Setting up indo-1

G Smith (prsgs@bath.ac.uk)
Mon, 10 Apr 1995 09:02:27 +0100 (BST)

Hello,

I am in the process of setting up an assay for intracellular calcium
mobilisation using indo-1 on a FACS vantage and have run up against a
couple of problems. If anyone could help me, it would be most appreciated.

The UV excited fluorescence signal is split by a 505nm dichroic mirror
and the two fluorescences are collected at 395+/-25nm (Fl4) and
525+/-25nm (Fl5). The dichroic mirror is a curious thing with a sort of
hinge, making it infinitely adjustable. I have aligned the system with
UV beads but get little or no signal in FL5. This may be due to the
spectrum of the beads.

With Indo-1 (3uM) loaded cells, I get a nice signal in FL4, but again no
signal in FL5. Considering that I will be looking for an increase in FL4
and a decrease in Fl5, this is not satisfactory. Is there an established
setting up protocol for this assay? I get nice changes using Fluo-3
alone, but I dont want to use this. I have a lot of noise in Fl5, and
this may be the source of the problem. If anyone has any ideas, Id be
pleased to hear them.

Thanks

Graham
Graham

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