Jul 09, 2024

Public workspaceRadiolabeled spermine uptake in cells

 Forked from Radiolabeled polyamine uptake in cells
DOI
dx.doi.org/10.17504/protocols.io.j8nlkorm5v5r/v1
  • 1Laboratory of Cellular Transport Systems, Department of Cellular and Molecular Medicine, KU Leuven, B-3000 Leuven, Belgium;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network Chevy Chase, MD 20815, USA;
  • 3KU Leuven
Stephanie Vrijsen
Open access
DOI: dx.doi.org/10.17504/protocols.io.j8nlkorm5v5r/v1
External link: https://doi.org/10.3390/biom13020337
Protocol Citation: Stephanie Vrijsen, Marine Houdou, Nathalie Jacobs, Peter Vangheluwe 2024. Radiolabeled spermine uptake in cells. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkorm5v5r/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 05, 2023
Last Modified: July 09, 2024
Protocol Integer ID: 91849
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's (ASAP)
Grant ID: ASAP-000458
Abstract
This protocol provides a technique to determine radiolabeled spermine uptake in cells, via the acquisition of counts per minute (CPM) using a Liquid Scintillation Counter.
Guidelines
Proper guidelines for working with radiolabeled materials should be followed at all times.
Materials
  • 14C-labeled spermine: 3139-50 µCi, ARC
  • Cold spermine: 85590-5G, Merck
  • RIPA Lysis and Extraction Buffer: 89900, Invitrogen
  • EcoLite Liquid Scintillation Cocktail: 01882475-CF, MP Biomedicals
Safety warnings
Attention
Radiation hazards
Cells are seeded in 12-well plates, such that 70-80% confluency is reached on the day of the assay. Seed out 2 'treatment' wells and 1 'background' well per cell line.


Remove the culture medium from all wells.

In the treatment wells: add Amount300 µL of medium, containing 5 µM radiolabeled spermine.
In the background wells: add Amount300 µL of medium, containing 5 µM radiolabeled spermine and 100 µM unlabeled (cold) spermine.

Incubate Temperature37 °C Duration00:30:00

30m
Aspirate the medium.

Wash wells 2x with Amount1 mL PBS (-/-).

Remove the last wash, then add 200 µl RIPA buffer to the wells.

Incubate Duration00:10:00 at TemperatureRoom temperature .


10m
Scrape the cells and pipette the lysates into scintillation vials, that are filled with Amount7 mL of scintillation fluid (Ecolite (+), MP). Rinse each well with Amount200 µL PBS (-/-) and pipette into the respective scintillation vial.

Mix scintillation vials well prior acquisition of counts per minute (CPM) in the Liquid Scintillation Counter.