Greg, Where do you get this 24h figure? Virtually the entire literature of intracellular staining for these cytokines after PMA/ionomycin has used 4-6h. Although your own personal experience is important, I think in this kind of forum you need to give the readers some perspective on published work. Go to virtually any review of intracellular cytokine staining and you will find a 4-6h time point used for the stimulation conditions above. Both of the below early works cited below did kinetics which support 4-6 h time points. In particular, some of our early work was performed on PBMC from eosinophilic donors which had high frequencies of IL-4/5 positive cells, making these kinetics easier to observe. I use a 6h time point routinely and since I study allergic diseases, I am most interested in getting optimal IL-4 and 5 detection. Prussin, C. and D. D. Metcalfe (1995). J Immunol Methods 188: 117-28. Picker LJ et al. Blood 1995 Aug 15;86(4):1408-19. Twenty four hours is generally not a good time point for intracellular staining after PMA/iono for the following reasons: 1. Down modulation of CD4 increases with time and is problematic at time points much beyond 6-8 h. 2. Cell death increases with time and is problematic after 8-10h. At 24 h, 30-50% of your cells are dead or dying. 3. IL-4 and IL-5 staining peak at around 6h, not 24h. 4. Autofluorescence may increase with extended time points. Often novices use the time points typical for ELISA assays, 24 h. There is a big difference between the kinetics of ELISA and intracellular staining . In the former, cytokine must be secreted and the cytokine "collects" in the supernatant- you are in a sense observing the summation of cytokine secretion. In intracellular staining there is no secretion. You are observing a transient phenomenon (intracellular cytokine before it has been secreted) that does not display this summation phenomenon. IL-4 is probably one of the most difficult cytokines to detect. My guess (assuming that your antibodies are known to recognize I/C IL-4 and IL-5 in Cynos) is that IL-4 and IL-5 are difficult cytokines for novice users to detect, even with all the simplicity that has been added by the commercially available kits. I would suggest you get a good positive control for IL-4 and IL-5 staining (Pharmingen sells some, others may?) and try those for starters. Read some of the review articles or Current Protocols in Immunology/Cytometry chapters. You may get more easily observed staining if you can gate on a memory cell population such as CD27 negative T cells. Essentially all IL-4 and IL-5 staining is limited that subset which is only 5-10% of all CD4 T cells. Reviews: Maino VC, Picker LJ Cytometry. 1998 Oct 15;34(5):207-15. Review. Prussin C. and P. Openshaw. 1998. Detection of intracellular cytokines by flow cytometry. In: Current Protocols in Immunology, J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W. Strober and R. Coico, eds. John Wiley & Sons, New York, NY. 6.24.1-6.24.11. There is also a chapter in Current Protocols in Cytometry for which I do not have a reference. > _______________________ > Calman Prussin > Laboratory of Allergic Diseases > NIAID/ National Institutes of Health > > ---------- > From: Hodge, Greg (HAEM) > Sent: Sunday, October 22, 2000 5:57 PM > To: Cytometry Mailing List > Subject: RE: Intracellular ifn-gamma and IL-4/5 > > > > > > ---------- > > From: andrew.head@covance.com > > Sent: Saturday, 21 October 2000 4:43 > > To: Cytometry Mailing List > > Subject: Intracellular ifn-gamma and IL-4/5 > > > > On the 21st October, Andy wrote: > > Dear flowers > > > > I am trying to type Th1 and Th2 cells in Cynolmolgus macaque > > lymphocytes using intracellular IFN-gamma, IL-4 and IL-5 > > Does anyone have any experience of this ? > > Using PMA activation(5 hours plus monensin) I have had some success > > with IFN-gamma, but no luck with IL-4 or 5.Am I supposed to use a > > different activator for these two monoclonals to work? or incubate > > for > > a different length of time? > > Any suggestions would be appreciated. > > > > Thanks > > Andy > > > -------------------------------------------------------------------------- > > --------------- > > > > Andy, > > I havent worked with Cynolmolgus macaque lymphocytes, but in the > > case of human lymphocytes you will get a better signal following 24 h > > incubation with PMA (25 ng/ml) + Ionomycin (1ug/ml) (with 1 ug/ml BA or > > Mon Golgi block), especially for IL-4, IL-5. > > Hope this helps. > > > > Regards, > > > > Greg Hodge PhD > > email: hodgeg@mail.wch.sa.gov.au > > > > > > ----------------------------------------------------- > > Confidentiality Notice: This e-mail transmission > > may contain confidential or legally privileged > > information that is intended only for the individual > > or entity named in the e-mail address. If you are not > > the intended recipient, you are hereby notified that > > any disclosure, copying, distribution, or reliance > > upon the contents of this e-mail is strictly prohibited. > > > > If you have received this e-mail transmission in error, > > please reply to the sender, so that we can arrange > > for proper delivery, and then please delete the message > > from your inbox. Thank you. > > >
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