Staining Human Dendritic Cells
Bob Craggs (Bob.R.I.Craggs@charnwood.gb.astra.com)
Thu, 15 Aug 1996 14:03:42 +0200
Greetings to the Cytometry list.
As a relatively new member of the list, this is my first
request for help.
Colleagues have asked me to perform phenotyping on human
blood-derived dendritic cells that they have produced in
culture after 8 days in the presence of GMCSF and IL4.
My first attempt revealed very high levels of
autofluorescence (detectable in both the FITC and PE
channels) in all the large cells (approx. 60% of the total
population) that they had scraped off their plates. This was
confirmed by fluorescent microscopy. With the gains set so
that the background of the unstained small cells was at the
left of my scale, the autofluorescence was at the end of the
2nd log of intensity on my Coulter XL cytometer. Consequently
only those markers with intensity greater than the
autofluorescence showed any 'specific' staining (e.g.: Class
II MHC & CD40).
I am following the methods and markers as published in the
following paper:
Sallusto & Lanzavecchia. J Exp Med 179: 1109-1118 (1994).
Romani et al. J Exp Med 180: 83-93 (1994).
Blauvelt et al. J Invest Dermatol 106: 1047-1052 (1996).
None of these authors make any reference to problems with, or
needs to quench, autofluorescence.
So please, has anyone any experience of similar problems and
knows how to solve them, or has anyone any suggestions that
might be worth a try? Are there methods for quenching
cytoplasmic staining while leaving surface staining intact?
Could it be something as simple as the cells taking-up the
Phenol Red from the RPMI medium?
Thanking you all in advance,
Bob Craggs,
Dept of Biochemistry,
Astra Charnwood,
Bakewell Road,
Loughborough, Leics,
LE11 5RH, U.K..
Tel: +44 1509 64 4055, FAX: +44 1509 64 5557,
E-mail: Bob.Craggs@Charnwood.GB.Astra.Com
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