On Fri, 26 Jan 1996 06:26:06 -0800 M.J. Tobin wrote:
 
snip...  I was wondering if anyone has particular
> comments on preparation of calcium standards to calibrate the
microscope.  A
> recent paper by Sanders et.al. (see end) describes lifetime imaging
of pH in
> cells using SNAFL-1, but the important point is that they describe
calibration
> of the microscope using labelled cells rather than solutions of
known pH. (is
> this common practice?)  They do this by loading the cells and making
them leaky
> to H+ ions.  Then by adjusting the pH of the surrounding medium, the
pH inside
> the cells is known.  They claim this is more accurate for
calibrations.  Does
> anybody know of a way of making cells 'leaky' to calcium ions and
thus
> measuring the reference solutions inside actual cells.
> Any comments would be helpful
>
> Mark Tobin
> Daresbury Laboratory
>
>
> Sanders R. et.al., (1995) Analytical Biochemistry 227, 302-308
 
Dear Mark
 
The idea is that the intracellular environment alters the sensitivity
of the probe so that calibrating inside the cell may be more
appropriate. However, there is the problem as to whether or not you
can accurately control the intracellular environment...
 
For calcium perople use ionomycin or Bromo-A23187, but in our cardiac
cells it has been hard for us to get Rmax (because the cells bleb) and
Rmin (because the calcium takes forever to fall to zero). A good
approach is to use a calibrating solution between Rmin and Rmax that
does not alter the resting level when an ionophore is added.
 
In my opinion, it is better to design experiments to look for changes
rather than relying on an absolute calibration. The calibration can be
given to allow others to compare their data but I wouldn't stake my
reputation on an absolute calibration if I were you...
 
To set the external solution pCa you can use EGTA buffers.
 
Hope this helps, good luck
 
Mark Cannell
SGHMS