CR1 (CD35) Expression sytometria at
Thu Oct 26 02:56:17 EST 2000


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.


Mika Korkeamäki
BioCity Turku

David L Haviland <David.L.Haviland at> 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?
> 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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