on 17/1/97 Murali Ramanathan wrote 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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