I don't know if there is an "accepted" method, but my approach is to run a test sort with beads similar in size to my cells, and tweak until that recovery is satisfactory. The only time I was able to calculate recovery directly was when we sorted GFP labelled lymphs--worked beautifully. I had a lot of trouble trying to sort GFP labelled bacteria, and one of the problems was that I had to use sub-micron beads for setup, and I couldn't verify their arrival in the wells--much too small and dim. Recently the PI informed me that the project was doomed anyway, because their clones that were supposedly expressing GFP turned out to be negative. (So I like to think my sorting worked for that experiment too!) Good luck-it's loads of fun! Steve On Thu, 1 Jul 1999, Doug Dooley wrote: > Fellow flowers: > What is the accepted standard for judging the success of "single cell" sorting into 96 well trays? That is, how does one verify that >95% of the wells received only one cell. We are sorting hematopoietic cells on to stroma. Therefore, I do not feel that we will be able to see the sorted cells afterwards. In fact, finding single cells in 96 well trays lacking stroma can be difficult. Is it "kosher" to use fluorescent beads as surrogates? Or, are beads not good representatives of the ability of the sorter to pick out single cells for deposition in a well. Your critical thoughts will be appreciated. Thanks. > Doug Dooley > Stem Cell Lab > American Red Cross, Portland, OR > dooleyd@pdxredx.org > +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ Steve G. Hilliard (706) 542-9474 University of Georgia Cell Analysis Facility flowman@uga.edu URL: http://floweb.cb.uga.edu/Floweb/ +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
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