Loading pyranine via purinergic receptors or hypotonic stress for measurement of cytosolic pH by imaging

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.