Dec 06, 2022
Fluorescently labeled polyamine uptake (via Flow Cytometry)
- 1KU Leuven
External link: https://doi.org/10.3390/biom13020337
Protocol Citation: Marine Houdou, Peter Vangheluwe 2022. Fluorescently labeled polyamine uptake (via Flow Cytometry). protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldp8qxl5b/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: December 02, 2022
Last Modified: April 15, 2024
Protocol Integer ID: 73483
Keywords: Fluorescenlty labeled polyamines, ASAPCRN
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
Make sure to stay protect from the light when manipulating fluorescent materials.
Materials
0.25% Trypsin-EDTA: Gibco, 25200056
Albumin Fraction V: Carl Roth, 8076.4
Dulbecco's Phosphate Buffered Saline modified without calcium chloride and magnesium chloride (DPBS (-/-)): Gibco, D8537
TrypLE Express Enzyme: Gibco, 12604021
Versene Solution: Gibco, 15040
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 12 well-plate that they reach 70%-80% confluency the day of the experiment.
Note
This protocol is made for a 12 well-plate format. Adaptations need to be done to scale up or down.
The day of the experiment, remove cell culture medium and add fluorescently labeled polyamines to the cells in a final volume of 500 µL / well .
Note
Concentration and incubation time may vary depending on the cell type. It's best to try different incubation times and doses to better appreciate the kinetics.
Note
Stay protect from the light.
Incubate the desired time at 37°C, 5% CO2, protected from the light.
Harvest cells and prepare samples for Flow Cytometry (FC).
Discard medium.
Wash with 500 µL /well Versene or DPBS (-/-).
Discard Versene or DPBS (-/-).
Add 200 µL /well 0.25% Trypsin or TrypLE. Incubate 00:05:00 at RT protect from the light.
5m
Add 500 µL /well 1%-Albumin Fraction V in DPBS (-/-), collect the cells and transfer in Eppendorf tubes on ice.
Centrifuge for 00:05:00 at 400 g and 4°C.
5m
Discard supernatants and resuspend cell pellets in 250-500 µL 1%-Albumin Fraction V in DPBS (-/-), depending on the size of the cell pellet.
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.