Hi John This is a guess. Have you looked at a FL1 vs. FL2 plot? It could be that that procedure creates a high autofluorescence, or non-specific fluorescence, or your cells just have a high autofluorescence. The autofluorescent 'banana' on the FL1/FL2 plot could then make your two populations become one broad peak when looking at just one parameter. the fact that your infected cells are a broad peak may mean you really have two populations under that peak. Also, put some of those permeabilized cells fro flow on a slide and see what you see. Hope that helps Artur On Thu, 14 Jan 1999, John Parker wrote: > > Hi Flowers, > > I am a relative neophyte to flow and I am having some problems developing > an assay to detect parvovirus (canine parvovirus infected cells). I have a > purified mouse mab against the capsid protein which I have directly > conjugated to Cy2. When I use this ab to stain infected cells on slides, I > see a very clean separation of infected from non-infected cells, almost all > the infected cell staining is in the nucleus and non-infected cells have no > or negligible background. I am using mv1Lu cells, a mink lung cell line. > However, when I trypsinize the cells from dishes and fix with 3% > paraformaldehyde, then permeabilize with 0.1% triton in PBSA , stain and do > flow. What I see is one broad peak which is shifted to the right on FL1 > when compared to a narrow peak of mock infected cells treated the same way > and stained with the same antibody. My interpretation of this is that I am > not fixing the antigen in the cells and that it subsequently leaks and > coats the surface of non-infected cells, hence the broad peak shifted to > the right. My questions are: > Is my interpretation reasonable, any other ideas? Any ideas on how I can > solve this problem or pin-point what is happening? > > Thanks in advance for your help > John >
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