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首页> 外文期刊>The Korean Journal of Physiology & Pharmacology >A Novel Nicotinamide Adenine Dinucleotide Correction Method for Mitochondrial Ca2+ Measurement with FURA-2-FF in Single Permeabilized Ventricular Myocytes of Rat
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A Novel Nicotinamide Adenine Dinucleotide Correction Method for Mitochondrial Ca2+ Measurement with FURA-2-FF in Single Permeabilized Ventricular Myocytes of Rat

机译：FURA-2-FF法测定大鼠单个通透性心室肌细胞线粒体Ca2 +的烟酰胺腺嘌呤二核苷酸校正新方法

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摘要

Fura-2 analogs are ratiometric fluoroprobes that are widely used for the quantitative measurement of [Ca2+]. However, the dye usage is intrinsically limited, as the dyes require ultraviolet (UV) excitation, which can also generate great interference, mainly from nicotinamide adenine dinucleotide (NADH) autofluorescence. Specifically, this limitation causes serious problems for the quantitative measurement of mitochondrial [Ca2+], as no available ratiometric dyes are excited in the visible range. Thus, NADH interference cannot be avoided during quantitative measurement of [Ca2+] because the majority of NADH is located in the mitochondria. The emission intensity ratio of two different excitation wavelengths must be constant when the fluorescent dye concentration is the same. In accordance with this principle, we developed a novel online method that corrected NADH and Fura-2-FF interference. We simultaneously measured multiple parameters, including NADH, [Ca2+], and pH/mitochondrial membrane potential; Fura-2-FF for mitochondrial [Ca2+] and TMRE for Ψm or carboxy-SNARF-1 for pH were used. With this novel method, we found that the resting mitochondrial [Ca2+] concentration was 1.03 μM. This 1 μM cytosolic Ca2+ could theoretically increase to more than 100 mM in mitochondria. However, the mitochondrial [Ca2+] increase was limited to ~30 μM in the presence of 1 μM cytosolic Ca2+. Our method solved the problem of NADH signal contamination during the use of Fura-2 analogs, and therefore the method may be useful when NADH interference is expected.
机译：Fura-2类似物是比例荧光探针，广泛用于[Ca 2 + ]的定量测量。但是，染料的使用本质上受到限制，因为染料需要紫外线（UV）激发，这也可能产生很大的干扰，主要是由烟酰胺腺嘌呤二核苷酸（NADH）自发荧光引起的。特别是，由于在可见光范围内没有可用的比例染料被激发，因此这一限制给线粒体[Ca 2 + ]的定量测量带来了严重的问题。因此，在定量测量[Ca 2 + ]期间，无法避免NADH干扰，因为大部分NADH都位于线粒体中。当荧光染料浓度相同时，两个不同激发波长的发射强度比必须恒定。按照这一原则，我们开发了一种新颖的在线方法，可以纠正NADH和Fura-2-FF干扰。我们同时测量了多个参数，包括NADH，[Ca 2 + ]和pH /线粒体膜电位。使用线粒体[Ca 2 + ]的Fura-2-FF和Ψ m 的TMRE或pH的羧基-SNARF-1。通过这种新方法，我们发现静止的线粒体[Ca 2 + ]浓度为1.03μM。从理论上讲，线粒体中1μM的胞质Ca 2 + 可以增加到100 mM以上。然而，在存在1μM胞质Ca 2 + 的情况下，线粒体[Ca 2 + ]的增加被限制为〜30μM。我们的方法解决了在使用Fura-2类似物过程中NADH信号污染的问题，因此，当预期出现NADH干扰时，该方法可能会有用。

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