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>our question is whether using ionomycin (as our positive control/at various
> concentrations) should the ratio of violet to blue return to near baseline
> after the influx of calcium?
>
In our hands, working mainly with mouse thymocytes, the ratio stays high
after the addition of ionomycin until such a time as the cells begin to
become "less healthy" as indicated by a decreasing forward scatter, slightly
increased side scatter. If you let the time course continue you can plot
nicely the return to near baseline of indo-1 fluorescence ratio correlating
with scatter change in a 3D type plot.
As a rough method of duplicating this effect, try adding a squirt (technical
measurement I use) of 70% ethanol to the end of one of your sample time
courses and you should see a similar pattern to the above ionomycin
treatment. This doesn't hang around in the sample lines, and can be a quick
way of checking whether you've got any loading if ever you don't get a
response from your agonist.
Hope this helps,
Geoff
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Geoffrey Osborne | ____ __ o Ahh!
Flow Cytometry (FACS LAB) | __ `\ <,_
John Curtin School of Medical Research, | __ (*)/ (*)
Australian National University, | ==============|
CANBERRA, AUSTRALIA. | |--|
Email: Geoff.Osborne@anu.edu.au | |--|...
Phone: 61 6 249 3694
FAX: 61 6 249 2595
-----Surfing the Web?: Try http://jcsmr.anu.edu.au/facshome.html------
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