calcium calibration

Alice L. Givan (Alice.L.Givan@Dartmouth.EDU)
04 Sep 97 17:06:20 EDT

Hello Flowers,
It's me again. I'd like to get some *honest* opinions on the methods people
are using for calibrating calcium concentration (with indo or other dyes).
1) Are people using EGTA for the Rmin value --- and, if so, do they load cells
with EGTA-AM or do they use plain EGTA after treatment of cells with
ionomycin?
2) does anyone else besides me have great trouble in getting the EGTA (Rmin)
value to be lower than the R value of resting cells?
3) Alternatively, are most people using the Chused method with Ca/EGTA buffers
in the presence of ionophores. Do people have better confidence in this
method than in the determination of Rmin and Rmax for the Grynkiewicz equation?
4) Does anyone (besides me) think that editors should not be insisting on
calcium concentration on flow plots and should be satisfied with the use of
fluorescence (indo) ratio -- as calcium concentration calibration is not an
exact science?

Thanks for your honest (as always) opinions on this!
Alice
Alice L. Givan
Englert Cell Analysis Laboratory
Dartmouth Medical School
Lebanon, New Hampshire
NH 03756 USA
tel 603-650-7661
fax 603-650-6130
e-mail givan@dartmouth.edu