Tim: The problem with putting the threshold too high is that the event is not digitized and therefore not classified and as a result it is randomly sorted with your cells. You will always get some debris appearing in your post-sort plots, most probably due to debris that was stuck to your cells when they were sorted. If you want to keep your investigators happy with not seeing debris in their post sort samples turn up the threshold after the sort. I have seen a much greater amount of debris in the post sort samples when I keep the threshold high during sorting for the reasons described below. There is a discussion of this in the sorting chapter in Current Protocols in Cytometry. Best Regards-Joanne Director, Flow Cytometry Core University of Virginia Jordan Hall Room 7065 1300 Jefferson Park Avenue Charlottesville, VA 22908-0734 flowcytometry@virginia.edu (434) 924-0274 Office (434) 982-1071 Fax On Fri, 14 Mar 2008 13:41:12 -0400 "Bushnell, Timothy" <Timothy_Bushnell@URMC.Rochester.edu> wrote: > > > I was hoping to start a discussion on the use of Threshold in sorting. > > > >For example, on our FACSAria, I like to threshold at 20,000, while the > default is 5,000. When I forgot to turn the threshold up, I know that > my actual 'cell' rate is much slower because of the cellular debris, > noise and such at the low end of the plots. Most of this doesn't affect > downstream applications, to the best of my knowledge, but when my > post-sorts show 10% in this debris range, I have some investigators get > concerned over the quality of their sort. > > > > Of course, run the sample with the higher threshold, and the problem > goes away, and investigators are happy. Thus, running at the higher > value is better for the 'look' of the plots and sorts ... and yet what > does that 'debris' really mean in the end of the day. > > > > So I'm curious as to how other groups treat thresholding of the sort > data, and if there are any reasons for me to go one way or the other? > > > > Thanks > > Tim > > > > Timothy Bushnell, Ph.D. > > Research Assistant Professor, Pediatrics and Oncology > > Co-Director, URMC Flow Cytometry Facility > > Office: 585-273-5535 > > Lab: 585-273-1361 > > Cell: 585-690-5157 > >Fax: 585-276-0233 > > http://www.urmc.rochester.edu/Aab/geneped/flow/ > > http://www.urmc.rochester.edu/wnyfug/ > > >Received on Tue Mar 18 14:18:00 2008
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