David,
My guess that the problem is the saponin permeabilization. I
haven't tried this particular combination, but when combining other surface
antigens with intracellular or PI I have often observed a reduction in
staining intensity. On the other hand some antigens seem to be less or not
at all effected. I would therefore think it is the CD35 + permeabilization.
George F. Babcock, Ph.D.
University of Cincinnati
College of Medicine &
Shriners Hospitals for Children
231 Bethesda Ave.
Cincinnati, OH 45267-0558
At 07:36 PM 10/20/00 +0100, David L Haviland wrote:
>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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