Radioactive DNA Fragmentation Assay

The principle of this assay is:
the cells' DNA is radioactively labeled by growing the cells in presence of 3H-Thymidine so that radioactive 3H-Thymidine is incorporated into the DNA. Then the labeled cells are incubated with or without the DNA fragmentation inducing agent. During this incubation the added agent (e.g. TNF-alpha induces cells to die by apoptosis and consequently the fragmentation of DNA while the DNA of untreated cells remains intact. After incubation (e.g. 48 h) the cells are harvested: during harvesting, the cells are washed out of the wells of the 96 well plate with bidest water: the cells and organelles burst and the cell's DNA is set free. The cell fragments and DNA are passed through a filter membrane (glassfiber). Only particles of smaller than 1,5 � can pass the filter. So, intact DNA (with a fragment length in the range of milimeters or even centimeters) will not be able to pass the filter but be collected on the filter membrane. DNA that was cleaved/degraded into fragments of about 5000 bp or less will be small enough to pass the pores of the filter and won't be collected on the filter. The filter membrane is dryed and the amount of radioactivity (what corresponds to the amount of intact DNA) counted in a scintillation counter. To calculate the percentage of DNA fragmentation you compare the counted radioactivity (counts per minute = cpm) of cells that were not treated with the cpm in cells that were treated with agent:

Fragmentation [%] = [cpm (untreated) - cpm (treated)] / cpm (untreated).

1. In a 25 cm2 flask: incubate cells (about 200 000 cells) for 16h - 24 h in the presence of 2 �i/ml 3H-Thymidine in 5 ml RPMI complete medium (i.e. 10 � of 1 �i/� stock solution);
2. Harvest cells (wash two times with RPMI), count cells
3. Seed 3500 - 5000 cells per wells in 96 well plate (in a volume of 100 �);
4. add agent (e.g. TNF-alpha in a volume of 100 � RPMI or just 100 � RPMI
5. Incubate plate for a certain time (e.g. 48h) at 37�;
6. Harvest cells: pipet supernatant into another plate, pipet 50 � of Trypsine/EDTA to the cells, incubate 37� until all cells are detached, pipet the supernatants back to the corresponding wells containing the detached cells, harvest with harvester;
7. Add scintillation fluid to the dried filter discs and count the radioacivity with the beta-counter;
8. Calculate DNA Fragmentation.