Radioactive Proliferation Assay

Monday, November 24, 2003

Description
The Proliferation Assay allows to determine the number of cells that are growing in the absence or presence of certain proliferation affecting agents, e.g. TNF-alpha or anti-Fas antibody (IPO-4).

Procedure
Harvest cells (wash two times with RPMI), count cells
seed 1000 - 2000 cells per well in 96 well plate (in a volume of 100 l);
add agent (e.g. TNF-alpha in a volume of 100 l RPMI.
Incubate plate for a certain time (e.g. 24 h) at 37C;
Add 1 Ci/well 3H-Thymidine (corresponds to 5 Ci/ml); e.g. add 20 l of 0.05 Ci/l 3H-Thymidine in RPMI complete medium;
Incubate plate for another time (24 h - 48h ) at 37C;
Harvest cells: pipet supernatant into another plate, pipet 50 l of Trypsine/EDTA to the cells, incubate 37C until all cells re detached, pipet the supernatants back to the corresponding wells containing the detached cells, harvest with harvester; dry the filter discs and add scintillation fluid.
Count the radioactivity in the beta-counter.
Calculate Inhibition of Proliferation.

CAUTION: Dispose radioactive materials into radioactive waste containers !!!

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Radioactive Proliferation Assay