Fluorescent pulse-chase labeling to monitor long-term mitochondrial degradation in primary hippocampal neurons

The accumulation of dysfunctional mitochondria is a hallmark of neurodegenerative diseases, yet the dynamics of mitochondrial turnover in neurons are unclear. Here, we describe a protocol to monitor the degradation of spectrally distinct, “aged” mitochondrial populations. We describe the preparation and transfection of primary rat hippocampal neuron cultures. We detail a mitochondrial-damaging assay, a SNAP pulse-chase labeling paradigm, and live imaging to visualize the mitochondrial network. Finally, we provide steps to quantify mitochondrial turnover via lysosomal fusion. For complete details on the use and execution of this protocol, please refer to Evans and Holzbaur (2020a).