Hi.
I am using Invitrogen's Fluo4 assay to measure intracellular calcium levels. I load my cells with the dye for an hour, then measure the fluorescence with a flow cytometer for a minute before I add thapsigargin to release ER calcium stores and then measure fluorescence for another 4 minutes.
My fluroescence vs. time plot has a peak after I add the thapsigargin, which I would expect because calcium is released and binds to the indicator, but then the fluroescence decreases after that (after about a minute after thapsigargin was added). I was just wondering why the fluroescence would taper off...???....I think this is also seen in other dyes.
Thanks for any insights in advance!
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