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Glad to chime in with Dearbhaile's comments about assumptions that
"unstimulated cells" mean "cytokine negative cells."
(But this provincial irish-american has to first ask, how in the emerald
world do you pronounce Dearbhaile? Is it the formal form of Darby, as made
familiar to most americans of my generation as the reluctant host of Disney's
Little People? Do you go by Bud or Biff or Skip as you probably would here?
Or Sweety, Punkin or Lil' Puddin'?)
Anyway Chuck, our experience so far is that you're right, unstimulated
lymphocytes don't show cytokine signals and so may serve as part of a
non-specific control panel. Monocytes, on the contrary, are easily
stimulated after isolation, and very commonly show TNF, IL1alpha or beta, IL8
and IL6. Anything which blocks adhesion can help to keep them quiet (we used
to culture them in teflon bags), but even maintained in whole blood for a few
hours ex vivo gives us signal.
Another useful control, in addition to competition with soluble cytokine or
"cold" antibody as described by Cal Prussin in this and other fora, or
isotype controls as are commonly used for everything, is to leave out the
Brefeldin A. It surprises me some, but even stimulated cells typically fail
to show cytokine signals unless you lock them up ( I don't have any parallel
experience with monensin, Cal?).
I suppose it says that the residence time for processed cytokine must be very
short, or that the BFA somehow aids in the availability of cytokines after
fixation/permeabilization beyond trapping. Anyway, it can be a useful trick
when doing preliminary characterizations of antibody behavior.
Clean Signals to you all,
Uncle Jack.
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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;
Web