Simultaneous measurement of water volume and pH in single cells using BCECF and fluorescence imaging microscopy

被引:33
作者
Alvarez-Leefmans, FJ
Herrera-Pérez, JJ
Márquez, MS
Blanco, VM
机构
[1] Wright State Univ, Dept Pharmacol & Toxicol, Dayton, OH 45435 USA
[2] Natl Inst Psychiat, Dept Neurobiol, Mexico City 14370, DF, Mexico
关键词
D O I
10.1529/biophysj.105.069450
中图分类号
Q6 [生物物理学];
学科分类号
071011 ;
摘要
Regulation and maintenance of cell water volume and intracellular pH (pHi) are vital functions that are interdependent; cell volume regulation affects, and is in turn affected by, changes in pHi. 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 pHi and volume in single cells loaded with the fluorescent probe BCECF. CWV is measured at the excitation isosbestic wavelength, whereas pHi is determined ratiometrically. The method has a time resolution of < 1 s and sensitivity to osmotic changes of similar to 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.
引用
收藏
页码:608 / 618
页数:11
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