>From what I understand Caltag, Beckman Coulter and Sigma have a blocking
component in their antibodies that inhibit the Fc receptor mediated binding of
this dye. To what extend any of their reagents will inhibit Fc receptor
labelling I do not know.
Regards
Gerhard
P.S.
The purdue mail archives can be most efficiently searched via
http://www.alltheweb.com. Searching on the purdue web site does not give the
same hits.
Looking for
+pe-cy5 +Fc +purdue
produced in hit 5 the relevant mail. If you complete the year (1997 instead of
97 due to the change of their directory structure) you get the mail below
Purdue Cytometry Mailing List: Re: PE-CY5 BINDING
Re: PE-CY5 BINDING Thomas Delohery (t-delohery@ski.mskcc.org) Tue, 19
Aug 1997 12:25:39 0530 Messages sorted by: [ date ] [ thread ] [ subject ] [
author ] Next message: KUKURUGA@medmail.med.umich.edu: "query: antisera
to 11-Beta-hydrosteroid
http://www.cyto.purdue.edu/hmarchiv/97/1917.html
Thomas Delohery (t-delohery@ski.mskcc.org)
Tue, 19 Aug 1997 12:25:39 +0530
Messages sorted by: [ date ] [ thread ] [ subject ] [ author ]
Next message: KUKURUGA@medmail.med.umich.edu: "query: antisera to
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Previous message: Hoffmann, H. J.: "RNA amounts by flow"
>There is a letter to the editor in BLOOD , September 1996,
>15:88(6):23558-61 by van Vugt, MJ et al which shows that PE-CY5
>binds SPECIFICALLY to the Fc(gamma)R1 receptor (CD64). These
>experiments using mouse B cells transfected with different human Fc
>receptors showed that only FcR1 cells bound PE-CY5. Whole blood
>showed much less binding than isolated cells. Their recommendation
>was that any antibody that you are using labelled with PE-CY5 be tested
>for FcR1 binding.
>In our hands blocking with heat inactivated normal human AB serum will
>significantly reduce the Fc binding in humans.
>
>Mike Keeney
>London Health Sciences Centre
>London Ontario Canada
along the same line:
Takizawa, F. Kinet, J.P. Adamczewski, M.
D 1993
T Binding of Phycoerythrin and Its Conjugates to Murine Low Affinity
Receptors for Immunoglobulin-G
%J Journal of Immunological Methods
%C PO Box 211, 1000 AE Amsterdam, Netherlands
%I Elsevier Science Publishers BV
%V 162
%N 2
%P 269-272
%@ 0022-1759
%K Phycoerythrin; Flow Cytometry; Fc Receptor; (Mouse); FC-RECEPTORS; MACROPHAGE
%X Conjugates of R-phycoerythrin are widely used for immunohistochemistry,
especially for two-color flow cytometry. Their use is however limited by
their apparent tendency to bind non-specifically. Using cells transfected
with cDNAs for the murine low affinity receptors for immunoglobulin G
(FcgammaRII and -III) and cells naturally expressing these receptors, we
demonstrate that R-phycoerythrin and its conjugates bind specifically and
inhibitably to FcgammaRII and -III. Immunofluorescence stainings of cells
bearing these receptors, such as macrophages, monocytes, neutrophils, mast
cells, subsets of T cells, and natural killer cells, may therefore not
reflect the binding of antibody to antigen, but rather the binding of
R-phycoerythrin to the receptors.
%W M Adamczewski, Niaid, Molec Allergy & Immunol Sect, Twinbrook II, RM
108, 12441 Parklawn Dr, Rockville, MD 20852
--
==============================================================================
Thomas Delohery |Internet: t-delohery@ski.mskcc.org
Supervisor, Flow Cytometry Core Facility | Phone: (212) 639-8729
Memorial Sloan-Kettering Cancer Center | Fax: (212) 794-4019
1275 York Ave. Box 98 |
New York, NY 10021 |
==============================================================================
-----Original Message-----
From: Douglas S. Smoot [SMTP:rin0dss@bumed30.med.navy.mil]
Sent: Thursday, April 12, 2001 9:10 PM
To: Cytometry Mailing List
Subject: PE-Cy5 Backgrounds
Just a quick questions. I have had trouble phrasing the search properly of
the e-mail discussions, but there was a discussion a few years ago
concerning high backgrounds for PE-Cy5 staining. (The samples are coming
up positive with Isotype staining - I know, Isotype staining might not be a
true negative, but that is a different discussion. :-)) Is there a solution
for the high background other than using some other fluorochrome like PerCp
which doesn't have this problem, as I understand it. Thank you in advance
for your help.
Doug
{-----------------------------------------------------------------------}
Douglas Smoot
NIDDK-Navy Transplantation & Autoimmunity Branch
Naval Medical Research Center
AFRRI Building 46, Room 2415
8901 Wisconsin Avenue
Bethesda, MD 20889-5603
voice: 301-295-1843
fax: 301-295-6484
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