Measurement of cell volume changes by fluorescence self-quenching
Measurement of cell volume changes by fluorescence self-quenching
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DOI：
10.1023/a:1016832027325
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发表时间：
2002-06-01
影响因子：
2.7
通讯作者：
Zeuthen, T
中科院分区：
文献类型：
--
作者：
Hamann, S;Kiilgaard, JF;Zeuthen, T
在高浓度下，某些荧光团经历自猝灭，即，荧光强度随着荧光团浓度的增加而降低。因此，自猝灭特性可用于测量脂质囊泡中的水体积变化。在细胞中，使用荧光自猝灭的水转运的定量测定由于需要相对高（mM）且通常有毒的加载浓度而变得复杂。在这里，我们报告了一个简单的方法，使用低（μ M）加载浓度的钙黄绿素-乙酰氧基甲酯（钙黄绿素-AM），以获得细胞内浓度的荧光团钙黄绿素适合于测量细胞水体积的变化，通过自猝灭。在体外和各种培养细胞类型中研究了在490 nm（其激发最大值）激发时钙黄绿素荧光强度与钙黄绿素浓度之间的关系。该关系是钟形的，在荧光团经历荧光自猝灭的浓度范围内具有负斜率。在培养的视网膜色素上皮细胞中，钙黄绿素荧光与细胞外渗透压呈线性相关。25 mOsm高渗激发对应于钙黄绿素荧光的降低，具有高信噪比（>15）。当在其等吸光波长（436 nm）激发时，用荧光团BCECF获得类似的结果。本研究结果表明，荧光自猝灭测量细胞水体积的快速变化的有用性。
At high concentrations, certain fluorophores undergo self-quenching, i.e., fluorescence intensity decreases with increasing fluorophore concentration. Accordingly, the self-quenching properties can be used for measuring water volume changes in lipid vesicles. In cells, quantitative determination of water transport using fluorescence self-quenching has been complicated by the requirement of relatively high (mM) and often toxic loading concentrations. Here we report a simple method that uses low (muM) loading concentrations of calcein-acetoxymethyl ester (calcein-AM) to obtain intracellular concentrations of the fluorophore calcein suitable for measurement of changes in cell water volume by self-quenching. The relationship between calcein fluorescence intensity, when excited at 490 nm (its excitation maximum), and calcein concentration was investigated in vitro and in various cultured cell types. The relationship was bell-shaped, with the negative slope in the concentration range where the fluorophore undergoes fluorescence self-quenching. In cultured retinal pigment epithelial cells, calcein fluorescence and extracellular osmolarity were linearly related. A 25-mOsm hypertonic challenge corresponded to a decrease in calcein fluorescence with high signal-to-noise ratio (>15). Similar results were obtained with the fluorophore BCECF when excited at its isosbestic wavelength (436 nm). The present results demonstrate the usefulness of fluorescence self-quenching to measure rapid changes in cell water volume.