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Hi Everybody,
We recently started doing intracellular
staining for cytokines on a FACScan with CellQuest
and are trying to resolve three issues.
1) We see substantial IL-2 and IFN-g positive
cells. Is there a rational, operator-independent
way of setting the markers so that the number of
positives can be identified? Is there software
that will do this?
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?
3) Does anybody have experience staining for TNF-beta (or alpha)
or IL-10?
Thanks in advance for any feedback.
Murali Ramanathan
Pharmaceutics
Re these questions:
I know of no software that will do this. I think you just have to set positives on isotype
matched controls. In fact, given that stimulation to induce ic expression of cks ususally causes a
great increase in intracellular non-specific binding, I have found it very necessary to fine-tune
for each experiment.
You just have to trust your Il-4 findings! - provided you have neat profiles for isotype matched
controls and that your unpermabilised cells are not positive relative to their controls.
IL-4 is so transiently produced that even in "Th2" responses you won't see very many. I would
suggest getting a positive control and running that to be sure. I don't know if there is a
BETTER way to detect Th2 cells: the advantage of this system is a single cell profile. If you
have the luxury of large numbers or cloned populations you can of course try ELISAs or PCR:
but again, the potency of IL-4 means that relatively little mRNA is detectable
Dearbhaile O' donnell,
Dept of Medicine and Therapeutics,
University College Dublin
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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;
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