Ca++ measurement
MK (LCMK@WEIZMANN.weizmann.ac.il)
Sat, 03 Jun 95 12:04:22 +0300
Raymond,
Fluo-3 is problematic with many cells.
The fact that Fluo-3 has only one emission wavelength precludes the use
of an algorithm that frees you from the dependence on the intracellular
concentration of the probe in order to derive Ca++ in the cytosol.
As is the case with most other probes, Fluo-3 is lost from the cytosol
to the external medium (leaks) and to other cellular compartments
(internalization). If your cells are not especially phlegmatic, this can
occur quite rapidly and you may find yourself with no signal to measure
within 20-30 min. Then, there is the matter of the calibration.
You will need to calibrate more than one time during a run, as the
depletion of Fluo-3 from your cells progresses. This can be done in the
following way, using the Bromide salt of A23187, which is NOT FLUORESCENT
itself: You need a minimmum of three points for your calibration curve
(according to the golden rule of "three" you can draw a straight line
through any three points on a plane, if you have a thick pencil...).
The "zero Ca++" point is achieved by incubating your cells for about 20
minutes before measurement in medium w/o added Ca++ supplemented with
1-2 mM Mg++, 1 mM EGTA and 1-5 micromolar A23187-bromide. The "maximal Ca++"
point can be achieved by adding either a permeabilising agent or A23187
(to a final concentration of 25 micromolar) to your cells IN THE MEASURING
TUBE, during or just 1-2 minutes prior to measurement with normal levels
of Ca++ in the external medium. In order to get a midway point you repeat
this with a lower concentration of Ca++ in the external medium.
Also, some people keep their cells in medium containing 1-2 micromolar
Fluo-3 AM and make the final washes with fresh medium just before
measurument. In conclusion, it becomes a royal pain. Fluo-3 is fine if
you are interested in qualitative results. If you need hard data you
should try to get a UV source and use Indo-1.
I've never heard of using "Fura-red" (I've never heard of "Fura-red",
as a matter of fact) in combination with Fluo-3 in order to measure
emission ratios, nor can I think what good would it do towards measuring
the intracellular Calcium concentrations, but I would be glad to learn
some about it.
I hope this is of some help.
MK (LCMK@WEIZMANN.WEIZMANN.AC.IL)
M. Kushnir
Cell Biology
WIS, Rehovot, Israel.
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