Re: Amplification techniques (WAS Question regarding CD40 surface expression level by FACS)

From: Regina Harley (rharley@medicine.umaryland.edu)
Date: Wed Aug 02 2000 - 08:03:50 EST


Regina H. Harley, M.S.
Lab Supervisor - Flow Cytometry Facility
Center for Vaccine Development
University of Maryland - Baltimore
685 West Baltimore Street
Baltimore, MD 21201
(410) 706-7376    FAX: (410) 706-6205
rharley@medicine.umaryland.edu

>>> ttown <ttown@luna.cas.usf.edu> 07/31/00 12:18PM >>>

The specific question is, do these percentages REALLY mean that out of
100 cells, x percentage express CD40?  I tend to think that many more
of
those cells actually express CD40 but that such low expression levels
of
CD40 are undetectable by FACS.  If this is the case, then what do
these
percentages really mean, and is there a reference(s)  for this?

Best wishes,

Terrence Town


This actually is a very good question and is relevant to any low
incidence/low percentage molecule (where the incidence is the number of
molecules per cell and the percentage is the number cells with the
molecule).

We are going to be working with a target that has both a low incidence
and a low percentage. Due to a number of technical issues, the only
fluorochromes we can use are PE and APC (and therefore can't use
Molecualr Probes FITC amp. kit). Does anyone have a working system of
amplification for these fluorochromes? We been trying various multistep
procedures but have been having problems with high background. Any
help/suggestions would be appreciated.

Thanks very much.

Regina



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