Fluorescently labeled polyamine uptake (via Flow Cytometry)
Marine Houdou, Peter Vangheluwe
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Abstract
Assess polyamine uptake capacities of a specific cell line after incubation with fluorescently labeled polyamines and mean fluorescence intensitiy acquisition via flow cytometry.
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.
Steps
Seed cells in 12 well-plate that they reach 70%-80% confluency the day of the experiment.
The day of the experiment, remove cell culture medium and add fluorescently labeled polyamines to the cells in a final volume of 500µL.
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 500µL Versene or DPBS (-/-).
Discard Versene or DPBS (-/-).
Add 200µL 0.25% Trypsin or TrypLE. Incubate 0h 5m 0s protect from the light.
Add 500µL 1%-Albumin Fraction V in DPBS (-/-), collect the cells and transfer in Eppendorf tubes on ice.
Centrifuge for 0h 5m 0sat 400 g and 4°C.
Discard supernatants and resuspend cell pellets in 250-500µL 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.
