From: Kushman, Mary Elizabeth (MEK0705@mail.ecu.edu)
Date: Tue Nov 17 1998 - 18:31:35 EST
I have recently become interested in the role of mitochondria as either a sink or source of calcium during cell signaling. The dye I have been working with, Rhod-2, has certainly been used to measure mitochondrial calcium via fluorescence imaging methods, but I have yet to see it used flow cytometrically. I have run some preliminary experiments with it, and I am not able to detect any discreet staining patterns for it (as I have for its "cousin", Rhodamine-123, which is very structurally similar), which concerns me, for I wonder if it even is entering the mitochondria at all. I am loading the dye for 45 minutes at 37 degrees C in a suspension of carcinoma cells. If any of you have had experience using this dye in flow cytometry, I would like to discuss the pros and cons of its usage in this context. I would also like general input about performing fluorescence microscopy on these cells loaded with this dye (which I probably should have done first!) before progressing any further with any more flow work. If the Rhod-2 isn't localizing to where its supposed to go in these types of cells, what's the point in carrying out any more flow experiments with it, you know???!!! Best Regards, Mary Kushman A Frazzled Master's Student :P East Carolina University Department of Biology Greenville, NC 27858
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