Quantitation of cytosolic [Ca2+] in whole perfused rat hearts using Indo-1 fluorometry.
Quantitation of cytosolic [Ca2+] in whole perfused rat hearts using Indo-1 fluorometry.
复制标题
使用 Indo-1 荧光测定法对整个灌注大鼠心脏中的胞质 [Ca2] 进行定量。
DOI：
10.1016/s0006-3495(93)81274-8
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发表时间：
1993
影响因子：
3.4
通讯作者：
Weiner,MW
中科院分区：
文献类型：
--
作者：
Brandes,R;Figueredo,VM;Camacho,SA;Baker,AJ;Weiner,MW
在完整组织中使用Indo-1荧光测定胞质钙[Ca 2 +]c受到在体内获得Indo-1校准参数的问题的限制。因此，本研究的目的是使用体外常数校准Indo-1，这些常数从含蛋白质的参比溶液中获得，旨在产生与体内相似的Indo-1光谱特性。由于波长依赖性的组织光吸收，在体外常数必须使用一种新的方法进行吸光度校正。根据两个检测波长下Indo-1荧光强度之间的关系计算校正因子。约28 mg/ml的蛋白质混合物具有与体内发现的（427 nm）相似的Indo-1等吸光度波长（430 nm），以及与体内发现的（吸光度校正后）相似的饱和Ca 2+的荧光比最大值。使用该蛋白质混合物的校准常数，在Langendorf灌注的大鼠心脏中计算的[Ca 2 +]c在心脏收缩期为187 nM，在心脏收缩期为464 nM。这种新的校准方法规避了相当大的实验问题，以前的方法，需要测量与细胞质完全耗尽和完全饱和的Ca 2+。
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+.