NFkB activation by FACS

David Basiji dbasiji at amnis.com
Thu Feb 9 19:42:45 EST 2006


Gerhard,
As you mentioned, you can use the ImageStream very effectively for NFkB
translocation assays. There will be a paper coming out by George, et.al. in
the Journal of Immulogical Methods within the next couple of months
describing NFkB an translocation in human dendritic cells. I'll post the
reference when it comes out.
Best,
David

David Basiji, Ph.D.
CEO
Amnis Corporation
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From: Nebe-Von-Caron, G [mailto:g.nebe-von-caron at unipath.com] 
Sent: Thursday, February 09, 2006 4:46 AM
To: cyto-inbox
Subject: RE: NFkB activation by FACS


There was an article by S. Foulds long time ago apparently doing just that
and from my understanding several people tried to repeat it w/o success. We
even offered her money to set it up at our lab at the time to no avail. The
problem is that you really want to see translocation and flow does not give
you that info unless you could set up some form of energy transfer. You
would be better off by doing on a LSC or the imagestream - if you have
one.However, you might try a modified method of cytoplasmic stripping
(Enhanced Expression of Intranuclear NF-[kappa]B in Primed Polymorphonuclear
Leukocytes in Systemic Inflammatory Response Syndrome Patients. Journal of
Trauma-Injury Infection & Critical Care. 54(2):253-260, February 2003)

Another way might be to use a probe sequence that identifies the
dissociation of the complex which is the activation of the NFkB but not the
translocation. I actually found a paper by a German group who had
synthesised such a probe including label and did show that successfully
using microscopy. It might be more interesting for looking at other
cytoplasm events. Being my usual self I don't remember the reference but
that I sent it to Laura Dash at Glaxo around 2000. So if we are lucky she
has kept the email and reads this one, otherwise I might have to try and
upload my old PST archives.
Good luck with it and please tell me if you succeed
Gerhard
 -----Original Message-----
From: Galen, Benjamin [mailto:benjamin.galen at mssm.edu] 
Sent: 07 February 2006 20:24
To: cyto-inbox
Subject: NFkB activation by FACS



Hi,
Thanks to your help last year, I have been successfully phenotyping (and
quantifying) the inflammatory infiltrate into mouse vaginal tissue after
certain treatments.
I would also like to detect NFkB activation in these same cells.  Could I
stain for the p65 subunit by FACS?  What would you recommend for a
permeablization protocol?
Would the collagenase digestion that I use to isolate the cells from tissue
interfere with the accuracy of this assay?
Thanks,
Benjamin Galen
Mount Sinai School of Medicine 

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