Greetings Flowfolk,
I need some input: I'm working on a project designed to sort rare-event
tumor cells (of epithelial origin) from peripheral blood samples. The
frequency of tumor cells in these specimens may be as low as 10E(-6) or
10E(-7).
Currently, I am using an anti-CD45-RPE/Cy5 mAb as a negative stain, and
have tried several different positive stains:
EpCAM which I have kit-labeled with AlexaFluor 488
Epithelial (Membrane) Antigen with an indirect RPE tag
and am about to try this same mAb as a direct FITC conjugate
The mAb that I kit-labled with AF488 shows up only about a half-decade
brighter on my FL1 channel than my isotype control; I lose too many of my
'precious' rare cells when I utilize the indirect system (due to the extra
washing steps); and I have yet to try the new direct-FITC mAb, since it
only arrived today!
My questions to the group are:
Can anyone suggest any other specific markers (immunological or
otherwise) to tumor cells or even epithelium? I'm looking specifically
for membrane targets since cell fixation is undesirable for the later
stages (post-sort) of this project. I'm hoping that a cocktail
of markers would help boost the signal of my rare cells.
Does anyone have any experience with signal amplification (the
only example of which I am remotely familiar with is Tyramide) that
could be used in suspensions of live cells?
Can anyone forward a good protocol for blocking Fc on my leucs?
And of course, any other helping hints about rare event work,
since I am relatively inexperienced with flow.
Best Regards,
Thomas
________________________
Thomas P. Ahern
University of Vermont/
Vermont Cancer Center
E-315 Given Building
Burlington, VT 05405
(802) 656-2218
tahern@zoo.uvm.edu
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