Problems with Apoptosis Kit

[Stefan Krautwald]

Hi,

I want to detect apototic events by using the FITC conjugate of
annexin V . My cells are adherent growing macrophages, which were
cultured in DMEM and 10 % FCS. Cells were treated for different times
and then scraped carefully with a rubber policeman. Cells were
collected by centrifugation (10 min., 1000 x g, 4 °C) and washed twice
with PBS. Quantification by Flow Cytometry were done by standard
techniques (User Manual).
Now my fundamental problem: all cells, the control too (untreated),
are apototic!!
I cannot detect a time course or concentration range. In all cases I
get identical results (95% apoptotic cells).


Any help would be much appreciated.

Thanks, Stefan

krautwald at ita.fhg.de