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
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