We have recently started to look at oxidative burst as a means of assesing phagocytosis. We use whole-blood method with DHR123 as a substrate which is converting to rhodamine during oxidative burst. Our problem is in high spontaneous metabolic activity of PMN's: in the polypropylene tube with lithium-heparin whole blood + DHR123 +NaCl 0,9% we have after incubation and lysis too high intensity of fluorescence FL1. Does anyone have experiences with this problem? Thanks for your help. Karin Malickova kmali@lf1.cuni.cz What you are seeing is fluorescence of DHR123. The references I looked at didn't mention it, but I found that loading cells with 1 um DHR123 produces 1-2 logs green fluorescence greater than cellular autofluorescence, depending on the cell type. Cells loaded with the same concentration of Rh123 had another log greater Fl1 fluorescence than the DHR loaded cells. Hope this helps. Doug Weidner, Ph.D. UT MD Anderson Cancer Center Houston, Texas, USA
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