DESCRIPTION of the method: The DNA Fragmentation Assay allows to determine the amount of DNA that is degraded upon treatment of cells with certain agents, e.g. with TNF-alpha or anti-Fas antibody (IPO-4). The principle of this assay is: Fragmentation [%] = [cpm (untreated) - cpm (treated)] / cpm (untreated). |
PROCEDURE In a 25 cm2 flask: incubate cells (about 200 000 cells) for 16h - 24 h in the presence of 2 礐i/ml 3H-Thymidine in 5 ml RPMI complete medium (i.e. 10 祃 of 1 礐i/祃 stock solution); Harvest cells (wash two times with RPMI), count cells Seed 3500 - 5000 cells per wells in 96 well plate (in a volume of 100 祃); add agent (e.g. TNF-alpha in a volume of 100 祃 RPMI or just 100 祃 RPMI Incubate plate for a certain time (e.g. 48h) at 37癈; Harvest cells: pipet supernatant into another plate, pipet 50 祃 of Trypsine/EDTA to the cells, incubate 37癈 until all cells are detached, pipet the supernatants back to the corresponding wells containing the detached cells, harvest with harvester; Add scintillation fluid to the dried filter discs and count the radioacivity with the beta-counter; Calculate DNA Fragmentation. CAUTION: Dispose radioactive materials into radioactive waste containers !!! |