Abstract：
:A procedure for calibration of fluorescence signals from adult rat heart cells loaded with the -AM ester of fura-2 is described. Calibration is complicated by dye compartmentation and potentially incomplete dye hydrolysis. These problems were overcome by subtracting from fluorescence transients the non-cytosolic (mitochondrial) component of fura-2 fluorescence plus any Ca-insensitive component of dye fluorescence, after selectively and sequentially quenching cytosolic and non-cytosolic dye with Mn. The Kd of fura-2 in cells loaded by the -AM ester, in cells depleted of ATP and equilibrated with Ca buffers, was found to be 371 +/- 39 nM at 37 degrees C. We found that calibration values for RMAX and RMIN derived from previously measured cells were of general validity, removing the need to measure RMAX and RMIN on every cell. Once these calibration values are determined, the calibration procedure to measure cytosolic Ca on any cell is a five minute procedure to determine compartmentation, using just one non-toxic and inexpensive solution. Finally, we have calculated how the errors intrinsic to the measurements translate into errors of the calculated Ca concentration and transient peak heights. These calculations allow reasonable parameters for data acquisition to be set.
journal_name
Cell Calciumjournal_title
Cell calciumauthors
Haworth RA,Redon Ddoi
10.1016/s0143-4160(98)90050-1subject
Has Abstractpub_date
1998-10-01 00:00:00pages
263-73issue
4eissn
0143-4160issn
1532-1991pii
S0143-4160(98)90050-1journal_volume
24pub_type
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