Measurement of changes in cell volume based on fluorescence quenching.
Measurement of changes in cell volume based on fluorescence quenching.
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基于荧光猝灭测量细胞体积的变化。
DOI：
10.1152/ajpcell.1997.272.4.c1405
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Bonanno,JA
中科院分区：
文献类型：
--
作者：
Srinivas,SP;Bonanno,JA
6-甲氧基-N-（3-磺丙基）-喹啉（SPQ）是一种Cl（-）敏感的荧光染料，其细胞内荧光被细胞内的有机阴离子和未知身份的蛋白质猝灭。然而，这些细胞内猝灭剂（ICQ）的浓度取决于细胞体积。在不存在Cl-的情况下，观察到的SPQ荧光的变化因此可以反映细胞体积的变化。这一概念已被应用于确定培养的角膜内皮细胞体积的相对变化，在响应于各向异性冲击，使用NO3-作为Cl-取代基。SPQ荧光随渗透压的降低而增加，反之亦然。需要20 mosM高渗休克来检测信噪比>25的SPQ荧光变化。假设动态淬灭ICQs，我们应用了一个扩展的Stern-Volmer方程，以开发一个简单的测量SPQ荧光和细胞体积的相对变化之间的关系。对于大的低渗性休克，观察到调节性音量降低（RVD）。RVD的速率可以通过暴露于低Na+林格溶液中的0.5 μ M短杆菌肽来增强（即，K+-NO3-流出），表明K+电导是RVD的速率限制。这些结果证明了使用荧光猝灭来真实的实时测量细胞体积变化的原理。由于SPQ对Cl-敏感，其作为猝灭探针的有用性受到限制。然而，结构-活性研究可以预期产生有用的Cl（-）-不敏感的类似物。
The intracellular fluorescence of 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ), a Cl(-) -sensitive fluorescent dye, is quenched by intracellular organic anions and proteins of unknown identity. The concentration of these intracellular quenchers (ICQs), however, is dependent on cell volume. In the absence of Cl-, changes in the observed SPQ fluorescence may therefore reflect changes in cell volume. This concept has been applied to determine relative changes in cell volume of cultured corneal endothelium in response to anisosmotic shocks, using NO3- as the Cl- substituent. SPQ fluorescence increased with decreasing osmolarity and vice versa. A 20 mosM hypertonic shock was needed to detect a change in SPQ fluorescence with a signal-to-noise ratio of >25. Assuming dynamic quenching by ICQs, we applied an extension of the Stern-Volmer equation to develop a simple relationship between the measured SPQ fluorescence and relative changes in cell volume. For large hyposmotic shocks, regulatory volume decrease (RVD) was observed. The rate of RVD could be enhanced by exposure to 0.5 microM gramicidin in low-Na+ Ringer solution (i.e., K+-NO3- efflux), indicating that K+ conductance is rate limiting for RVD. These results demonstrate the principle of using fluorescence quenching to measure changes in cell volume in real time. Because SPQ is sensitive to Cl-, its usefulness as a quenching probe is limited. However, a structure-activity study can be expected to yield useful Cl(-)-insensitive analogs.