Dear Jan, although it is difficult to judge where the problem can be found some short remarks; 1. It is possible to use almost all cell type for intracellular staining. Do you have a positive control? A protein of, produced for the adenovirus (to check your transfection)? Or a general intracellular protein, like actin (to check staining protocol e.g.. fixation and permeabilization). 2. Are the cells stimulated to produce IL-15? PMA/ionomycin should work. 3. Does the antibody recognize the fixed protein? and 4. A short Pubmed search learns that intracellular IL-15 detection is possible: International Immunology, Vol. 14, No. 8, pp. 917-924, August 2002. Sugiura et.al. show intracellular detection on cells cultured in chamber slides. When this works it also should be possible in flow cytometry. I will be glad to help with further trouble shooting. Don't hesitate to contact me. Kind regards, Joost Schuitemaker http://home.wanadoo.nl/flowcytometry -----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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