Murali Ramanathan asked: ...-We recently started doing intracellular staining for cytokines... >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? 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. --------------------------------------------------- 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 ---------------------------------------------------
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:49:18 EST