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On Thu, 15 Aug 1996, Bob Craggs wrote:
> Date: Thu, 15 Aug 1996 14:03:42 +0200
> From: Bob Craggs <Bob.R.I.Craggs@CHARNWOOD.GB.ASTRA.COM>
> To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
> Subject: Staining Human Dendritic Cells
>
>
>
> 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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