Jun 30, 2025
Lysosomal pH detection assay
- Bishal Basak1,2,
- Erika L.F. 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 L.F. Holzbaur 2025. Lysosomal pH detection assay . protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3wr47v25/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: June 20, 2025
Last Modified: June 30, 2025
Protocol Integer ID: 220644
Keywords: detection of lysosomal ph, lysosomal ph detection, lysosomal ph, ph, live cortical neuron, neuron
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000350
Abstract
Detection of lysosomal pH in live cortical neurons.
Protocol materials
pHLys Red (Lysosomal Acidic pH Detection Kit - Green/Red)DojindoCatalog #L266
LysoPrime Green (Lysosomal Acidic pH Detection Kit - Green/Red)DojindoCatalog #L266
Troubleshooting
Plate neurons
Plate 120,000-150,000 neurons in an imaging dish at days in vitro (DIV) 0
Prepare reagents
Reconstitute pHLys Red (from Lysosomal Acidic pH Detection Kit) using 20 µL DMSO; vortex and store at -20 °C pHLys Red (Lysosomal Acidic pH Detection Kit - Green/Red)DojindoCatalog #L266
On the day of imaging, prepare 1:2000 fresh dilution of LysoPrime Green in fresh maintenance media (MM) LysoPrime Green (Lysosomal Acidic pH Detection Kit - Green/Red)DojindoCatalog #L266
Note
Maintenance media composition can be obtained from: dx.doi.org/10.17504/protocols.io.81wgby723vpk/v1
Prepare 1:1000 fresh dilution of reconstituted pHLysRed in fresh MM
Equilibrate fresh MM in an incubator kept at 37 °C and keep fresh imaging media (IM) for warming at 37 °C
Note
Imaging media composition can be obtained from:
Imaging steps
30m
At DIV 6 or 7, aspirate out conditioned media from the imaging dish, wash the neurons once with fresh MM
Add 1.8 mL of fresh MM and then add 200 µL of the diluted LysoPrime Green solution (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 twice with fresh MM
Add 1.8 mL of fresh MM and then add 200 µL of the diluted pHLys Red solution (prepared in step 4)
Incubate the neurons in an incubator at 37 °C for 00:30:00
Post incubation, aspirate out the media, and washed twice with fresh IM
Add 2 mL of pre-warmed IM and in the lysosomes quickly image neurons live under a confocal microscope to check for labeling of both the dyes.
Note
This step is optional, but is recommended to check for proper labeling with the pHLysRed dye, whose fluorescence in the lysosomes will rapidly decrease upon addition of Bafilomycin A1 in the later steps.
Aspirate out IM, and wash the neurons twice with fresh MM.
Add 2 mL fresh MM containing 75 nM of Bafilomycin A1 to partially deacidify lysosomes
Incubate the neurons in an incubator at 37 °C for 00:30:00
Aspirate out MM, add 2 mL of warm IM and quickly image neurons live under a confocal microscope with a heating chamber kept at 37 °C