Re: Setting up indo-1

Dennis_Young@CIS.ucsd.edu
Mon, 10 Apr 1995 13:17:00 -0700

I use calf thymus nuclei (CTN) stained with DAPI (or Hoechst) and PI (or EB)
(PI/EB are excited by UV and/or 488). They are so bright you should see signal
even with the correct filters in place. You should try taking out the filters,
(just use the filter ring) since it sounds like your FL-5 is not aligned
properly.
For single laser Indo-1, I just use the FL-1 and FL-2 and 405/490 filters with
450 dichroic.
I always see higher CV's (@4.5 or so with CTN) with dual lasers.
Be sure to rinse sample tubing extensively or you'll stain your loaded cells!
I've also found it helpful to wash between samples with EtOH (1 min) and H20 (1
min) to remove residual Calcium activators.

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* Dennis J. Young Voice : (619) 543-3928 *
* Flow Cytometry Core Facility FAX : (619) 543-7487 *
* University of California, San Diego USA e-mail: djyoung@ucsd.edu *
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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

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