Try a Pubmed search using "flow cytometry" rather than FACS, there are many papers. We have worked extensively with anti- human IL-5 ICS. See Prussin C. and D.D. Metcalfe. 1995. Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies. J Immunol Methods 188: 117-128. or Elsen L., T. Nutman, D.D. Metcalfe and C. Prussin. 1995. Flow cytometric analysis for cytokine production identifies Th1, Th2, and Th0 cells within the human CD4+ CD27- lymphocyte subpopulation. J Immunol 154:4294-4301. Bottom line: there are fewer IL-5 producers than IL-4 producers, although this depends on the stimulus and other factors. Typically, after PMA/iono we see 3-5% IL-4 positive cells and about one-tenth that number of IL-5 positives (0.5%). That being said, IL-5 stains brighter than IL-4, so these cells are fairly easy to pick up, provided you have a low noise fixation and staining system. Low noise is the key. If your background staining is 1% you will not see the IL-5 producers above the noise. Because the numbers of IL-4 or IL-5 positive cells is so low, you must validate your data with blocking controls. I assume you use a bright fluorochrome for the IL-5 such as PE or APC. In the "bad old days, before there were commercially available directly conjugated mAbs, I spent months trying to get IL-4 staining to work, whereas IL-5 worked the first time out. All the DNAX anti-IL-5 clones (TRFK-5, 39D10, 5A10) work well and are widely available. > ---------- > From: Ress, S, Stan, Dr > Sent: Tuesday, October 16, 2001 15:15 > To: Cytometry Mailing List > Subject: intracellular IL-5 vs IL-4 > > > Hi all, > > A recurring problem in evaluating TH1/2 status is low levels of > detectable IL-4 in stimulated culture sups (ELIZA), many researchers > measure IL-5 for this reason, which is more readily > detectable. I wondered if the same applied to intracellular IL-5 > detected by FACS. Certainly, our own limited experience, and > published papers, seem to indicate low intracellular IL-4. > > A pubmed search for i.c. IL-5 and FACS yielded only 1 paper that > looked at TH2-associated cytokines in schistosome granuloma, here > IL-4 was shown by FACS, IL-5 was not detectable by FACs (although it > was histochemically) and this was attributed to "technical difficulty > detecting i.c. Il-5 by flow...most likely due to in-vitro stimulation > conditions" in the discussion. > > I wonder if others have tried this with similar negative results? > Has anyone found higher IL-5 than IL-4 by FACS? > > I would appreciate any comments or references. > > Stan > >
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:01:35 EST