The other issue that comes up is autofluorescence and brightness of cytokine staining. CHO cells and many tumor lines have much more autofluorescence than do fresh ex vivo lymphoid cells. Your IL-15 signal will have to be brighter than the autofluorescence. That leads me to a second point: per cell production. One reason that intracellular staining works so well for some T cell cytokines that are produced at low levels (i.e. IL-4 & 5) is that there are few T cells producing the cytokine, but those that are produce a good deal and stain quite brightly. The brightness actually pulls the signal out over the noise of the autofluorescence/non-specific staining. For example, imagine you detect 100 pg/ml of both IL-4 and IL-15, the former in CD4 T cells, the latter in transfected CHO cells. For the T cells that 100 pg is being produced by say 2.5% of the cells, whereas with the CHO cells, the IL-15 is being produced by 100% of the cells. Thus, for the T cells the per cell cytokine production is 40 times greater and will thus have 40 times brighter fluorescence. Obviously, this is a contrived example, but you get the idea. Calman > -----Original Message----- > From: Jan Grawé [mailto:jan.grawe@rudbeck.uu.se] > Sent: vrijdag 22 november 2002 10:03 > To: cyto-inbox > Subject: Cytokine measurement in transfected cell line > > > > Dear All, > > I have a user who wishes to detect cytokine production in a > transfected cell line, see below . Use of at least one commercial > permeabilisation kit has failed. > Literature and email archive searches have yielded virtually no > information, so any insights are appreciated! > > Jan Grawé > > >Hi, > > > >I am trying to develop an intracellular staining protocol to detect > >intracellular protein in a tumor cell line > >by flow cytometry. I have checked the literature and the technique > >seems to be used almost entirely for > >lymphoid cells. Does this mean that other types of cells are > >difficult to permeabilize? > > > >Does anyone have any experience detecting intracellular protein > >within tumor cells using flow cytometry? > > > >I am transducing the cell line MB49 (murine bladder carcinoma) with > >recombinant adenovirus expressing IL-15, > >and I would like to detect IL-15 by intracellular staining. > > > >I would really appreciate any insight and/or comments. > > > >Thanks, > >Christina Ninalga > > > -- > -------------------------------- > Jan Grawé > Cellanalyslab > Rudbecklaboratoriet/C5 > Uppsala Universitet > Dag Hammarskjölds väg 20 > 75185 Uppsala > > tel: +(0)18-4714656 > mobil:+(0)70-2577874 > fax: +(0)18-552739 > epost: jan.grawe@rudbeck.uu.se > http://www.rudbeck.uu.se/cellanalys > >
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