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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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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web