Jun 20, 2025
Autophagy 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
Protocol Citation: Bishal Basak, Erika Holzbaur 2025. Autophagy detection assay. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqqkz3gk5/v1
Manuscript citation:
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 20, 2025
Protocol Integer ID: 218190
Keywords: autophagy detection, autophagosome detection, lysotracker green in neuron, using dojindo dapred dye, dojindo dapred dye, lysotracker green, autophagosome
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000350
Abstract
Autophagosome detection using Dojindo DAPRed dye and Lysotracker Green in neurons
Protocol materials
DAP RedDojindoCatalog #D677
Lysotracker GreenThermo Fisher ScientificCatalog #L7526
Troubleshooting
Plate 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 reagents
On the day of imaging, equilibrate 8 ml of fresh maintenance media (MM) per imaging dish in an incubator
at 37 °C
Note
Maintenance media composition can be obtained from: dx.doi.org/10.17504/protocols.io.81wgby723vpk/v1
Prepare intermediate dilution of DAP Red in MM: for each imaging dish, make 500 µL of fresh MM containing 1 µL of of DAP RedDAP RedDojindoCatalog #D677
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
1h
At DIV 6 or 7, aspirate out conditioned media from the imaging dish, wash the neurons once with fresh MM
Add 1.5 mL of fresh MM and then add 0.5 mL of MM with the intermediate dilution of DAP 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 followed by 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