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Simultaneous Measurement of Water Volume and pH in Single Cells Using BCECF and Fluorescence Imaging Microscopy

Francisco J. Alvarez-Leefmans ^*, José J. Herrera-Pérez , Martín S. Márquez  and Víctor M. Blanco ^*

^* Department of Pharmacology and Toxicology, Wright State University, Dayton, Ohio 45435, and Department of Neurobiology, National Institute of Psychiatry, Mexico 14370 D/F, Mexico

Correspondence: Address reprint requests to Francisco J. Alvarez-Leefmans, Dept. of Pharmacology and Toxicology, Wright State University, 3640 Colonel Glenn Hwy., Dayton, OH 45435. E-mail: francisco.alvarez-leefmans{at}wright.edu.

Regulation and maintenance of cell water volume and intracellular pH (pH_i) are vital functions that are interdependent; cell volume regulation affects, and is in turn affected by, changes in pH_i. Disruption of either function underlies various pathologies. To study the interaction and kinetics of these two mechanisms, we developed and validated a quantitative fluorescence imaging microscopy method to measure simultaneous changes in pH_i and volume in single cells loaded with the fluorescent probe BCECF. CWV is measured at the excitation isosbestic wavelength, whereas pH_i is determined ratiometrically. The method has a time resolution of <1 s and sensitivity to osmotic changes of 1%. It can be applied in real time to virtually any cell type attached to a coverslip, independently of cellular shape and geometry. Calibration procedures and algorithms developed to transform fluorescence signals into changes in cell water volume (CWV) and examples of applications are presented.

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