Dear Hege,
I have not tried Fura Red and Fluo-3 ( I use INDO 1), but you should see an
increase in Fluo-3 at the same time as a decrease in Fura Red fluorescence
when you stimulate the cells. The changes might be quite subtle if you use
a log amplifier, and would be more apparent if you used linear amplifiers.
I'd also make sure that compensation was switched off.
I would check that the cells are loaded, then stimulate them with ionomycin,
which is receptor-independent and works with all cell types (?). If this
works then you should titrate your PHA/FMLP. If ionomycin doesn't work, you
should check that the cells are viable and in a suitable medium (no
preservatives or fixatives, physiological levels of Calcium) before trying
it again.
To calculate the ratio I would (of course) recommend my own software,
FCSPress, available from http://www.fcspress.com, which calculates ratio as
well as allowing you to make kinetic plots of various statistics (eg percent
responding, mean and median ratio) against time.
By the way, the ratio to plot is Fluo3 divided by Fura Red, which increases
with increasing calcium concentration.
Cheers,
Ray
Ray Hicks
134 High Street, Harston
Cambridge. CB2 5QD UK
e-mail mailto:RayH@FCSPress.com
Web http://www.FCSPress.com
Tel +44 797 453 8647
+44 1223 871081
Fax +44 870 7408595
> From: "Fjerdingstad, Hege" <Hege.Fjerdingstad@no.nycomed-amersham.com>
> Date: Mon, 25 Jun 2001 13:16:04 +0100
> To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
> Subject: Ca2+
>
>
>
> Hi!!
> I have some problem measuring Ca2+ with my Epics Elite. I load the cells
> for 45 min with Fura red and Fluo-3. After washing the cells I run them
> with an argon 488 nm laser. I try to stimulate both ECV-304 cells and MNC
> with PHA / FMLP or histamin without seeing anything. I'm looking for the
> ratio between Fura red and fluo-3 against time. Do anyone out ther have a
> procedure for staing and also for doing the calculation??
> Regards
> Hege Brincker Fjerdingstad
>
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:01:23 EST