Oct 14, 2022
Radiolabeled polyamine uptake in cells
- 1KU Leuven
External link: https://doi.org/10.3390/biom13020337
Protocol Citation: Marine Houdou, Nathalie Jacobs, Peter Vangheluwe 2022. Radiolabeled polyamine uptake in cells. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm2x85g3p/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: October 04, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 70812
Keywords: ASAPCRN, radiolabeled polyamine uptake capacity, polyamine uptake capacity in cell, polyamine uptake in cell, polyamine uptake, dpm
Abstract
This protocol provides a technique to determine the radiolabeled polyamine uptake capacity in cells, via the acquisition of disintegrations per minute (DPM) using a Liquid Scintillation Counter.
Guidelines
Proper guidelines for working with radiolabeled materials should be followed at all times.
Safety warnings
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, 1 'quick wash' well and 1 'untreated' well per cell line and treatment dose.
Note
| A | B | C | D | |
| treatment | treatment | quick wash | untreated | |
Remove the culturing medium in 'treatment wells' and add 500 µL of medium, containing the desired concentration of radiolabeled polyamines. Leave regular culturing medium in the 'quick wash' and 'untreated' wells.
Incubate 37 °C 00:30:00
30m
4 minutes before reaching the 30-minute mark, perform a Quick-wash step as follows: add 500 µL of medium, containing the desired concentration of radiolabeled polyamines in the 'quick wash' wells. 00:00:00 immediately remove the treatment medium, and wash with 500 µL of DPBS (-/-) containing 50 micromolar (µM) of the respective unlabeled (=cold) polyamine at 4 °C . Continue with 2 more washing steps with 800 µL of DPBS (-/-) 4 °C .
Proceed with washing the other wells ('treatment' and 'untreated'). Remove the treatment medium, and wash with cold 500 µL of DPBS (-/-) containing 50 micromolar (µM) of the respective cold polyamine at 4 °C . Continue with 2 more washing steps with 800 µL of DPBS (-/-) at 4 °C .
After the last wash, add 200 µL 0.1% SDS-DPBS (-/-) per well to lyse the cells.
Incubate 00:10:00 at Room temperature .
10m
Scrape the cells and pipette the lysates into scintillation vials, that are filled with 7 mL of scintillation fluid (Ecolite (+), MP). Rinse each well with 200 µL DPBS (-/-) and pipette into the respective scintillation vial.
Mix scintillation vials well prior acquisition of disintegrations per minute (DPM) in the Liquid Scintillation Counter.