Loading pyranine via purinergic receptors or hypotonic stress for measurement of cytosolic pH by imaging
Loading pyranine via purinergic receptors or hypotonic stress for measurement of cytosolic pH by imaging
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DOI：
10.1152/ajpcell.1998.275.4.c1158
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发表时间：
1998-10-01
影响因子：
5.5
通讯作者：
Grinstein, S
中科院分区：
文献类型：
--
作者：
Gan, BS;Krump, E;Grinstein, S
虽然广泛用于测量细胞内pH，但荧光素的衍生物如2 '，7'-双（2-羧乙基）-5（6）-羧基荧光素（BCECF）具有次优的灵敏度，并且可以产生有毒的光产物。这些限制可以克服使用pH敏感的荧光染料8-羟基芘-1，3，6-三磺酸（吡喃），它具有改善的光谱特性。然而，在将这种高度亲水性染料递送到细胞内部时遇到的困难限制了吡喃碱的使用。我们描述了一种基于嘌呤能P-2x 7受体的可逆激活的细胞内递送吡喃的策略，其允许染料渗透到其他完整的细胞中。当通过这种方法加载到J774或RAW细胞中时，吡喃不仅比BCECF更敏感（动态范围类似于7倍大），而且保留得更好，毒性更小。吡胺分布在整个细胞质中，但在内膜区室中检测不到。反复照射导致BCECF负载的细胞起泡和功能反应性丧失，而吡喃碱负载的经100次照射的细胞保持健康和反应性。也可以通过短暂暴露于低渗溶液将吡喃装载到不表达P-2x 7受体的细胞中。用这种方法标记的细胞的性质与通过嘌呤能受体加载的细胞的性质相似，并且与加载BCECF的细胞的性质相比是有利的。因此，吡喃提供了一个有用的替代荧光素衍生物的细胞内pH值的测量，特别是当使用显微镜数字成像所需的高激发强度。
Although used extensively for the measurement of intracellular pH, derivatives of fluorescein such as 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) have suboptimal sensitivity and can generate toxic photoproducts. These limitations can be overcome using the pH-sensitive fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonic acid (pyranine), which has improved spectroscopic properties. However, the use of pyranine has been limited by the difficulties encountered in delivering this highly hydrophilic dye to the cell interior. We describe a strategy for intracellular delivery of pyranine based on the reversible activation of purinergic P-2x7 receptors, which allow permeation of the dye into otherwise intact cells. When loaded into J774 or RAW cells by this method, pyranine is not only more sensitive than BCECF (the dynamic range is similar to 7-fold greater), but is retained better and is less toxic. Pyranine was distributed throughout the cytosol but was not detectable in endomembrane compartments. Repeated illumination resulted in blebbing and loss of functional responsiveness of cells loaded with BCECF, whereas comparably irradiated cells loaded with pyranine remained healthy and responsive. Pyranine can also be loaded into cells not expressing P-2x7 receptors by brief exposure to a hypotonic solution. The properties of cells labeled by this method are similar to those loaded via purinergic receptors and compare favorably with those of BCECF-loaded cells. Pyranine thus provides a useful alternative to fluorescein derivatives for the measurement of intracellular pH, particularly when using the high excitation intensities required for microscopic digital imaging.