Just as we calibrate parameters for imaging in different situations (such as checking if a cell sample has been illuminated properly or not, simulating a code for mean square displacement and comparing it our experimental values, etc.,) do we take the Kd values of indicators for granted?
Or simply, what's the next step after using the indicator and getting the movies? What comes next?
This all depends on your question. If you are assessing activity using Ca signals, then the specific Ca concentration may not matter. If you are assessing relative changes in Ca influx as a result of some manipulation, then exact Ca concentration may not matter (though you would want to use as linear a dye as possible). If you are quantifying the Ca dependence of intracellular signalling pathways, then you are well advised to verify the Kd. If you look through the literature, you will see that Kd values vary from what Molecular Probes says. It kind of depends on your question if those differences matter.
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