Radioactive Proliferation Assay

DESCRIPTION of the method:
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).

The principle of this assay is:
A certain number of cells is seeded in the wells of a 96 well plate. At the same time either a proliferation affecting agent is added or not. The cells are incubated for a certain time (e.g. 24 h) at 37C. Then 3H-Thymidine is added to the wells and incubated for another period of time (e.g. 24h). Cells that are incubated without any growth inhibiting agent will grow: during each cell division the cells will incorporate 3H-Thymidine into its DNA. The more cell divisions (or the higher the proliferation rate) the more radioactivity will be incorporated into DNA. Cells that are incubated in the presence of the growth inhibiting agent (e.g. TNF-alpha will incorporate less radioactivity. After incubation 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 m 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. The higher the proliferation rate of the cells was during incubation, the more cells are harvested and the more radioactive DNA will be collected on the filter. The filter membrane is dryed and the amount of radioactivity (what corresponds to the number of cells in the well or the number of cell divisions during incubation) is counted in a scintillation counter. To calculate the inhibition of proliferation in presence of a growth inhibitor you compare the counted radioactivity (counts per minute = cpm) of cells that were not treated, cpm (untreated), with the cpm in cells that were treated with agent, cpm (treated):

Inhibition of Proliferation [%] = [cpm (untreated) - cpm (treated)] / cpm (untreated).


PROCEDURE
  1. Harvest cells (wash two times with RPMI), count cells
  2. seed 1000 - 2000 cells per well in 96 well plate (in a volume of 100 l);
  3. add agent (e.g. TNF-alpha in a volume of 100 l RPMI.
  4. Incubate plate for a certain time (e.g. 24 h) at 37C;
  5. 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;
  6. Incubate plate for another time (24 h - 48h ) at 37C;
  7. 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.
  8. Count the radioactivity in the beta-counter.
  9. Calculate Inhibition of Proliferation.

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