CR1 (CD35) Expression
sytometria at pp.nic.fi
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.
David L Haviland <David.L.Haviland at uth.tmc.edu> sanoi:
> 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 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
> If everything seems to be going so well, you have obviously
> overlooked something.
Ilmainen Internet @ http://www.nic.fi/
More information about the Cytometry