Flu-4 is a widely used calcium sensing dye (200+ publication in pubmed; where it has been used in neurons). I wonder in how much time this dye will reach a saturation point (stable signal) ? Is there any other dye which makes it possible in neurons. I wonder if it is ever possible in a living cell? Thank you!
Probably between 30 and 60 min at room temperature. Less time is not enough for the amount of light you probably need for measurements but more time is detrimental as the dy probably gets into subcellular organelles where calcium concentration is much larger than in cytosol. If you encounter problems is probably due to the amount of light excitation in confocal systems. Reduce it as much as possible.
Well you can try all the following things together and check if cells become loaded in 5 min (which is unlikely):
Increase dye concentration by 5-10 fold(from 4 microM to 20 or even 40 microM).
Increase loading temperatura to 37 ºC (inside an incubator and adding pre-heated médium containing the solved dye)
Increase excitation light by several fold.
Use glass coverslips instead of plastic coverslip
Increase digital camera adquisition time from ideal 100 ms to 1000 ms
Increase camera gain
Increase binning from 1 pixel to 4x4 pixels or even more
Decrease background cut-off to zero
Good luck
Carlos
I
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