 |
 |
 |
 |
I'm wondering if your filter setup is optimal for Indo-1. It would
seem that a 505 dichroic is going to allow essentially all of the
fluorescence to enter only one of the PMTs (see Molecular Probes
catalogue, p. 114)
We do calcium measurements using Indo-1 on an ACAS 570 and the filter
setup we use is a 445 LP to split the fluorescence between the PMTs,
a 485/45 filter in front of one PMT and a 405/45 for the other PMT. THis
seems to work quite nicely.
Ray
++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
Raymond B. Hester, Ph.D.
Flow Cytometry Lab
The University of South Alabama "Life is too short to be small."
Mobile, AL 36688-0002
Email: rhester@jaguar1.usouthal.edu Disraeli
Voice: (334) 460-6029
FAX: (334) 460-6073
++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
On Mon, 10 Apr 1995, G Smith wrote:
> 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
>
|
|
|
|