From: Martin Poot (MARTIN@probes.mhs.compuserve.com)
Date: Thu Dec 28 1995 - 14:17:29 EST
Dear Netters, 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
This archive was generated by hypermail 2.1.6 : Thu Jan 01 2004 - 17:30:41 EST
