Lucy, Yes, indo-1 is the indicator of choice. It's ratiometric aspect does allow one to average effects, while also improving sensitivity. Also, as a UV excitable dye, it leaves the 488 laser to allow for subset labeling if necessary (one could also correlate 633 excitable dyes, assuming that's available). Points to remember: 1) Dye loading is most important. Dye concentration, medium, temp, condition of the cells (viability) will dramatically effect your results. 2) Proper controls are essential. Cells in good shape, well loaded, then treated with an ionophore (like ionomycin) serve as a "positive" control, where the change in CA++ is maximized (and so the ratio change). Cells treated with a CA++ chelator (like EGTA) will serve as the "negative," where no amount of stimulation will cause a ratio conversion. 3) Cross-contamination is the the biggest problem in measuring CA++. Even a minuscule amount of ionomycin on the sample sipper tube can ruin your results. Be very aware of adequate cleaning and flushing between samples. 4) Without special sample delivery systems, CA++ effects measurable over minutes is the best one can do. That, however, is okay for most applications. The Time Zero module (Cytek) can help to get below a minute. In-line mixing systems have been designed that allow for measuring very fast reactions . . . look that up if you're interested. On both of these issues, and others, there is plenty of literature, including protocol outlines in Current Protocols. MAK. -- Mark A. KuKuruga, Managing Director University of Michigan Core Flow Cytometry <http://www.cancer.med.umich.edu/flow_cytometry> phone: 734-647-3216 fax: 734-936-7376 kukuru@umich.edu Lucy Brown wrote: > Hello to all, > > I have a user who would like to look at Ca+2 levels. I have never run > this before and would like to know what others think is the best > reagent for this measurement. We have a UV laser (Coherent Innova > 300) on our MoFlo so we can run Indo1. In my reading, Indo1 seems > like the dye of choice since using the ratio of the 2 emissions for > bound and unbound Ca+2 eliminates problems with loading differences > and size differences of a population of cells. What do you > experienced cytometrists think? Thanks for your input. > > Lucy Brown > Beckman Research Institute/City of Hope > 626-359-8111 X3306 > lbrown@coh.org
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