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Does anyone out there have any experience with detection of NFkB using flow
cytometric methods? Presently, we are using rabbit polyclonal antibodies (Santa
Cruz) to the p50 and p65 subunits followed by goat anti-rabbit fitc conjugate.
The cell line I am using is called RAW (murine macrophage) and I am using a
fixation/pemeabilization method that calls for 2 min. 1% paraformaldehyde/ 20
ug/ml lysophosphatidyl choline, 10 min. absolute MEOH, and 5 min. .1% Nonindent
P-40. From the way the cells look under the scope, it appears that I have
whole, permeable cells after this fixation. This is ok for measurement of both
nuclear and cytoplasmic NFkB. I am interested in translocation of NFkB into the
nucleus as well, so it would be advantageous for me to be able to isolate the
nuclei before staining.
If anyone else is into this type of research and/or has any methods,
suggestions, etc., please respond.
Thanks in advance,
Connie Wagner
Monsanto Environmental Health Lab
645 S. Newstead Ave.
St. Louis, MO 63110 phone: (314) 694-7971
fax: (314) 694-7938
email: cawagn@ccmail.monsanto.com
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