> -----Original Message----- > From: Jan Grawé [SMTP:jan.grawe@rudbeck.uu.se] > Sent: Friday, 22 November 2002 7:33 PM > To: Cytometry Mailing List > Subject: Cytokine measurement in transfected cell line > > Jan, > > We regularly stain for i/c cytokine production in lung and gut epithelial > cell lines. > We use BD's i/c cytokine method. Culture cells overnight in the presence > of Brefeldin A. Centrifuge cells then FACSPerm for 5 mins, followed by > BSA/PBS wash. PharMingen's moabs are ideal for cell lines but titre the > antibodies. Usual concentrations we use are around 1:10. Have not tried > IL-15, but works fine for IL-1, IL-4, IL-6, IL-8, IL-10, IL-12, TNFa, > GM-CSF. Controls are important (pos and neg) at least until protocol > established. > > Regards, > > Greg Hodge PhD > Senior Medical Scientist, > Flow Cytometry Laboratory > Haematology Department, > Women's and Children's Hospital > 72 King William Rd, > North Adelaide, > South Australia 5006 > > ph 08 81616089 > fax 08 81616043 > hodgeg@mail.wch.sa.gov.au > > -------------------------------------------------------------------------- > --------------------------- > > > > 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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