Oct 21, 2025

Public workspaceLive-Imaging of GCase Activity in iPSC-Derived Dopaminergic Neurons Using the Lyso-FQ Probe

DOI
https://dx.doi.org/10.17504/protocols.io.x54v95onzl3e/v1
  • Roxanne Larivière1,
  • Edward A. Fon1
  • 1Montreal Neurological Institute - McGill University
Roxanne Larivière
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DOI: https://dx.doi.org/10.17504/protocols.io.x54v95onzl3e/v1
Protocol Citation: Roxanne Larivière, Edward A. Fon 2025. Live-Imaging of GCase Activity in iPSC-Derived Dopaminergic Neurons Using the Lyso-FQ Probe. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v95onzl3e/v1
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: October 20, 2025
Last Modified: October 21, 2025
Protocol Integer ID: 230294
Keywords: GCase activity, lysosomes, iPSC-derived DA neurons, imaging of gcase activity, assessing gcase activity, lysosomal gcase probe green, gcase activity, permeable reporter for gcase activity, fq gcase substrate, dopaminergic neuron, derived dopaminergic neuron, glucocerebrosidase activation, cell imaging, monitoring of gba1 activity, gba1 activity, cell imaging with the opera phenix
Funders Acknowledgements:
Michael J. Fox Foundation for Parkinson's research
Grant ID: MJFF-025378
Abstract
This protocol describes the procedure for assessing GCase activity in iPSC-derived dopaminergic neurons using the Lyso-FQ GCase substrate (Biolegend 421948). The assessment is performed via live-cell imaging with the Opera Phenix high-content imaging system and analyzed using Harmony software.
This LysoFQ-GBA (Lysosomal GCase Probe Green) probe, a cell-permeable reporter for GCase activity, fluoresces upon glucocerebrosidase activation, facilitating the detection and monitoring of GBA1 activity.




Materials



Troubleshooting
Safety warnings
Be extremely gentle when dispensing into the wells, as dopaminergic neurons are prone to detaching with forceful pipetting
CBE treatment
16h
For the negative control, incubate neurons overnight at Temperature37 °C with Concentration25 micromolar (µM) of the GCase inhibitor Conduritol B epoxide (CBE)

16h
Labeling lysosomes with LysoTracker Deep Red
30m
Prepare a LysoTracker Deep Red working solution at Concentration50 nanomolar (nM) in media. Remove the existing media from the wells and add Amount60 µL of the LysoTracker solution. Incubate for Duration00:30:00 at Temperature37 °C .

30m
Preparing LysoFQ stock solution
While the cells are incubating, prepare the Lyso-FQ stock solution (Biolegend 421948). Reconstitute one Amount500 µg vial of Lyso-FQ in Amount77.3 µL of DMSO for a Concentration5 millimolar (mM) stock, then aliquot into Amount3 µL single-use stock vials.
Store aliquots atTemperature-80 °C , protected from light, and avoid repeated freeze-thaw cycles.

Addition of Lyso-FQ
1. Prepare the Lyso-FQ working solution by diluting the stock 1:1000 in FluoroBrite DMEM.
2. Carefully remove the LysoTracker Deep Red solution from the wells and gently add Amount50 µL of the Lyso-FQ working solution to each well.
3. Proceed with imaging
Imaging using the Opera Phenix high-content imaging system
2h 30m
1. Insert the prepared plate into the Opera Phenix chamber.
2. Utilize a 40× water immersion objective. Capture images using the 488 nm and 647 nm laser channels. Acquire 15-18 fields of view per well every Duration00:15:00 for a total duration ofDuration02:30:00 . Maintain the plate under standard cell culture conditions ( Temperature37 °C , 5% CO2) throughout the imaging session.

2h 30m
Image Analysis Workflow in Harmony Software

  1. Load Images Open the Data Analysis tab in the Harmony software and load the desired image dataset.
  2. Create an Analysis Sequence
  3. Identify Cells Use the Alexa 647 channel with the P method to detect cells. Parameters: Area > 100 µm², Splitting Sensitivity: 0.70, Common Threshold: 0.55). Save this population as "Cells".
  4. Select Cell Population Apply Common Filters to the "Cells" population to refine it, creating a new population named "Cells Selected".
  5. Calculate Morphological Properties For the "Cells Selected" population, use the Standard Method to calculate the Area.
  6. Calculate Intensity Properties
  • For the Alexa 647 channel, use the Standard Method to calculate the mean intensity for the "Cells Selected" population.
  • For the Alexa 488 channel, use the Standard Method to calculate the mean intensity for the "Cells Selected" population.
7. Define Results Output
  • Output: Cells Selected - Intensity Cell Alexa 647 Mean
  • Output: Cells Selected - Intensity Cell Alexa 488 Mean
8. Run the Analysis
9. Export Data Download the results as a CSV file.
Assess GCase activity
  • From the table generated by Harmony, calculate the ratio of the PFB-FDGlu mean fluorescence intensity to the LysoTracker Deep Red (647) baseline reading (at t=0) for each time point.
  • Enter these calculated values into Prism to generate the GCase activity curves.