Measurement of cell volume changes by fluorescence self-quenching

Steffen Hamann; J.F. Kiilgaard; Thomas Litman; F.J. Alvarez-Leefmans; B. R. Winther; Thomas Zeuthen

doi:10.1023/A:1016832027325

Measurement of cell volume changes by fluorescence self-quenching

Steffen Hamann, J.F. Kiilgaard, Thomas Litman, F.J. Alvarez-Leefmans, B. R. Winther, Thomas Zeuthen

135 Citations (Scopus)

Abstract

At high concentrations, certain fluorophores undergo self-quenching, i.e., fluorescence intensity decreases with increasing fluorophore concentration. Accordingly, the self-quenching properties can be used for measuring water volume changes in lipid vesicles. In cells, quantitative determination of water transport using fluorescence self-quenching has been complicated by the requirement of relatively high (mM) and often toxic loading concentrations. Here we report a simple method that uses low (muM) loading concentrations of calcein-acetoxymethyl ester (calcein-AM) to obtain intracellular concentrations of the fluorophore calcein suitable for measurement of changes in cell water volume by self-quenching. The relationship between calcein fluorescence intensity, when excited at 490 nm (its excitation maximum), and calcein concentration was investigated in vitro and in various cultured cell types. The relationship was bell-shaped, with the negative slope in the concentration range where the fluorophore undergoes fluorescence self-quenching. In cultured retinal pigment epithelial cells, calcein fluorescence and extracellular osmolarity were linearly related. A 25-mOsm hypertonic challenge corresponded to a decrease in calcein fluorescence with high signal-to-noise ratio (>15). Similar results were obtained with the fluorophore BCECF when excited at its isosbestic wavelength (436 nm). The present results demonstrate the usefulness of fluorescence self-quenching to measure rapid changes in cell water volume.

Original language	English
Journal	Journal of Fluorescence
Volume	12
Issue number	2
Pages (from-to)	139-145
Number of pages	7
ISSN	1053-0509
DOIs	https://doi.org/10.1023/A:1016832027325
Publication status	Published - 2002

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@article{3bcb4c4074c511dbbee902004c4f4f50,

title = "Measurement of cell volume changes by fluorescence self-quenching",

abstract = "At high concentrations, certain fluorophores undergo self-quenching, i.e., fluorescence intensity decreases with increasing fluorophore concentration. Accordingly, the self-quenching properties can be used for measuring water volume changes in lipid vesicles. In cells, quantitative determination of water transport using fluorescence self-quenching has been complicated by the requirement of relatively high (mM) and often toxic loading concentrations. Here we report a simple method that uses low (muM) loading concentrations of calcein-acetoxymethyl ester (calcein-AM) to obtain intracellular concentrations of the fluorophore calcein suitable for measurement of changes in cell water volume by self-quenching. The relationship between calcein fluorescence intensity, when excited at 490 nm (its excitation maximum), and calcein concentration was investigated in vitro and in various cultured cell types. The relationship was bell-shaped, with the negative slope in the concentration range where the fluorophore undergoes fluorescence self-quenching. In cultured retinal pigment epithelial cells, calcein fluorescence and extracellular osmolarity were linearly related. A 25-mOsm hypertonic challenge corresponded to a decrease in calcein fluorescence with high signal-to-noise ratio (>15). Similar results were obtained with the fluorophore BCECF when excited at its isosbestic wavelength (436 nm). The present results demonstrate the usefulness of fluorescence self-quenching to measure rapid changes in cell water volume.",

author = "Steffen Hamann and J.F. Kiilgaard and Thomas Litman and F.J. Alvarez-Leefmans and Winther, {B. R.} and Thomas Zeuthen",

year = "2002",

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language = "English",

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pages = "139--145",

journal = "Journal of Fluorescence",

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TY - JOUR

T1 - Measurement of cell volume changes by fluorescence self-quenching

AU - Hamann, Steffen

AU - Kiilgaard, J.F.

AU - Litman, Thomas

AU - Alvarez-Leefmans, F.J.

AU - Winther, B. R.

AU - Zeuthen, Thomas

PY - 2002

Y1 - 2002

N2 - At high concentrations, certain fluorophores undergo self-quenching, i.e., fluorescence intensity decreases with increasing fluorophore concentration. Accordingly, the self-quenching properties can be used for measuring water volume changes in lipid vesicles. In cells, quantitative determination of water transport using fluorescence self-quenching has been complicated by the requirement of relatively high (mM) and often toxic loading concentrations. Here we report a simple method that uses low (muM) loading concentrations of calcein-acetoxymethyl ester (calcein-AM) to obtain intracellular concentrations of the fluorophore calcein suitable for measurement of changes in cell water volume by self-quenching. The relationship between calcein fluorescence intensity, when excited at 490 nm (its excitation maximum), and calcein concentration was investigated in vitro and in various cultured cell types. The relationship was bell-shaped, with the negative slope in the concentration range where the fluorophore undergoes fluorescence self-quenching. In cultured retinal pigment epithelial cells, calcein fluorescence and extracellular osmolarity were linearly related. A 25-mOsm hypertonic challenge corresponded to a decrease in calcein fluorescence with high signal-to-noise ratio (>15). Similar results were obtained with the fluorophore BCECF when excited at its isosbestic wavelength (436 nm). The present results demonstrate the usefulness of fluorescence self-quenching to measure rapid changes in cell water volume.

AB - At high concentrations, certain fluorophores undergo self-quenching, i.e., fluorescence intensity decreases with increasing fluorophore concentration. Accordingly, the self-quenching properties can be used for measuring water volume changes in lipid vesicles. In cells, quantitative determination of water transport using fluorescence self-quenching has been complicated by the requirement of relatively high (mM) and often toxic loading concentrations. Here we report a simple method that uses low (muM) loading concentrations of calcein-acetoxymethyl ester (calcein-AM) to obtain intracellular concentrations of the fluorophore calcein suitable for measurement of changes in cell water volume by self-quenching. The relationship between calcein fluorescence intensity, when excited at 490 nm (its excitation maximum), and calcein concentration was investigated in vitro and in various cultured cell types. The relationship was bell-shaped, with the negative slope in the concentration range where the fluorophore undergoes fluorescence self-quenching. In cultured retinal pigment epithelial cells, calcein fluorescence and extracellular osmolarity were linearly related. A 25-mOsm hypertonic challenge corresponded to a decrease in calcein fluorescence with high signal-to-noise ratio (>15). Similar results were obtained with the fluorophore BCECF when excited at its isosbestic wavelength (436 nm). The present results demonstrate the usefulness of fluorescence self-quenching to measure rapid changes in cell water volume.

U2 - 10.1023/A:1016832027325

DO - 10.1023/A:1016832027325

M3 - Journal article

SN - 1053-0509

VL - 12

SP - 139

EP - 145

JO - Journal of Fluorescence

JF - Journal of Fluorescence

IS - 2

ER -