Protocol for assessing real-time changes in mitochondrial morphology, fission and fusion events in live cells using confocal microscopy

Changes in mitochondrial size, shape, and subcellular position, a process collectively known as mitochondrial dynamics, are exploited for various cancer traits. Modulation of subcellular mitochondrial trafficking and accumulation at the cortical cytoskeleton has been linked to the machinery of cell movements, fueling cell invasion and metastatic spreading. Here, we detail a technique to track changes in mitochondrial volume using a commercial CellLight™ Mitochondria-RFP/GFP reporter and live confocal microscopy. This allows a real-time study of mitochondrial dynamics in live cells. For complete details on the use and execution of this protocol, please refer to Bertolini et al. (2020).