Hi guys,
I'm trying to use CC2-DMPE/DiSBAC2(3) FRET pairs to image membrane potential and firstly i just try to show classical increase in FRET ratio(460nm increase, decrease in 580nm) in HEK293 cells after introduction of high K+ buffer.
But instead of access to a fluorescence plate reader, i only have access to confocal microscope.
However i couldn't see any increase in 460nm even after I tried optimizing loading concentration. when i started with imaging CC2DMPE fluorescence, it does get quenched when DisBAC2(3) is added. But if i put high K+ buffer into it, the 460nm fluorescence still doesn't change at all.
I've been doing it at room temp (23degree celsius). should i do it at 37?
or did i underload or overload the cells?
Thanks guys
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