Mark, Ionomycin is a radical insult on the ion CA+2 channels and in many of our experiments with many cell types rarely drop to baseline over our time parameter. However, one should always run a biological control which should return your cells to baseline. Good controls are; X-linking the biotinylated Anti-CD3 with avidin or using CD2/2R (Becton Dickinson) which is a clone mixture which naturally causes x-linking. In all biological control systems these should return to baseline. Stephen P. Perfetto, MS.,MT. (ASCP) Walter Reed Army Institute of Research Department of Molecular Diagnostics and Pathogenesis 1600 East Gude Drive Rockville, MD. 20850 _______________________________________________________________________________ Subject: ca++ flux quandry From: KUKURUGA@medmail.med.umich.edu at Internet_Gateway Date: 12/17/97 3:12 PM Here's a good one (maybe) . . . Jurkat cells, loaded with indo, hit with ionomycin (1ug/ml). The ratio inverts showing good ca++ flux, yet after a minute or so, the ratio returns to baseline. So, what does it mean? Is the ionomycin concentration perhaps too high, and therefore toxic, thus causing cell death? What do you think? Also . . . same cells, transfected with whatever, but co-transfected to be neomycin resistant . . . under above conditions (indo loaded, ionomycin treated)the ratio moves in the unbound direction, exxentially backwards from expected. Could this be due to the resistance, and a loading issue? Any thoughts will be appreciated. MAK. --- Mark A. KuKuruga, Managing Director University of Michigan Flow Cytometry kukuruga@medmail.med.umich.edu
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