Apr 15, 2026

Fluorescently labeled polyamine uptake (via confocal microscopy)

DOI
https://dx.doi.org/10.17504/protocols.io.5qpvo9kd9v4o/v1
  • 1KU Leuven;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Sarah van Veen
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DOI: https://dx.doi.org/10.17504/protocols.io.5qpvo9kd9v4o/v1
Protocol Citation: Sarah van Veen, Peter Vangheluwe 2026. Fluorescently labeled polyamine uptake (via confocal microscopy). protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo9kd9v4o/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: December 11, 2024
Last Modified: April 15, 2026
Protocol  Integer ID: 115016
Keywords: ASAPCRN, visualizing polyamine uptake, polyamine uptake in cell, labeled polyamine uptake, polyamine uptake, polyamine transport dynamic, study of polyamine transport dynamic, confocal microscopy, genetic modifications on polyamine uptake, labeled polyamine, intracellular fluorescence, tagged polyamine, polyamine, fluorescence, subcellular localization, extracellular signal
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000458
Fonds Wetenschappelijk Onderzoek (FWO)
Grant ID: G011424N
Abstract
This method involves visualizing polyamine uptake in cells using fluorescently labeled polyamines and confocal microscopy. Cells are incubated with fluorescently tagged polyamines to allow uptake, followed by washing to remove extracellular signal. Confocal microscopy is then used to detect and quantify intracellular fluorescence, enabling the study of polyamine transport dynamics, subcellular localization, and the effects of specific treatments or genetic modifications on polyamine uptake.
Materials
PBS-T:
  • PBS
  • 0.5% Tween-20

Materials
  • BODIPY-putrescine (Cat# SCT248, Merck)
  • BODIPY-spermidine (Cat# SCT249, Merck)
  • BODIPY-spermine (Cat# SCT250, Merck)
  • DAPI (Cat# 62248, Thermo Fisher) 
  • Dulbecco's Phosphate Buffered Saline without calcium chloride and magnesium chloride (DPBS) (Cat# 14190144, GIBCO)
  • FluorSave‱ Reagent (Cat# 345789, Millipore)
  • Glycine (Cat# G8898, Sigma-Aldrich)
  • Paraformaldehyde, 4% in PBS (Cat# J61899.AP, Thermo Fisher)
  • Triton X-100 (Cat# 93443, Sigma-Aldrich)
  • Tween-20 (Cat# A4974.0500, PanReac AppliChem)
Safety warnings
Follow institutional guidelines for the disposal of biological and chemical waste.
Seed cells onto cover slips placed in a 12-well or 24-well plate. Allow the cells to attach and grow for approximately 24:00:00 .
1d
The day of the experiment, remove cell culture medium and add fluorescently labeled polyamines to the cells in a final volume of 1000 µL / well .
Note
Concentration and incubation time may vary depending on the cell type. It is recommended to test various incubation times and doses to better understand the kinetics.

Incubate at 37 °C with 5% CO₂ for the desired duration, ensuring protection from light.
Fix cells with 4% PFA in PBS for 00:30:00 at 37 °C .
30m
Wash cells 3 times in PBS-T (00:05:00 each wash) at Room temperature on a rocker.
5m
Incubate cells with DAPI (1:5000 in PBS-T) for 00:15:00 at Room temperature , protected from light, on a rocker.
15m
Wash cells 3 times in PBS-T (00:05:00 each wash) on a rocker, protected from light.
5m
Wash cells Overnight with PBS-T at 4 °C on a rocker, protected from light.
1d
Mount coverslips onto glass slides using mounting medium (FluorSave‱ Reagent).
Allow the slides to set Overnight at Room temperature .
1d
Acquire images using a Zeiss LSM880 confocal microscope or equivalent at 63x magnification.