Date: Fri, Feb 28, 1997 1:07 PM EDT From: ThatJack Subj: Re: Intracellular % Formula To: cyto-inbox 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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