Apr 06, 2026
Fluorescently labeled polyamine uptake (via flow cytometry)
- Sarah van Veen1,2,
- Marine Houdou1,2,
- Peter Vangheluwe1,2
- 1KU Leuven;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
External link: https://doi.org/10.3390/biom13020337
Protocol Citation: Sarah van Veen, Marine Houdou, Peter Vangheluwe 2026. Fluorescently labeled polyamine uptake (via flow cytometry). protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld4eynl5b/v1
Manuscript citation:
Houdou M, Jacobs N, Coene J, Azfar M, Vanhoutte R, Haute CVd, Eggermont J, Daniëls V, Verhelst SHL, Vangheluwe P, Novel Green Fluorescent Polyamines to Analyze ATP13A2 and ATP13A3 Activity in the Mammalian Polyamine Transport System. Biomolecules 13(2). doi: 10.3390/biom13020337
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: April 06, 2026
Last Modified: April 06, 2026
Protocol Integer ID: 314559
Keywords: ASAPCRN, Fluorescently labeled polyamines, labeled polyamine uptake, polyamine uptake, labeled polyamine, acquisition via flow cytometry, flow cytometry, fluorescence, uptake capacity
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000458
Disclaimer
Protocol Particulars Video
The video below is a supplement with extra context and tips, as part of the Aligning Science Across Parkinson's (ASAP) Protocol Particulars video interview series, featuring conversations with protocol authors.
Abstract
Assess polyamine uptake capacities of a specific cell line after incubation with fluorescently labeled polyamines and mean fluorescence intensitiy acquisition via flow cytometry.
Guidelines
Ensure fluorescent materials are protected from light during manipulation.
Materials
- BODIPY-putrescine (Cat# SCT248, Merck)
- BODIPY-spermidine (Cat# SCT249, Merck)
- BODIPY-spermine (Cat# SCT250, Merck)
- Bovine Serum Albumin (BSA) Fraction V (Cat# 8076.4, Carl Roth)
- Dulbecco's Phosphate Buffered Saline without calcium chloride and magnesium chloride (DPBS) (Cat# 14190144, GIBCO)
- TrypLE (Cat# 12604021, GIBCO)
Safety warnings
Follow institutional guidelines for the disposal of biological and chemical waste.
Before start
- Prepare appropriate dilution of fluorescently labeled polyamine in cell culture medium.
- Prepare Flow Cytometry (FC) buffer made of 1% Albumin Fraction V in DPBS (-/-).
- Prepare Eppendorf tubes and FC tubes by labeling them and keeping them on ice.
Seed cells in a 12-well plate so they reach 70-80% confluency on the day of the experiment.
The day of the experiment, remove cell culture medium and add fluorescently labeled polyamines diluted in media to the cells in a final volume of 1000 µL / well .
Note
Keep protected from light.
Incubate at 37 °C with 5% CO₂ for 2h, ensuring protection from light.
Harvest cells and prepare samples for Flow Cytometry (FC).
Discard medium.
Wash with 500 µL /well PBS.
Discard PBS.
Add 200 µL /well TrypLE. Incubate 00:05:00 at RT protected from light.
5m
Add 500 µL /well 2% of BSA (0.7% solution) in PBS.
Centrifuge for 00:05:00 at 300 g and 4°C.
5m
Discard supernatants and resuspend cell pellets in 500 µL in BSA+PBS+DAPI (1:5000).
Prepare single color samples.
Filter cell suspension through Nylon filter into FC tubes.
Keep on ice until acquisition at the flow cytometer. Record 10,000 live events per sample.