Oct 13, 2025
Protocol — LysoTracker® Red DND-99 Live-Cell Staining 26 HCS Analysis
- Sara Lucas1,
- Giuseppe Uras1,
- Sofia Koletsi2,
- federico fierli2,
- david chau2,
- anthony schapira2
- 1UCL;
- 2University College London, University of London
Protocol Citation: Sara Lucas, Giuseppe Uras, Sofia Koletsi, federico fierli, david chau, anthony schapira 2025. Protocol — LysoTracker® Red DND-99 Live-Cell Staining 26 HCS Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlky211g5r/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: 229673
Keywords: ASAPCRN, cell lysosomal staining, content confocal imaging
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP000420
Abstract
Live-cell lysosomal staining with LysoTracker® Red DND-99 followed by high-content confocal imaging (Opera Phenix® Plus) and automated quantification in Harmony®/Columbus®.
Guidelines
- Acquire channels sequentially: DAPI (Hoechst) — 405 nm laser ~30% / 100 ms; 568 nm (LysoTracker) — ~70% / 100 ms.
- Avoid saturation; keep instrument settings consistent across plates.
Analysis — Harmony®/Columbus®
- Find Nuclei on Hoechst channel (tune intensity/size to exclude debris/clumps).
- Find Cytoplasm around each nucleus using 568 channel; apply intensity filter 200–100,000 a.u.
- Find Spots in cytoplasm on 568 channel to detect LysoTracker®-positive puncta (tune size/intensity).
- Compute total spots per cell: normalise spot counts by nuclei count in each field.
- Export per-well spot-per-cell metrics for statistics.
Materials
- LysoTracker® Red DND-99 (Life Technologies®, Cat# L7528)
- Hoechst 33342 (ThermoFisher Scientific®, Cat# H3570)
- Complete culture medium; pre-warmed PBS
- 96-well imaging plates (optical bottom)
- Opera Phenix® Plus HCS System (RRID:SCR_021100)
- Harmony® High-Content Imaging 26 Analysis Software (RRID:SCR_018809)
Troubleshooting
Safety warnings
Handle fluorescent dyes with care; protect from light. Follow institutional live-cell handling guidelines.
Before start
- Equilibrate plate and medium to 37°C and 5% CO₂.
- Prepare LysoTracker® working solution: 60 nM in complete medium (fresh).
- Prepare Hoechst 33342 working solution: 1 µg/mL in medium.
Procedure — Live staining
Add LysoTracker® Red working solution to wells (final 60 nM). Incubate 37°C for 50 min (protected from light).
During the final 10 min, add Hoechst 33342 to 1 µg/mL.
Gently wash once with pre-warmed PBS to remove unbound dye.
Add phenol-red-free medium (optional) for imaging.
Imaging — Opera Phenix® Plus
Objective: 63× water; Mode: Confocal.
Fields per well: 5–10 random.
Analysis — Harmony®/Columbus®
Acquire channels sequentially: DAPI (Hoechst) — 405 nm laser ~30% / 100 ms; 568 nm (LysoTracker) — ~70% / 100 ms.
Avoid saturation; keep instrument settings consistent across plates.
Find Nuclei on Hoechst channel (tune intensity/size to exclude debris/clumps).
Find Cytoplasm around each nucleus using 568 channel; apply intensity filter 200–100,000 a.u.
Find Spots in cytoplasm on 568 channel to detect LysoTracker®-positive puncta (tune size/intensity).
Compute total spots per cell: normalise spot counts by nuclei count in each field.
Export per-well spot-per-cell metrics for statistics.
Expected results
Robust punctate 568-nm signal marking acidic organelles; per-cell spot counts enabling group comparisons.
Troubleshooting
Weak staining → verify dye concentration and incubation time; avoid over-washing.
High background → reduce exposure or dye concentration; ensure sequential acquisition.