Hello, If you think that you would get better results by measuring PI in FL3-A, but you are not doing it because of the lack of pulse processing in FL3. Perhaps you could change the FL2 and FL3 wires vice versa and get the FL3-A and FL3-W. Greetings, Mika Korkeamäki BioCity Turku David L Haviland <David.L.Haviland@uth.tmc.edu> sanoi: > > Hi: > > I have a user that is looking at CR1 (CD35) as a function of cell cycle, so what > has been attempted was first surface staining for CD35 with a Monoclonal direct > FITC conjugate followed by 1% PF fixation, then saponin permeabilization. Then > PI (5ug/ml) is incubated with RNAse-A for 20 mins. Separate samples (CD35 and > PI alone) are run to adjust compensation as the Calibur doesn't have an FL3-Area > option so we collect in FL2. > > The problem is that when CD35 is run alone, we see expression that is 3rd decade > expression. When the dual labeled sample is run we see a significant drop > (order of magnitude) in the expression of CD35. Does anyone know if it is a > matter of FRET between FITC and PI, something about epitopes and CD35, or have > any suggestions? I've tried this before with other markers, Class-I HLA (W6/32) > and CD44 and did not see a drop in signal which makes me wonder if what we are > observing is unique to CD35. > > Any thoughts? > > TIA > David > =========== > David L. Haviland, Ph.D., Asst. Prof. Immunology > University of Texas - Houston, H.S.C. > Institute of Molecular Medicine, R907 > 2121 W. Holcombe Blvd., Houston, TX 77030 > 713.500.2413-Voice//713.500.2424-FAX > ----------------- > If everything seems to be going so well, you have obviously > overlooked something. > ========== > > > -- ________________________________________________________________ Ilmainen Internet @ http://www.nic.fi/
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