Bill
---------- Forwarded message ----------
Date: Tue, 3 Jun 1997 16:50:01 -0400 (EDT)
From: William George Telford <apoptosi@umich.edu>
To: cyto-inbox
Subject: Re: FTIC + PI
Hi Lyndal...
Sorry I didn't respond sooner but I've been out of town. One of the major
pitfalls of using single dye methods with fixed cells to detect apoptosis
is that you often don't get a nice, clean apoptotic peak. The state of
the apoptotic cells with respect to DNA content after DNA fragmentation is
quite variable depending on the cell type. We were fortunate to initially
try the method with mouse lymphoid cells, which give a nice peak with a
relative index of about 0.5 with respect to the G0/G1 diploid peak. We
later found that cycling mouse lymphocytes and human cells often gave a
peak well below this, often at the threshold of instrument sensitivity on
the linear scale. Some people have tried to use log scaling to quantify
these very dim apoptotic cells, but they are often not very clearly
defined even then. If you are seeing this type of apoptotic peak (with
the cells smeared up at the minimum linear threshold), then you might be
measuring both apoptotic cells and cellular debris, giving you an
exaggerated number of apparent apoptotic events.
So, what to do? Try analyzing the cells on a log scale; this might allow
you to detect multiple subdiploid populations, one or ore of which should
be apoptotic. You can also analyze your cells for both DNA fluorescence
and forward scatter, and then exclude events with overly low FS signal;
this will allow you to separate apoptotic cells from debris, etc. that
have the same apparent DNA fluorescence. You might also want to try Dr.
Darzynkiewicz's protocol where he uses phosphate/citrate buffers of
different concentrations during the DNA staining process in an attempt to
control the amount of DNA loss following fixation. I don't have the
references with me (I'm still out of town!) but I can give them to you
once I get back.
The other option is to try another apoptosis detection method! Single dye
detection methods have sort of "fallen into disfavor" recently for the
variability of the apoptotic regions depending on cell type. We still use
them but recognize their limitations (fortunately,most of my work is with
mouse lymphocytes, where they work quite well ;-). You might want to
independeently confirm your results with a different method (two dye
membrane exclusion, FITC-annexin, etc.). It is also a good idea (although
a pain) to sort the cells you think are apoptotic and check them for DNA
fragmentation by electrophoresis, or morphology by confocal or EM.
If you want, send or FAX me a copy of the histograms you are getting, and
I can compare them to what we've seen before. Our address here is:
Hospital for Special Surgery
Flow Cytometry Core Facility
Research Building Room 312
535 E. 70th Street
New York, NY 10021
Our phone and FAX numbers are:
phone: (212) 606-1925
FAX: (212) 717-1192
Good luck! Let me know how things go.
Cheers!
Bill