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首页> 外文期刊>Journal of Biochemical and Biophysical Methods >Dual probe fluorescence monitoring of intracellular free calcium during ischemia in mouse heart by using continuous compensation for pH dependence of the dissociation constant of Fura-2, and the interference of myoglobin.
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Dual probe fluorescence monitoring of intracellular free calcium during ischemia in mouse heart by using continuous compensation for pH dependence of the dissociation constant of Fura-2, and the interference of myoglobin.

机译:通过连续补偿Fura-2的解离常数对pH的依赖性以及对肌红蛋白的干扰,对小鼠心脏缺血期间的细胞内游离钙进行双探针荧光监测。

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摘要

Mitochondrial damage is the main source of cellular injury upon ischemia-reperfusion, and calcium loading has been implicated in this phenomenon. The use of optical probes for calcium monitoring of the intact heart is hampered by internal filter effects of intracellular hemoproteins, endogenous fluorescence, and their sensitivity to pH. We describe here a method for measurement of intracellular free calcium in isolated myoglobin-deficient perfused mouse hearts under conditions of large intracellular pH fluctuations by simultaneous fluorescence monitoring of the calcium-probe Fura-2 and the pH probe BCECF through dual wavelength excitation of both probes. In myoglobin-containing mouse heart endogenous chromophores interfere with Fura-2 fluorometry. It is shown that a paradoxical decrease in Fura-2 fluorescence occurs during ischemia in isolated mouse hearts. Simultaneous recording of BCECF fluorescence (calibrated against pH measurement with phosphorus NMR) and data reduction based on continual recalculation of the apparent dissociation constant of the calcium-probe complex revealed that a marked increase in intracellular free calcium occurs, and that the Fura-2 fluorescence decrease was caused by an increase in dissociation constant due to intracellular acidification. Intracellular free calcium rose almost linearly during a 20-min period of ischemia and returned to basal values rapidly upon the commencement of perfusion.
机译:线粒体损伤是缺血再灌注时细胞损伤的主要来源,并且钙负荷与该现象有关。细胞内血红蛋白的内部过滤作用,内源性荧光及其对pH的敏感性阻碍了使用光学探针监测完整心脏的钙。我们在这里描述了一种方法,该方法通过同时荧光监测钙探针Fura-2和pH探针BCECF,通过两个探针的双波长激发,在大的细胞内pH波动条件下,在孤立的肌红蛋白缺陷型灌注小鼠心脏中测量细胞内游离钙的方法。在含肌红蛋白的小鼠心脏中,内源性发色团会干扰Fura-2荧光测定法。结果表明,在孤立的小鼠心脏缺血过程中,Fura-2荧光出现了自相矛盾的下降。同时记录BCECF荧光(根据磷NMR的pH测量进行校准)和基于连续重新计算钙-探针复合物的表观解离常数的数据减少显示,细胞内游离钙显着增加,并且Fura-2荧光减少是由于细胞内酸化引起的解离常数增加所致。在缺血20分钟内,细胞内游离钙几乎呈线性上升,并在灌注开始后迅速恢复至基础值。

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