Re: sorting rare events- 1 in a million or more

From: Ann Atzberger (ann.atzberger@EMBL-Heidelberg.de)
Date: Wed Aug 14 2002 - 11:39:49 EST


Hi Todd,

you haven't really given any info regarding what kind of cells you need to
sort. As mentioned in previous posts the best option is to pre-enrich if
possible. If not possible you need a strategy:
one of the things I do for rare cell sorting is to put as wide a gate as
possible across the area where the target cells are expected to fall and
include 5% to 10% of the neg population. This is simply to recover as much
of the target cells as possible and also to acquire enough cell to keep
them  happy in culture.
The cells usually go back into culture and are resorted for purity a few
days later. If your cells are not going into culture you could if you have
enough rerun them through the sorter again to purify.
Just how pure do the cells need to be? you do not always need 99% purity-
depends on the application. A bit more info if possible would help.
Do some trial runs by diluting samples 1:100, 1:1000 etc see how far you
can go and tell us.

regards
Ann


At 10:23 12.08.02 -0400, you wrote:
>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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