Determination of intracellular pH in the in vitro hippocampal slice preparation by transillumination spectrophotometry of Neutral red

Thomas J. Sick; Tim S. Whittingham; Joseph C. LaManna

doi:10.1016/0165-0270(89)90050-2

Determination of intracellular pH in the in vitro hippocampal slice preparation by transillumination spectrophotometry of Neutral red

Thomas J. Sick, Tim S. Whittingham, Joseph C. LaManna

Neurology

Research output: Contribution to journal › Article › peer-review

11 Scopus citations

Abstract

A method is presented for measuring intracellular pH (pHi) spectrophotometrically in hippocampal slices using the pH dye indicator, Neutral red (NR). Measurements of pHi by NR were compared directly with the creatine kinase (CK) equilibrium method. Slices were bathed in artificial cerebrospinal fluid buffered with bicarbonate/CO₂. Intracellular pH in hippocampal slices was found to be more alkaline (approximately 0.3 pH units) than buffer pH and far more alkaline (0.5 pH units) than extracellular pH. Resting intracellular alkalinity was observed by using both the NR and the CK equilibrium methods. The method may be useful for studies of pH regulation in intact functioning tissues in vitro where rapid and repeated measurements are necessary or where cell size precludes measurements with pH-sensitive microelectrodes.

Original language	English (US)
Pages (from-to)	25-34
Number of pages	10
Journal	Journal of Neuroscience Methods
Volume	27
Issue number	1
DOIs	https://doi.org/10.1016/0165-0270(89)90050-2
State	Published - Feb 1989

Keywords

Brain slice
Creatine kinase equilibrium
Hydrogen ion
Neutral red
pH
Spectrophotometry

ASJC Scopus subject areas

Neuroscience(all)

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10.1016/0165-0270(89)90050-2

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@article{1c05ac97664c4738890bbf61efe8b381,

title = "Determination of intracellular pH in the in vitro hippocampal slice preparation by transillumination spectrophotometry of Neutral red",

abstract = "A method is presented for measuring intracellular pH (pHi) spectrophotometrically in hippocampal slices using the pH dye indicator, Neutral red (NR). Measurements of pHi by NR were compared directly with the creatine kinase (CK) equilibrium method. Slices were bathed in artificial cerebrospinal fluid buffered with bicarbonate/CO2. Intracellular pH in hippocampal slices was found to be more alkaline (approximately 0.3 pH units) than buffer pH and far more alkaline (0.5 pH units) than extracellular pH. Resting intracellular alkalinity was observed by using both the NR and the CK equilibrium methods. The method may be useful for studies of pH regulation in intact functioning tissues in vitro where rapid and repeated measurements are necessary or where cell size precludes measurements with pH-sensitive microelectrodes.",

keywords = "Brain slice, Creatine kinase equilibrium, Hydrogen ion, Neutral red, pH, Spectrophotometry",

author = "Sick, {Thomas J.} and Whittingham, {Tim S.} and LaManna, {Joseph C.}",

note = "Funding Information: The authors thank Mr. Solomon M. Pikarsky from the University of Miami for assistance with computer routines used for acquisition and analysis of spectral data and Mr. Hussein Assaf for assistance with experiments carried out at Case Western Reserve University. Experiments were supported in part by PHS Grants NS05820 and HL38657 (T.J.S.), NS22077 (J.C.L.) and NS22219 (T.S.W.).",

year = "1989",

month = feb,

doi = "10.1016/0165-0270(89)90050-2",

language = "English (US)",

volume = "27",

pages = "25--34",

journal = "Journal of Neuroscience Methods",

issn = "0165-0270",

publisher = "Elsevier",

number = "1",

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T1 - Determination of intracellular pH in the in vitro hippocampal slice preparation by transillumination spectrophotometry of Neutral red

AU - Sick, Thomas J.

AU - Whittingham, Tim S.

AU - LaManna, Joseph C.

N1 - Funding Information: The authors thank Mr. Solomon M. Pikarsky from the University of Miami for assistance with computer routines used for acquisition and analysis of spectral data and Mr. Hussein Assaf for assistance with experiments carried out at Case Western Reserve University. Experiments were supported in part by PHS Grants NS05820 and HL38657 (T.J.S.), NS22077 (J.C.L.) and NS22219 (T.S.W.).

PY - 1989/2

Y1 - 1989/2

N2 - A method is presented for measuring intracellular pH (pHi) spectrophotometrically in hippocampal slices using the pH dye indicator, Neutral red (NR). Measurements of pHi by NR were compared directly with the creatine kinase (CK) equilibrium method. Slices were bathed in artificial cerebrospinal fluid buffered with bicarbonate/CO2. Intracellular pH in hippocampal slices was found to be more alkaline (approximately 0.3 pH units) than buffer pH and far more alkaline (0.5 pH units) than extracellular pH. Resting intracellular alkalinity was observed by using both the NR and the CK equilibrium methods. The method may be useful for studies of pH regulation in intact functioning tissues in vitro where rapid and repeated measurements are necessary or where cell size precludes measurements with pH-sensitive microelectrodes.

AB - A method is presented for measuring intracellular pH (pHi) spectrophotometrically in hippocampal slices using the pH dye indicator, Neutral red (NR). Measurements of pHi by NR were compared directly with the creatine kinase (CK) equilibrium method. Slices were bathed in artificial cerebrospinal fluid buffered with bicarbonate/CO2. Intracellular pH in hippocampal slices was found to be more alkaline (approximately 0.3 pH units) than buffer pH and far more alkaline (0.5 pH units) than extracellular pH. Resting intracellular alkalinity was observed by using both the NR and the CK equilibrium methods. The method may be useful for studies of pH regulation in intact functioning tissues in vitro where rapid and repeated measurements are necessary or where cell size precludes measurements with pH-sensitive microelectrodes.

KW - Brain slice

KW - Creatine kinase equilibrium

KW - Hydrogen ion

KW - Neutral red

KW - pH

KW - Spectrophotometry

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