We have been sorting virus specific B cells from peripheral blood. When we single cell sort them, we have the ability to expand them into clones that differentiate and secrete antibody, so that we can double check to see if they really are specific for the selecting antigen. We find that with starting frequencies of 1 in 10,000 or 1 in 100,000 we generally end up with 1 in 10 really being confirmed as specific after the sort. Therefore, the sorts are either highly efficient (10,000-fold enrichment) or terribly nonspecific (90% noise) depending on the way you look at it (depending on whether you are a donut or the hole kind of person). With 1 in 1,000,000 frequency you will be looking mostly at noise. There is some literature on similar work in which investigators have sorted with a single peptide antigen conjugated to two colors, sorting only dual colored cells, which is reputed to greatly enhance the specificity of the sort. Jim Crowe Vanderbilt -----Original Message----- From: Todd Belanger [mailto:tbelanger@vaccinex.com] Sent: Mon 8/12/2002 9:23 AM 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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