Correcting for artifacts in complex aqueous solutions when using the pH-sensitive dye 2',7'-bis-(2-carboxyethyl)- 5-(and -6)carboxyfluorescein.
Correcting for artifacts in complex aqueous solutions when using the pH-sensitive dye 2',7'-bis-(2-carboxyethyl)- 5-(and -6)carboxyfluorescein.
复制标题
使用 pH 敏感染料 2,7-双-(2-羧乙基)-5-(和-6)羧基荧光素时校正复杂水溶液中的伪影。
DOI：
10.1006/abio.1998.3005
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Dandala,S
中科院分区：
文献类型：
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作者：
Cardullo,RA;Dandala,S
pH敏感性荧光指示剂染料2′，7 ′-双-（2-羧乙基）-5-（和-6）羧基荧光素（BCECF）通常用于测量细胞内的pH。令人惊讶的是，没有研究已经进行，看看是否各种溶液参数调节BCECF的荧光强度，即使粘度文物已被报道为特定的Ca 2+选择性染料。在这份报告中，我们证明，即使是轻微的增加，在一些不同的代理商的浓度显着降低激发荧光强度，在两个波长通常用于确定溶液的pH值。溶液粘度不同，使用一些不同的代理商，包括甘油，蔗糖，聚乙二醇，聚乙烯吡咯烷酮，和甲基纤维素。在一般情况下，有一个可检测的和显着的减少，最大荧光激发比的粘度增加，虽然效果是更显着的牛顿溶液比非牛顿溶液。在pH 6.5、7.0和7.3下观察到相同的一般效应，发现BCECF特别敏感的pH水平范围。为了校正这些人为的低值，我们使用不同的激发波长组合来确定哪些可以用于准确测量pH值，同时最大限度地减少人为因素。选择激发波长，使得在470和435 nm处收集激发比，允许定量测量pH的显著信号，同时伪影几乎被消除。
The pH-sensitive fluorescent indicator dye 2′,7′-bis-(2-carboxyethyl)-5-(and -6)carboxyfluorescein (BCECF) is routinely used to measure intracellular pH within cells. Surprisingly, no studies have been performed to see if various solution parameters modulate the fluorescence intensity of BCECF even though viscosity artifacts have been reported for particular Ca2+selective dyes. In this report we demonstrate that even minor increases in the concentration of a number of different agents significantly decrease the excitation fluorescence intensity at two wavelengths routinely used to determine solution pH. Solution viscosity was varied using a number of different agents including glycerol, sucrose, polyethylene glycol, polyvinylpyrrolidone, and methylcellulose. In general, there was a detectable and significant decrease in the maximum fluorescence excitation ratio as the viscosity was increased, although the effect was more dramatic with Newtonian solutions than with non-Newtonian solutions. This same general effect was seen at pH 6.5, 7.0, and 7.3, a range of pH levels where BCECF is found to be particularly sensitive. To correct for these artifactually low values we used different combinations of excitation wavelengths to determine which could be used to accurately measure pH while minimizing the artifact. Choosing excitation wavelengths so that excitation ratios were collected at 470 and 435 nm allowed a significant signal to quantitatively measure pH while the artifact was nearly abolished.