Lynn, We have also been looking into this in the context of both low-frequency MHC tetramer-binding and peptide-stimulated intracellular cytokine staining events. I've come to the conclusion that positive events occurring at frequencies as low as 0.05%, and probably lower, can be statistically significant if: 1) you have a negative control with sufficiently low background staining (0.01%) and 2) you have collected a large enough number of events (this example would require collection of 100,000 gated events from both the negative control and the test sample). We routinely collect 50,000 CD8+ lymphocyte events for MHC class I tetramers, which in most cases allows us to detect <0.1% staining (if the background is low enough). This was arrived at by doing a Chi-square analysis of background events vs. test events. I would also be interested to hear about other ways of determining the minimum number of events needed to achieve significance. In his intracellular cytokine staining talk at the Methods in Cytokine Biology symposium, Calman Prussin mentioned the importance of collecting large numbers of events and including multiple controls-perhaps he would be willing to expand on those comments? Thanks for posting the symposium announcement on the list, Calman, and for organizing the event- it was a very worthwhile day. Mandy ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Mandy Cromwell, Ph.D. New England Regional Primate Research Center Department of Immunology Harvard Medical School One Pine Hill Drive Southborough, MA 01772 (508)624-8022 >Hello all, > >This question arises from a rather heated discussion following a seminar >yesterday, in which the speaker claimed that flow cytometry is not >useful for analyzing cells that are less than 1-2% of the starting >population. I am sure that with all of the sorting, multicolor analysis, >and multiparameter gating that people do, we can prove this assertion >wrong. > >If you have experience or publications with analysis of events well >below 1% of the starting cell population, could you please share some >examples or references? > >Thanks in advance for your help! >********************************************** >Lynn B. Dustin, Ph.D. >Center for the Study of Hepatitis C >Rockefeller University >Box 64 >1230 York Ave. >New York, New York 10021 >Phone: 212-327-7067 >email: dustinl@mail.rockefeller.edu Mandy
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