I have been asked by several to post a summary of the response to my query on Ca++ flux using Flou 3 and Fura Red so here goes. 1. We had originally thought that we would be able to see Ca++ flux from intracellular stores but most responses said that the response was too small and we needed to look for loading of extracellular Ca++, a couple suggested adding 1mM Ca++, others just CA++ containing media. 2. Concentrations of the dyes looked too high. Suggested concentrations were around 5-10uM Fura Red and 2-6uM Fluo 3. 3. Staining period may be different for different cells. Try loading for different time periods and at different temperatures. 4. Use ionomycin or a-23187 as a positive control stimulant. 5. Try to stay away from having Mg++ in the media as it may block the Ca++ flux to a degree. We have taken all of these into account and things seem to be working now. We are using Ca++ containing media, concentrations of dyes are now 10uM Fura and 6uM Fluo, our original staining conditions are still in place, and we have used ionomycin as a + control and get a response that goes off scale. The stimulant of choice for us now gives a good signal and all is well. Thanks for the advice and hope this helps someone else. Brian Newsom Director, Flow Cytometry Baylor College of Medicine Center for Cell and Gene Therapy
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