Thank you to all who responded. I had a couple of requests to post the replies, so here they are. They mostly fell into three major groups. 1) Some preservative could be affecting the results. We always dialize the Abs that are going into the incubation step to get rid of the Na-azide, so I don't think this is the problem. 2) Incorrect compensation. Well, I don't have proof but there's three of us here with about 30 years of combined experience, so you'll just have to take my word that the compensation is being done properly. 3) Try adding other Abs to see the effect. One suggestion was to try adding mIgG Abs to see the effect but I don't think that would tell us anything, but it did give us the idea (which was suggested by someone else after we came up with it - always good to get outside confirmation <g>) to add unlabeled CD4/CD8 to see whether it is a binding issue or a quenching issue. If we get a decrease with the unlabeled antibodies, then it's a binding issue, i.e., the binding of other Abs internally is interfering with the binding of the anti-IFNg Ab. If we don't get a decrease with the unlabeled but do with the labeled, then it's a quenching issue, i.e. the *fluorescence* of the other IC Abs is interfering with the fluorescence of the IFNg Ab. We haven't yet had the chance to try this experiment but I'll be happy to post the results, once we get around to it, if anyone is interested. There were a few other responses: Try using plate-bound anti-CD3 and anti-CD28 to stimulate instead of PMA/Iono. However, we're trying to compare our results to some others obtained with PMA/Iono so we really need to stay with this procedure. Problems with uneven (i.e. not consistent from sample to sample) cell numbers. This doesn't seem to be the problem. Thanks again to all who responded. cheers- Regina Regina H. Harley, M.S. Lab Supervisor - Flow Cytometry Facility Center for Vaccine Development University of Maryland - Baltimore 685 West Baltimore Street Baltimore, MD 21201 (410) 706-7376 FAX: (410) 706-6205 rharley@medicine.umaryland.edu
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