Quantifying Mitochondrial Axonal Transport

To image mitochondrial axonal transport, cultured RGCs were either nontransduced or transduced with the viruses above on day 1. After 5 days, mitochondria were labeled with 50 nM MitoTracker Deep Red FM (Invitrogen, Carlsbad, CA, USA) for 15 minutes and imaged every 1.5 seconds for 3 minutes by confocal microscope with incubation (LSM880; Carl Zeiss Meditec, Dublin, CA, USA) over a wide field of view. From the time-lapse images, we generated kymographs using the ImageJ (National Institutes of Health, Bethesda, MD, USA) Multiple Kymograph plug-in for ImageJ submitted by J. Rietdorf and A. Seitz (European Molecular Biology Laboratory, Heidelberg, Germany) to categorize mitochondrial state and calculate speed of the transport as described previously.²³ (link) In all time courses, mitochondria with a total absolute distance of >10 µm defined over a period of 3 minutes were included as motile. Any mitochondria 50 µm from the cell body or the end of the neurite were excluded. Although it is not possible to definitively differentiate between axons and dendrites at this stage, the longest neurite and main branch were chosen as the candidate axon. Additionally, cells were plated at low density to avoid contact between cells, and neurites touching neighboring cells were excluded from analysis.