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We tried to quantitate the number of cells with fragmented nuclei during
induction of apoptosis by staining DNA in a detergent containing buffer. We
got a flurry of DNA containing small particles. This has also been found by
Michael Nuesse and coworkers (GSF at Oberschleissheim bei Muenchen,
Germany), who have reported that result at the 8th Annual Heidelberger
Cytometry Symposion (abstracts, in German only, ISSN: 0949-5347).
Michael Nuesse and I agreed that it is impossible to quantitate apoptosis
this way. There is no regular pattern in how nuclei fragment during
apoptosis. As a result, you cannot infer the number of nuclei that gave
rise to an apoptotic fragment of a given relative size if you know the size
and the number of these fragments. In other words, there is no way of
knowing, when you find, say, hundred fragments of one-tenth of the size of a
nucleus, whether those originated from 10 nuclei splitting into ten
fragments each, or whether a hundred nuclei split off one piece of one-tenth
of their size (and some other pieces of any other undetermined sizes), or
whether any other conceivable pattern occured.
The only way out, I can conceive now, is to first fix your cells in ethanol,
then wash them in a high phosphate/citrate buffer according to Darzynkiewicz
and coworkers (1994) Anal.Biochem. 218, 314-319. In this procedure every
cell will remain one particle. Those who underwent apoptosis will have
breaks in their DNA, which will lead to loss of some of it during the wash
step. After DNA staining the cells which had DNA breaks will appear as
"sub-G1" particles. This worked great in our hands.
Wishing good all good luck with this, and any other endeavor, in 1996 !
Martin Poot
martin@probes.mhs.compuserve.com
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