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You must make sure that you include isotype matched controls on each
experiment. We set markers on these controls for each sample to give 1%
positives, then use this marker to see how many cells in that gated
population are +ve with the anticytokine antibody. There is no short cut!
>2) There are a few IL-4 positives and I am not sure that I want to trust
those low percentages? Is there a
> better way to detect Th2 cells?
The low percentage of cells would be expected from previous limiting
dilution studies of the frequency of antigen specific cells. The surprise
is that so many cells are positive for 'Th1' cytokines, the majority of
which are probably bystanders. We have compared other methods and find
RT-PCR very sensitive, but it won't tell you the things that flow will. For
example, in our hands, IL-10 is co-produced by IFN-g+ cells and not (like
IL-4 and IL-5) by IFN-g negative cells, so it isn't behaving the same as
other 'Th2' cytokines.
>3) Does anybody have experience staining for TNF-beta (or alpha) or IL-10?
see our recent papers:
Openshaw PJM, Murphy EE, Hosken NA, Manio V, Davis K and O'Garra A. (1995)
Heterogeneity of intracellular cytokine synthesis at the single cell level
in polarised T helper 1 and T helper 2 populations. J. Exp. Med. 182:1357-1367.
Hussell T, Spender LC, Georgiou A, O'Garra A and Openshaw PJM.(1996) Th1 and
Th2 cytokine induction in pulmonary T-cells during infection with
respiratory syncytial virus. J. Gen. Virol. 77:2447-2455.
TNF-a stains well in much the same cells that stain for IFN-g, but with
subtle variations.
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Peter J Openshaw [Medicine] (p.openshaw@ic.ac.uk)
Imperial College School of Medicine at St.Mary's
Norfolk Place, LONDON W2 1PG
Fax: +44 171 724 7349 Tel: +44 171 594 3854/3
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web