From: Calman Prussin (CPRUSSIN@atlas.niaid.nih.gov)
Date: Mon Sep 09 1996 - 07:50:06 EST
Question: are you using PE for CD4/ CD8 or for a mysterious memory marker optimal only in PE? It may be simplest to try IFN in FITC if possible. However, if this is still not to your liking, consider using biotinylated anti-IFN-gamma and then coming back with streptavidin Pe/Cy5. Biotinylated mAbs, in the context of intracellular staining have a lot of background due to: 1.) endogenous biotin in the cell 2.) they are just plain stickier. IFN is a very bright stainer, so even if you don't block the endogenous biotin (see J.I., 154 4292-4301) you will still have an acceptable level of signal. Don't forget to eliminate sources of free biotin that will bind up your straptavidin, such as serum. Calman Prussin NIAID/ NIH >---------- >From: Ress, S, Stan, Dr >Sent: Sunday, September 8, 1996 10:26 >To: Cytometry Mailing List >Subject: 3-col surface marker/i.c. cytokine staing > > >Hi all, > >We would like to stain human PBL for simultaneous dual color surface >markers, and IFN gamma intracellular expression, using a coulter >epics profile 2. We would like to quantitate IFN -G production by >memory T cells and CD4/8 subsets in this way. Most of the commercial >mca to surface markers will be FITC/PE combinations, does anyone know >of any available IFN-gamma MCA conjugated to a third fluorochrome >which could be used in these experiments? I understand that for I.C. >work PE should be used, not FITC, but this won't work as PE-conjugated >MCA will be used for surface marker as indicated. >Any advice? > >Thanks, > >Stan. >Stan Ress >Clinical Immunology laboratory >Dept of Medicine >University of Cape Town >Cape Town, South Africa > >e-mail: sress@uctgsh1.uct.ac.za >Phone: Intern + 2721-4066201 >FAX : Intern + 2721-4486815 >
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