ASSESSMENT OF FURA-2 FOR MEASUREMENTS OF CYTOSOLIC FREE CALCIUM
ASSESSMENT OF FURA-2 FOR MEASUREMENTS OF CYTOSOLIC FREE CALCIUM
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DOI：
10.1016/0143-4160(90)90060-8
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发表时间：
1990-02-01
期刊：
影响因子：
4
通讯作者：
HERMAN, B
中科院分区：
文献类型：
--
作者：
ROE, MW;LEMASTERS, JJ;HERMAN, B
Fura-2已经成为最受欢迎的荧光探针，用来监测完整活细胞胞浆游离钙的动态变化。在这篇文章中，我们描述了目前公认的在活细胞中使用Fura-2的许多限制，以及可以绕过其中一些问题的某些方法。其中许多问题是细胞特有的，包括：(A)胞浆酯酶对Fura-2乙酰氧甲基酯键的不完全水解，以及Fura-2/AM分子上可能存在抗酯酶的甲酯复合体或商业Fura-2/AM制剂中其他未知污染物的存在；(B)Fura-2在非细胞质隔间(即细胞质细胞器)中的隔离；(C)标记细胞的染料丢失(主动或被动)；(D)重金属对Fura-2荧光的猝灭；(E)荧光不对钙敏感的Fura-2物种的光漂白和光化学形成；(F)Fura-2的吸收和发射光谱的变化，以及Fura-2的Kd值作为探针环境的极性、粘度、离子强度或温度的函数；以及(G)准确校准细胞内的Fura-2信号。这些问题的解决方案包括：(A)用Fura-2五钾盐标记细胞(通过刮擦加载、微量注射ATP透气剂)，以绕过酯水解问题；(B)在低温或4度之后标记细胞。C预冷，以防止细胞内细胞器隔离；(C)在低于生理温度(即15-33度)的情况下进行实验。C)和使用比率定量来弥补染料泄漏造成的不准确；(D)添加N，N，-N‘’，N-四(2-吡啶甲基)乙二胺(TPEN)来螯合重金属；(E)使用低激发能和高灵敏度检测器，以最大限度地减少光漂白或形成荧光的非钙敏感形式的Fura-2；和(F)使用340 nm和365 nm(而不是340 nm和380 nm)进行比率成像，这减少了极性、粘度和离子强度伪影对计算的钙浓度的潜在影响，提供了从细胞中渗出染料的测量方法，Fura-2光漂白速度，并可用于在完整细胞中执行Fura-2荧光的原位校准；然而，使用该波长对缩小了比率的动态范围，从而使其对光子检测中的噪声更敏感。如果不考虑这些潜在的问题，可能会导致对胞浆游离钙的错误估计。通过准确地评估这些潜在伪迹的贡献，可以使用Fura-2来准确地估计完整活细胞中的胞浆游离钙水平。
Fura-2 has become the most popular fluorescent probe with which to monitor dynamic changes in cytosolic free calcium in intact living cells. In this paper, we describe many of the currently recognized limitations to the use of Fura-2 in living cells and certain approaches which can circumvent some of these problems. Many of these problems are cell type specific, and include: (a) incomplete hydrolysis of Fura-2 acetoxymethyl ester bonds by cytosolic esterases, and the potential presence of either esterase resistant methyl ester complexes on the Fura-2/AM molecule or other as yet unidentified contaminants in commercial preparations of Fura-2/AM; (b) sequestration of Fura-2 in non-cytoplasmic compartments (i.e. cytoplasmic organelles); (c) dye loss (either active or passive) from labeled cells; (d) quenching of Fura-2 fluorescence by heavy metals; (e) photobleaching and photochemical formation of fluorescent non-Ca2+ sensitive Fura-2 species; (f) shifts in the absorption and emission spectra, as well as the Kd for Ca2+ of Fura-2 as a function of either polarity, viscosity, ionic strength or temperature of the probe environment; and (g) accurate calibration of the Fura-2 signal inside cells. Solutions to these problems include: (a) labeling of cells with Fura-2 pentapotassium salt (by scrape loading, microinjection of ATP permeabilization) to circumvent the problems of ester hydrolysis; (b) labeling of cells at low temperatures or after 4.degree. C pre-chill to prevent intracellular organelle sequestration; (c) performance of experiments at lower than physiological temperatures (i.e. 15-33.degree. C) and use of ratio quantitation to remedy inaccuracies caused by dye leakage; (d) addition of N,N,-N'',N-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) to chelate heavy metals; (e) use of low levels of excitation energy and high sensitivity detectors to minimize photobleaching or formation of fluorescent non-Ca2+ sensitive forms of Fura-2; and (f) the use of 340 nm and 365 nm (instead of 340 nm and 380 nm) for ratio imaging, which diminishes the potential contributions of artifacts of polarity, viscosity and ionic strength on calculated calcium concentrations, provides a measure of dye leakage from the cells, rate of Fura-2 photobleaching, and can be used to perform in situ calibration of Fura-2 fluorescence in intact cells; however, use of this wavelength pair diminishes the dynamic range of the ratio and thus makes it more sensitive to noise involved in photon detection. Failure to consider these potential problems may result in erroneous estimates of cytosolic free calcium. By accurately assessing the contribution of each of these potential artifacts, it is possible to use Fura-2 to accurately estimate cytosolic free calcium levels in intact living cells.