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. ==========
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