Unraveling T cell mitochondrial dynamics using imaging flow cytometry

Abstract

Abstract Mitochondria are highly mobile, dynamic subcellular organelles that mediate energy generation in mammalian cells. In response to changes in energy and stress, mitochondria can either join or divide, a process known as fusion or fission, respectively. Mitochondrial morphology plays a critical role in the metabolic state of T cells and can affect T cell function in health and disease. Despite increased interest in mitochondria dynamics, a robust and efficient method to simultaneously image mitochondria morphology, analyze T cell phenotypes and sort cells has not been feasible. Here, we used the state-of-the-art BD CellView™ Image Technology on the BD FACSDiscover™ S8 Cell Sorter to detect changes in mitochondria morphology with high resolution and profile T cell phenotypes associated with specific mitochondrial shapes. With the imaging capabilities of the BD FACSDiscover™ S8 System, we visualized live mitochondria and nuclei using three-color staining in a high-throughput manner. We differentiated effector T cells in vitro and imaged increased fission-like mitochondria in these cells. With the cell sorting feature of the BD FACSDiscover™ S8 System, we sorted effector T cells based on their cell surface phenotype and distinct mitochondrial characteristics and evaluated their mitochondrial gene signature. Thus, we provide an advanced and comprehensive workflow to unravel mitochondrial dynamics and isolate cells with a particular phenotype for downstream in-depth characterization enabled by the BD FACSDiscover™ S8 System. As many cellular processes are linked to mitochondrial dynamics, this technique provides a powerful tool to understand how this critical organelle is regulated in a variety of human diseases.