Abstract
Recent advances in flow cytometry facilitate the detection of subcellular components, such as organelles and vesicles. Fluorescence-activated mitochondria sorting (FAMS) is a flow cytometry-based technique that allows for quantitative analysis and sorting of mitochondria as individual organelles from various tissues and in vitro cell culture. This manuscript details three novel applications of this technique to study mitochondrial function on an organelle-specific level, which is not possible with other approaches. Specifically, we detail the further development and versatility of this nanoscaled flow cytometry approach, including assays to quantitatively assess mitochondrial subpopulations, mitochondrial protein translocation, and both free-floating and EV-encapsulated secreted mitochondria. We demonstrate a multi-parameter quantitative assay for the analysis of mitochondrial autophagy using antibodies targeting the proteins PINK1 and Parkin corresponding to ΔΨM and further show how these can be assessed for mtDNA content on a single organelle level. Further, we establish parameters for the size and surface marker-based analysis of EVs, many of which contain identifiable and respiring mitochondria, as well as free-floating respiratory-competent mitochondria. These results display the versatility of nanoscaled flow cytometry in terms of both sample input and target organelle and provide an important methodological means for the quantitative assessment of mitochondrial features.