Todd,
I would highly recommend that you try to do some sort of pre-enrichment
instead of just trying to sort out these very rare events. Otherwise you
will have to sort for days to get an appreciable number of cells! (Imagine
that you run at a flow rate of 50K cells/sec. If target cells are 1 in
10million, you will detect 18 cells of interest per hour!) Negative selection using
magnetic
separation works well. Even panning could help significantly. If you
can't pre-enrich and you have the time you will still need to sort twice;
the first sort as an enrichment sort and the second for purity.
Good luck.
David McFarland
GlaxoSmithKline
----- Forwarded by David C McFarland/DEV/PHRD/SB_PLC on 13-Aug-2002 08:44
-----
"Todd Belanger" <tbelanger@vaccinex.com>
12-Aug-2002 10:23
To: "Cytometry Mailing List"
cc:
Subject: sorting rare events- 1 in a million or more
Hi,
I am new to sorting (but I have ten years of flow experience) and we just
purchased a FACSVantage/DiVa. Some of our projects require sorting rare
cells at levels of 1 in a million or ten million. Some of the researchers
say it could be one in 100 million (which seems quite impossible to me).
Does anyone have any pointers or particularly good papers that would help
me
in this task? How low can you go (in terms of rare events) and still be
relatively confident in what you sorted? Currently the researchers I will
be
doing the sort for has two markers- PI to discriminate live cells and a
FITC
conjugated marker. I know more markers would be better for discriminating
rare events but their doesn't seem to be any for this particular
experiment.
Any and all help would be greatly appreciated.
Todd
Todd J. Belanger
Lab Manager
Cellular Immunology
Vaccinex, Inc.
1895 Mt. Hope Ave.
Rochester, NY 14620
email: tbelanger@vaccinex.com
www.vaccinex.com
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