Jan 10, 2025

Public workspaceMeasuring lysosomal exocytosis by Flow cytometry

DOI
dx.doi.org/10.17504/protocols.io.n92ld8oyxv5b/v1
  • 1RVC;
  • 2The Michael J. Fox Foundation for Parkinson’s Research (MJFF) and the Aligning Science Across Parkinson’s (ASAP) Initiative
Susanne Herbst
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DOI: dx.doi.org/10.17504/protocols.io.n92ld8oyxv5b/v1
Protocol Citation: Susanne Herbst, Patrick Lewis 2025. Measuring lysosomal exocytosis by Flow cytometry. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld8oyxv5b/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: In development
We are still developing and optimizing this protocol
Created: October 16, 2024
Last Modified: January 10, 2025
Protocol Integer ID: 110083
Keywords: ASAPCRN, Lysosome, Exocytosis, Flow cytometry
Funders Acknowledgements:
The Michael J. Fox Foundation for Parkinson’s Research (MJFF) and the Aligning Science Across Parkinson’s (ASAP) Initiative
Grant ID: .
Abstract
Protocol for measuring lysosomal exocytosis by flow cytometry.
Materials
Staining buffer: 5% FCS in PBS (filtered)
Blocking buffer: 5% FCS in PBS containing FcR block at 1:100 dilution (Anti-Mouse CD16/CD32; BD Biosciences Cat# 553141, RRID:AB_394656)
Antibody staining solution: Dilute 0.5 ul anti-mouse-LAMP1-AF647 (Rat-anti-mouse-LAMP-1-AF647 (BioLegend Cat# 121610, RRID:AB_571990) ) in 25 ul staining buffer per sample.
Viability stain: eg Fixable Viability Dye eFluor-450, # 65-0863-18, eBioscience

Sample preparation
Sample preparation
Seed cells as required and stimulate to induce exocytosis. Prepare ~ 5x10^5 cells per sample.
Detach cells. Keep all samples on ice from now on.
Staining
Staining
Pellet cells.
Resuspend in 75 ul blocking buffer (5% FCS in PBS - optional: containing FcR blocker) and incubate for 10 min on ice.
Add 25 ul anti-LAMP1-AF647 antibody staining solution containing 0.5 ul antibody and incubate for 30 min on ice. Protect from light.
Add 300 ul PBS and pellet cells.
Resuspend cells in 100 ul dead/live staining solution and incubate for 10 min on ice. Protect from light.
Add 300 ul PBS and pellet cells.
Resuspend cells in 300 ul 2 % PFA/PBS.
Sample aquisition and analysis
Sample aquisition and analysis
Acquire 10'000 events per sample.
After removing doublets, analyse the mean and medium LAMP1 fluorescence intensity in the live cell gate.
Controls
Controls
10m
10m
Dead/live single-colour control
To prepare a live/dead single stain control, heat half of the sample at Temperature65 °C for Duration00:10:00 min. Put back on ice immediately, mix with non-heat treated cells and stain as above.

LAMP1 single-colour control
Remove part of the stimulated samples and omit dead/live stain.

10m