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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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