RE: Intracellular ifn-gamma and IL-4/5

From: Calman Prussin (CPRUSSIN@niaid.nih.gov)
Date: Tue Oct 24 2000 - 08:44:07 EST


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
> >
> >
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