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Intracellular pH-determination by fluorescence measurements.

J W Visser, A A Jongeling, H J Tanke

The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society

|January 1, 1979

Summary

This study developed a fluorescence method to measure intracellular pH (pHi) in single cells. The technique accurately determined cell pH, offering a new tool for biological research.

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Digital fluorescence microscopy.

Current protocols in cytometry·2008

Area of Science:

Biophysics
Cell Biology
Analytical Chemistry

Background:

Intracellular pH (pHi) is a critical parameter regulating cellular functions.
Accurate measurement of pHi in individual cells is essential for understanding cellular physiology.
Existing methods for pHi determination have limitations in precision and applicability to single cells.

Purpose of the Study:

To develop and validate a novel fluorescence-based method for determining the intracellular pH (pHi) of individual cells.
To assess the accuracy and reliability of the developed method across a range of pH values and buffering capacities.
To explore the potential of flow cytometry for high-throughput pHi measurements.

Main Methods:

Utilized fluorescein fluorescence excitation spectra, which exhibit pH-dependent properties.
Treated individual rat bone marrow cells with fluorescein diacetate (FDA) to generate intracellular fluorescein.
Compared fluorescence excitation spectra of cells with those of known pH fluorescein solutions.

Main Results:

The developed method accurately determined pHi in cells suspended in media with pH ranging from 4.0 to 8.1.
Cells in media with high or normal buffering capacities exhibited pHi values matching the external media pH.
Cells in low buffering capacity media maintained a stable intracellular pH of 6.7 +/- 0.2.
Preliminary data suggest the feasibility of using flow cytometry for pHi determination.

Conclusions:

A reliable fluorescence-based method for measuring individual cell intracellular pH (pHi) has been established.
The method demonstrates accuracy and stability, particularly in low buffering conditions.
Flow cytometry presents a promising avenue for rapid, high-throughput pHi analysis of cellular populations.

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