Re: apoptosis by flow
Eric Miller (miller@icrf.icnet.uk)
Wed, 3 Jan 1996 09:54:56 +0000 (GMT)
This is a problem we have tried to solve. In a classic plot of a cell
culture undergoing apoptosis the typical appearance of apoptotic cells is
lower FSC and higher SSC. This, however, takes no account of the
apoptotic bodies which have a low FSC and low SSC, thus can be lost when
thresholding on FSC. We use an end-labelling assay for apoptosis,
displaying SSC against green fluorescence in an ungated dot plot to
distinguish apoptotic cells and bodies. From this plot it appears that
apoptotic bodies have a similar fluorescence to apoptotic cells. The
picture is complicated when necrosis appears..........!
Eric P Miller
Edinburgh Medical Oncology Unit
"I am Pentium of Borg. Division is futile......you will be approximated."
Star Trek - Generations. Two Captains, one density.
On Wed, 20 Dec 1995 hwortis_sup@opal.tufts.edu wrote:
> Is this a problem that other people have solved?
> We have been using flow to analyze apoptosis in lymphocytes. We
> have been using P.I. staining and using either ethanol or hypotonic lysis
> to obtain nuclei and stained nuclear fragments. The problem is whether
> to use FSC/SSC gates. If the answer is yes, which particles do you omit
> from the gate?
> It seems that there is a real problem of analysis. Each
> apoptotic nucleus may form several fragments. Therefore there
> is no defined relationship between the number of fragments and the number of
> apoptotic cells. Do other flowers recognize this as a problem? If so
> how do you solve it?
>
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