Brandes R, Figueredo V M, Camacho S A, Baker A J, Weiner M W
Cardiovascular Research Institute, University of California, San Francisco.
Biophys J. 1993 Nov;65(5):1973-82. doi: 10.1016/S0006-3495(93)81274-8.
Fluorometric determination of cytosolic calcium, [Ca2+]c, using Indo-1 in intact tissue, is limited by problems in obtaining calibration parameters for Indo-1 in vivo. Therefore, the goal of this study was to calibrate Indo-1 using in vitro constants, obtained from protein-containing reference solutions designed to produce similar Indo-1 spectral properties to those in vivo. Due to wavelength-dependent tissue light absorbance, the in vitro constants had to be absorbance-corrected using a novel method. The correction factor was calculated from the relationship between the Indo-1 fluorescence intensities at the two detection wavelengths. A mixture of proteins at approximately 28 mg/ml had a similar Indo-1 isosbestic wavelength (430 nm) to that found in vivo (427 nm), and a similar fluorescence ratio maximum with saturating Ca2+ to that found in vivo (after absorbance correction). Using calibration constants from this protein mixture, calculated [Ca2+]c in a Langendorf perfused rat heart was 187 nM during diastole, and 464 nM in systole. This new calibration method circumvented the considerable experimental problems of previous methods which required measurements with the cytosol fully depleted and fully saturated with Ca2+.
在完整组织中使用吲哚-1（Indo-1）通过荧光法测定胞质钙浓度[Ca2+]c受到体内获取吲哚-1校准参数问题的限制。因此，本研究的目的是使用从含蛋白质的参比溶液获得的体外常数来校准吲哚-1，这些参比溶液旨在产生与体内相似的吲哚-1光谱特性。由于组织吸光度与波长有关，体外常数必须使用一种新方法进行吸光度校正。校正因子是根据两个检测波长下吲哚-1荧光强度之间的关系计算得出的。浓度约为28 mg/ml的蛋白质混合物具有与体内（427 nm）相似的吲哚-1等吸收波长（430 nm），并且在Ca2+饱和时具有与体内相似的最大荧光比率（吸光度校正后）。使用该蛋白质混合物的校准常数，在Langendorff灌注的大鼠心脏中，舒张期计算得到的[Ca2+]c为187 nM，收缩期为464 nM。这种新的校准方法规避了先前方法中相当多的实验问题，先前方法需要在胞质完全耗尽Ca2+和完全饱和Ca2+的情况下进行测量。
