Radiolabeled polyamine uptake in cells

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.

Remove the culturing medium in 'treatment wells' and add 500µL of medium, containing the desired concentration of radiolabeled polyamines. Leave regular culturing medium in the 'quick wash' and 'untreated' wells.

Incubate 37°C 0h 30m 0s

4 minutes before reaching the 30-minute mark, perform a Quick-wash step as follows: add 500µL of medium, containing the desired concentration of radiolabeled polyamines in the 'quick wash' wells. immediately remove the treatment medium, and wash with 500µL of DPBS (-/-) containing 50micromolar (µM) of the respective unlabeled (=cold) polyamine at 4°C . Continue with 2 more washing steps with 800µL of DPBS (-/-) 4°C .

Proceed with washing the other wells ('treatment' and 'untreated'). Remove the treatment medium, and wash with cold 500µL of DPBS (-/-) containing 50micromolar (µM) of the respective cold polyamine at 4°C . Continue with 2 more washing steps with 800µL of DPBS (-/-) at 4°C .

After the last wash, add 200µL 0.1% SDS-DPBS (-/-) per well to lyse the cells.

Incubate 0h 10m 0s at Room temperature .

Scrape the cells and pipette the lysates into scintillation vials, that are filled with 7mL of scintillation fluid (Ecolite (+), MP). Rinse each well with 200µL DPBS (-/-) and pipette into the respective scintillation vial.

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