Jun 30, 2025
Mitophagy detection assay
- Bishal Basak1,2,
- Erika Holzbaur1,2
- 1Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
- University of Pennsylvania
External link: https://doi.org/10.1038/s41467-025-62379-5
Protocol Citation: Bishal Basak, Erika Holzbaur 2025. Mitophagy detection assay. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkrpj6v5r/v1
Manuscript citation:
Basak, B., Holzbaur, E.L.F. Mitochondrial damage triggers the concerted degradation of negative regulators of neuronal autophagy. Nat Commun 16, 7367 (2025). https://doi.org/10.1038/s41467-025-62379-5
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 13, 2025
Last Modified: June 30, 2025
Protocol Integer ID: 218197
Keywords: detection of mitophagy flux, mitophagy detection, mitophagy flux, using dojindo mtphagy dye, dojindo mtphagy dye
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000350
Abstract
Detection of mitophagy flux in neurons using Dojindo Mtphagy dye and Lysotracker Green
Protocol materials
Mtphagy DyeDojindoCatalog #MD01
Lysotracker GreenThermo Fisher ScientificCatalog #L7526
Troubleshooting
Before start
Make maintenance media (MM) and imaging media (IM) for cortical neurons
Plate 120,000-150,000 neurons in an imaging dish at days in vitro (DIV) 0
Note
Protocol for primary neuron isolation and in vitro culture can be obtained from:
Preparing reagants
On the day of imaging, equilibrate 12 ml of fresh maintenance media (MM) per imaging dish in an incubator at 37 °C
Note
Maintence media composition can be obtained from: dx.doi.org/10.17504/protocols.io.81wgby723vpk/v1
Prepare intermediate dilution of DMP Red (commercially available as Mtphagy DyeDojindoCatalog #MD01 ) in MM: for each imaging dish, make 500 µL of fresh MM containing 0.5 µL of DMP Red
Prepare intermediate dilution of Lysotracker Green in MM: for each imaging dish make 50 µL of fresh MM containing 0.1 µL of Lysotracker Green Lysotracker GreenThermo Fisher ScientificCatalog #L7526
Prepare imaging media (IM) with Lysotracker Green- for each imaging dish make 50 µL of fresh MM containing 0.1 µL of of Lysotracker Green
Note
Imaging media composition for 50 ml:
45% Glucose 660 µL
B-27 1 mL
Hibernate E Up to 50 mL
Imaging steps
3h 30m
At DIV 6 or 7, aspirate out conditioned media from the imaging dish, wash the neurons once with fresh MM
Add 1.6 mL of fresh MM and then add 0.4 mL of MM with the intermediate dilution of DMP Red (prepared in step 3)
Incubate the neurons in an incubator at 37 °C for 00:30:00
30m
Post incubation, aspirate out the media, and wash once with fresh MM
Add 2 mL of fresh MM containing 3 nM Antimycin A or an equivalent volume of 100% ethanol
Incubate the neurons in an incubator at 37 °C for 01:00:00
1h
Post incubation, aspirate out the media, and replace it with fresh MM with no Antimycin A or ethanol
Incubate the neurons in an incubator at 37 °C for 01:30:00
1h 30m
Post incubation add 50 µL of MM with the intermediate dilution of Lysotracker Green (prepared in step 4)
Incubate the neurons in an incubator at 37 °C for 00:30:00
30m
Post incubation, aspirate out the media, and wash once with fresh IM
Add 2 mL of fresh IM followed by 50 µL of IM with the intermediate dilution of Lysotracker Green (prepared in step 5)
Image neurons live under a confocal microscope with a heating chamber kept at 37 °C