Oct 13, 2025
Protocol — DQ™ Red BSA Lysosomal Activity Assay HCS Analysis
- Sara Lucas1,
- Giuseppe Uras1,
- federico fierli1,
- Sofia Koletsi2,
- david chau2,
- anthony schapira2
- 1UCL;
- 2University College London, University of London
Protocol Citation: Sara Lucas, Giuseppe Uras, federico fierli, Sofia Koletsi, david chau, anthony schapira 2025. Protocol — DQ™ Red BSA Lysosomal Activity Assay HCS Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz92ergx1/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 13, 2025
Last Modified: October 13, 2025
Protocol Integer ID: 229674
Keywords: ASAPCRN, lysosomal proteolysi, assay
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP000420
Abstract
Functional readout of lysosomal proteolysis using self-quenched DQ™ Red BSA, followed by confocal imaging and spot-based quantification.
Guidelines
- Acquire DAPI channel for nuclei and 568-nm channel for DQ-BSA signal (sequential acquisition).
- Use exposure settings similar to LysoTracker® acquisition; avoid saturation.
Analysis — Harmony®/Columbus®
- Find Nuclei on Hoechst channel; adjust thresholds to exclude debris/clumps.
- Find Cytoplasm using the 568 channel; apply intensity filter 200–100,000 a.u.
- Find Spots on the 568 channel for DQ-BSA puncta; set split sensitivity 0.8 and detection sensitivity 0.5 (tune as needed).
- Extract spot intensity metrics and normalise by nuclei count to obtain per-cell measures.
- Export per-well values for statistical analysis.
Materials
- DQ™ Red BSA (Life Technologies®, Cat# D12051)
- Hoechst 33342 (ThermoFisher Scientific®, Cat# H3570)
- 4% Paraformaldehyde (PFA) in PBS (for fixation)
- Complete medium; pre-warmed PBS
- Imaging plates; Opera Phenix® Plus (RRID:SCR_021100)
- Harmony® High-Content Imaging Analysis (RRID:SCR_018809)
Troubleshooting
Safety warnings
PFA is hazardous; use in a fume hood with appropriate PPE. Protect fluorescent probes from light.
Before start
- Prepare DQ-BSA working solution at 10 µg/mL in complete medium (fresh).
- Prepare Hoechst 33342 at 1 µg/mL.
Procedure — Loading 26 Fixation
Add DQ-BSA (final 10 µg/mL) to wells; incubate 37 °C for 1 h.
Wash gently with warm PBS to remove uninternalised probe.
Add Hoechst 33342 (final 1 µg/mL) for 5 min at 37 °C; wash with pre-warmed PBS.
Fix with 4% PFA (e.g., 10–15 min at RT); wash with PBS.
Imaging — Opera Phenix® Plus
Objective: 63×; Mode: Confocal.
Acquire DAPI channel for nuclei and 568-nm channel for DQ-BSA signal (sequential acquisition).
Use exposure settings similar to LysoTracker® acquisition; avoid saturation.
Analysis — Harmony®/Columbus®
Find Nuclei on Hoechst channel; adjust thresholds to exclude debris/clumps.
Find Cytoplasm using the 568 channel; apply intensity filter 200–100,000 a.u.
Find Spots on the 568 channel for DQ-BSA puncta; set split sensitivity 0.8 and detection sensitivity 0.5 (tune as needed).
Extract spot intensity metrics and normalise by nuclei count to obtain per-cell measures.
Export per-well values for statistical analysis.
Expected results
Discrete cytoplasmic puncta with measurable 568-nm fluorescence proportional to proteolytic activity.
Troubleshooting
Low signal → confirm probe storage and working concentration; extend incubation to 2 h if needed.
Diffuse signal → reduce loading concentration or time; verify wash steps.