Hi Jill, FYI - we do try and separate end user recovery from sorter yield to monitor where cells go. The sorter yield on any setup might best be done by sampling the waste stream and calculating how many of the possible sort window events were actually deflected. For example, if we're sorting 8% of the total cells, a 80% yield lets us expect 8.0 - 6.4 = 1.6% still in the waste after sorting. We often monitor "real" aborts with the counters as a sobriety check. On the Vantage SEs, the numbers agree very closely. On the older systems, deadtime aborts resulted in more cells not being accounted for and "lost" in the the sorting process... We don't monitor the waste evey time. We do it periodically under a variety of conditions to get feedback on the accuracy of the counters. Once we know the system efficiency by monitoring the waste, we can them determine how many sorted events (ie, removed from the waste) are recoverable. As you know, this varies considerably with cell type, condition, and investigator. Most labs here recover from 75% to 99% of the cells we assume were "sorted" into their catch tubes. A simple example: If an investigator comes with 1e6 cells, and wants to sort 8% (+), we might answer their "how many will I get back" questions by estimating 80% or better on the instrument yield and 90% on their recovery from the catch tubes. They would get ~ 80,000 (+) run, 64,000 (+) sorted, and 57,600 (+) recoverd after centrifugation. 57,6000/80,000 is only 72% recovery, so giving the user reality checks is usually appreciated. We carefully titrate flow rates to control aborts whenever yield is an issue, and experience has taught us which cells may be "problem cells" in terms of recovery from catch tubes. Joe On Tue, 7 Dec 1999, Jill Martin wrote: > > Hi Gang, > Just to get this in black and white, as most non sorting > experts look at the numbers on the Vantage and assume that is what they > are going to get. No one listens to me. Should the end user be > responsible for doing a cell count on the sorted cells? > > Jill > > > Jill Martin, Manager > Molecular Biology & Flow Cytometry Core > Johnson Research Building > Mayo Clinic Scottsdale > > 480-301-6071 (Voice) > 480 301-7017 (FAX) > email: jmartin@mayo.edu or martin.jill@mayo.edu > > > Joe Trotter Director, Flow Cytometry Mailstop Imm-20 The Scripps Research Institute 10666 North Torrey Pines Rd. La Jolla, California 92037
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