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What you can do is to add your BD CD14-PE
1- at 4 degrees after having incubated your cells with the MA at 37
2- at 37 degrees after having incubated your cells with MA at 37
If both still are negative for PE then probably the epitope of the BD
CD14 has been internalised (Dako's not being internalised). At this stage
you can permeate the cells (you can use FACSLyse for 10 min on your
cells) and re-incubate them with BD CD14-PE;
if still again your cells are negative,then your MA probably competes
with BD CD14 for the same epitope.
Good Luck and tell us what you get,
Christian Awaraji
Department of Pathology and Laboratory Medicine
American University of Beirut - Medical Center
e-mail:Christian Awaraji <ca00@aub.edu.lb>
voice:+961-3-771314
> Subject: phagocytosis
> Author: paukovic@mbcmr.unimelb.edu.au at INTERNET
> Date: 04/02/97 01:34
>
>
> Hey ho Flow-ers,
>
> We have an established phagocytosis protocol, which HAS worked well for us.
> The organism being used is Mycobacterium avium-FITC labelled (MA-FITC),
> which is phagocytosed by HIV- and HIV+ monocytes. (a commercial quenching
> agent is being used.) Of recent times we have switched to a Becton Dickinson
> (BD) CD14-PE antobody (Ab) from a DAKO CD14-PE. The problem encountered is
> that where as once I used to get staining at 37C with DAKO CD14, the BD Ab
> fails to do this at the same temperature, however the BD Ab stains
> beautifully at 4C.
> So the dilemma?
> Has anyone come across inhibition of binding of BD Abs, (or any other Ab for
> that matter) when working with MA at 37C (or any temp). Is it common for BD
> Ab to become inactive if kept at 37 for only 10mins (in our protocol) with
> MA or any other organism?? Does MA have properties which inactivate this
> particular BD CD14-PE...at 37C?? Is there a chance of MA digesting PE at
> 37C, rendering it non fluorescent but still bound??
> (and oh yes, the antibody hasn't expired yet)
> Fanx... In advance
> Geza Paukovics - Flow Laboratory - AIDS Pathogenesis Research Unit
> Macfarlane Burnet Centre .***. .***. .**
> PO Box 254 _--_|\ * | | | * * | |
> Fairfield, VIC 3078 / \ * * | | | * * | | |
> AUSTRALIA. \_.--._/ * * | | | * | | | *
> ph. (+61 3) 9282 2132, O '***' '***'
> FAX (+61 3) 9282 2100
>
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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;
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