Hi Maryalice I just wanted to let you know about an alternative to intracellular staining (ICS) for flow cytometry analysis of cytokine secreting cells, such as mouse IL-10. The MACS Cytokine Secretion Assay uses a cell surface capture system to catch secreted cytokines as they are released from the cell. After a subsequent labeling step with a fluorochrome-conjugated cytokine-specific antibody, the cells can be analyzed by flow cytometry. The cells remain viable with this assay and can be further enriched with MACS beads based on cytokine secretion. The enriched cells can be cultured for use in further experiments. The flow analysis results are comparable to ICS but with the MACS enrichment step, assay sensitivity in greatly enhanced, with detection of antigen-specific T cells as low as 1 in 10E6. A number of investigators in the NIH community and other large research centers around the country are using this system successfully (see attached reference list). If you have further interest, our website cytokine page gives an overview of the assay systems. http://www.miltenyibiotec.com/index.php?site=products-cytokine I have attached for your review, the datasheet for the mouse IL-10 assay. We currently have PE-conjugated kits for human and mouse IFNg, IL-2, IL-4 and IL-10. We also have some FITC and APC conjugates for measuring dual cytokine secretion from the same cell or for use in combination with Tetramer analysis. AS an aside, we will present a TUTORIAL on the Cytokine Secretion Assay at the ISAC meeting in San Diego, on Monday, May 6 at 3:30 PM in Pacific Salon One. I hope to see you there. Please feel free to call me with further questions. Roger A. Burger, PhD Technical Marketing Scientist Miltenyi Biotec Inc. Auburn, CA (530 )887-5351 www.miltenyibiotec.com > -----Original Message----- > From: Stetler-Stevenso, Maryalice (NCI) [mailto:stetler@mail.nih.gov] > Sent: Wednesday, February 20, 2002 11:02 AM > To: Cytometry Mailing List > Subject: IL-6 and IL-10 in mice > > > I need to measure IL-6 and IL-10 in B cells in transgenic > mice. We don't > want to stimulate them- they should have higher levels without > stimulation. Has anyone done this before in mice and if so- > what did you > use- momensin or Brefeldin A and for how long? Any tips for these 2 > specific cytokines and for mice in general would be helpful. > > Maryalice Stetler-Stevenson, M.D., Ph.D. > Chief, Flow Cytometry Unit > Laboratory of Pathology, NCI, NIH > > Sometimes you're the windshield, sometimes you're the bug.
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