Published: Vol 4, Iss 2, Jan 20, 2014 DOI: 10.21769/BioProtoc.1027 Views: 22662
Reviewed by: Lin FangFanglian He
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Abstract
Intracellular pH (pHi) is an important physiological determinant of enzyme activity and cellular function (Kurkdjian and Guern, 1989). All proteins depend on a tightly regulated pH to maintain their structure and function. Protonation–deprotonation events can dictate the charge of biological surfaces and are integral steps in many metabolic reactions (Casey et al., 2010). Moreover, the proton gradient across the mitochondrial membrane is used to generate cellular energy and support other mitochondrial processes. As a result, cells have developed multiple mechanisms to maintain a narrow range of pHi in response to extra- and intracellular fluctuations in pH (Orij et al., 2012). Here, we describe a protocol for pHi measurement in live cells that uses fluorescent microscopy and the pH sensitive dye 2’,7’-Bis-(2-Carboxyethyl)-5-(and-6-)-Carboxyfluorescein Acetoxymethyl Ester (BCECF-AM). This method was recently used to determine the effects of intracellular pH changes on global histone acetylation levels (McBrian et al., 2013).
Materials and Reagents
Equipment
Software
Procedure
In situ calibration
Recipes
CaCl2.2H2O | 2.65 g/L |
MgSO4 (anhydrous) | 0.9767 g/L |
KCl | 4 g/L |
NaCl | 68 g/L |
NaH2PO4 | 0.122 g/L |
EBSS | 1x (from 10x stock) |
Glucose | 1 g/L (from 100 g/L stock) |
NaHCO3 | 24 mM (for pH 7.4) |
Acknowledgments
This protocol has been modified from several previous publications as described in the references section, and recently used in McBrian et al. (2013).
References
Article Information
Copyright
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Behbahan, I. S., McBrian, M. A. and Kurdistani, S. K. (2014). A Protocol for Measurement of Intracellular pH. Bio-protocol 4(2): e1027. DOI: 10.21769/BioProtoc.1027.
Category
Biochemistry > Other compound > Ion
Cell Biology > Cell-based analysis > Ion analysis
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