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> 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
Hi
A dichroic with a sort of hinge eh? It sounds to me that the dichroic is
at the wrong angle. I use indo-1 with my FACS Vantage and get very good
results. I use the same setup as you with regard to filters BUT I use the
dichroic supplied with the instrument intended for splitting off the
signals from a red secondary laser (I think), it is labelled FL4/5 640 LP
but works well as short pass dichroic with a cut-off of about 500nM.
The signal in the FL5 channel should be stronger than that in FL4.
Hope this helps.
Simon Monard
FACSman
Dept Medicine
Cambridge, UK
tel 01223 330149
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