Hi everyone: I was wondering if anyone has experience performing intracellular cytokine flow cytometry in rats. I've been using the technique on human and murine cells for a couple of years now but I just recently needed to do the same with rat cells. We've tried some mAbs and were successful in seeing IL-10 and IFN-gamma staining in ConA (primary activation for 3 days) and anti-CD3 (secondary activation for 16 hours) activated Lewis rat CD4+ T cells. We haven't yet had great success with IL-4 (We've been trying Pharmingen's clone). We really want to look at TNF-alpha, however, but cannot find a clone which works for intracellular flow with rat cells. Does anyone know of a clone I can use for rat TNF-alpha and of a better way to stain for IL-4 (I am going to assume for now that Pharmingen's clone is good and that perhaps we are not seeing IL-4 because strain differences between rats although if someone has a clone they have had a lot of success with please do tell) . Also, I need a large quantity of neutralizing anti-rat TNF-alpha antibodies for in vivo work. Does anyone know where I can get a hybridoma? All the best to all, please reply directly to brian.rabinovich@utoronto.ca Take care, Brian ************************************ Brian A. Rabinovich Department of Immunology University of Toronto Princess Margaret Hospital 610 University Avenue, Room 9-303 Toronto, Ontario M5G 2M9 Tel: 416 946 4501 ext.4994 fax: 416 946 2065 ***********************************
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