein (DsRed-mito) were mixed together, induced to fuse, and analyzed by the Flow NanoAnalyzer. An individual particle simultaneously exhibiting green and red fluorescence was identified as an event of heterotypic fusion, and the efficiency of heterotypic fusion was used as a surrogate for overall fusion efficiency.
