Neuronal free calcium measurement using BTC/AM, a low affinity calcium indicator

Krzysztof L. Hyrc; Joanna M. Bownik; Mark P. Goldberg

doi:10.1016/S0143-4160(98)90126-9

Neuronal free calcium measurement using BTC/AM, a low affinity calcium indicator

Krzysztof L. Hyrc, Joanna M. Bownik, Mark P. Goldberg

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

12 Scopus citations

Abstract

BTC is a low affinity calcium indicator (K(d) ~ 7-26 μM) featuring many desirable properties for cellular calcium imaging, including long excitation wavelengths (400/485 nm), low sensitivity to Mg²⁺, and accuracy of ratiometric measurement. To assess the usefulness of this indicator in cultured neurons, we examined properties of BTC and its acetoxymethyl ester, BTC/AM. BTC/AM had substantial calcium-independent fluorescence at all excitation wavelengths. BTC/AM was readily loaded into neurons and was rapidly hydrolysed. There was little dye compartmentalization, as assessed by digitonin lysis, Co²⁺ quenching of BTC fluorescence and by confocal microscopy. Despite adequate loading, BTC gradually became unresponsive to [Ca²⁺](i) when cultures were examined under routine imaging conditions. This effect was a function of the cumulative fluorescence illumination and could be minimized by attenuating light intensity or duration. Ratio imaging after exposure of neuronal cultures to 1-50 μM ionomycin revealed distinct sensitivity ranges for BTC and Fura-2. BTC reported graded neuronal [Ca²⁺](i) responses to glutamate receptor stimulation with N-methyl-D-aspartate in the range 10-50 μM, whereas Fura-2 did not distinguish between these stimuli. Under appropriate loading and illumination conditions, bath-loaded BTC/AM may be well suited for measurement of moderate to high calcium concentrations in cultured neurons.

Original language	English (US)
Pages (from-to)	165-175
Number of pages	11
Journal	Cell Calcium
Volume	24
Issue number	3
DOIs	https://doi.org/10.1016/S0143-4160(98)90126-9
State	Published - Sep 1998
Externally published	Yes

ASJC Scopus subject areas

Molecular Biology
Physiology
Cell Biology

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10.1016/S0143-4160(98)90126-9

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title = "Neuronal free calcium measurement using BTC/AM, a low affinity calcium indicator",

abstract = "BTC is a low affinity calcium indicator (K(d) ~ 7-26 μM) featuring many desirable properties for cellular calcium imaging, including long excitation wavelengths (400/485 nm), low sensitivity to Mg2+, and accuracy of ratiometric measurement. To assess the usefulness of this indicator in cultured neurons, we examined properties of BTC and its acetoxymethyl ester, BTC/AM. BTC/AM had substantial calcium-independent fluorescence at all excitation wavelengths. BTC/AM was readily loaded into neurons and was rapidly hydrolysed. There was little dye compartmentalization, as assessed by digitonin lysis, Co2+ quenching of BTC fluorescence and by confocal microscopy. Despite adequate loading, BTC gradually became unresponsive to [Ca2+](i) when cultures were examined under routine imaging conditions. This effect was a function of the cumulative fluorescence illumination and could be minimized by attenuating light intensity or duration. Ratio imaging after exposure of neuronal cultures to 1-50 μM ionomycin revealed distinct sensitivity ranges for BTC and Fura-2. BTC reported graded neuronal [Ca2+](i) responses to glutamate receptor stimulation with N-methyl-D-aspartate in the range 10-50 μM, whereas Fura-2 did not distinguish between these stimuli. Under appropriate loading and illumination conditions, bath-loaded BTC/AM may be well suited for measurement of moderate to high calcium concentrations in cultured neurons.",

author = "Hyrc, {Krzysztof L.} and Bownik, {Joanna M.} and Goldberg, {Mark P.}",

note = "Funding Information: KLH is on leave from the Institute of Molecular Biology, Jagiellonian University, Krakow, Poland. We thank Sandy P. Althomsons and Qingyan Zhu for expert assistance with cell cultures and Stefano Sensi for helpful discussion. This work was supported by NIH grants NS32140, NS32626, NS37230 (to MPG), and NS28995 (to Chung Y. Hsu) and was done during the tenure of a Grant-in-Aid Award (to MPG) from the American Heart Association and William Randolph Hearst Foundation.",

year = "1998",

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doi = "10.1016/S0143-4160(98)90126-9",

language = "English (US)",

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AU - Hyrc, Krzysztof L.

AU - Bownik, Joanna M.

AU - Goldberg, Mark P.

N1 - Funding Information: KLH is on leave from the Institute of Molecular Biology, Jagiellonian University, Krakow, Poland. We thank Sandy P. Althomsons and Qingyan Zhu for expert assistance with cell cultures and Stefano Sensi for helpful discussion. This work was supported by NIH grants NS32140, NS32626, NS37230 (to MPG), and NS28995 (to Chung Y. Hsu) and was done during the tenure of a Grant-in-Aid Award (to MPG) from the American Heart Association and William Randolph Hearst Foundation.

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N2 - BTC is a low affinity calcium indicator (K(d) ~ 7-26 μM) featuring many desirable properties for cellular calcium imaging, including long excitation wavelengths (400/485 nm), low sensitivity to Mg2+, and accuracy of ratiometric measurement. To assess the usefulness of this indicator in cultured neurons, we examined properties of BTC and its acetoxymethyl ester, BTC/AM. BTC/AM had substantial calcium-independent fluorescence at all excitation wavelengths. BTC/AM was readily loaded into neurons and was rapidly hydrolysed. There was little dye compartmentalization, as assessed by digitonin lysis, Co2+ quenching of BTC fluorescence and by confocal microscopy. Despite adequate loading, BTC gradually became unresponsive to [Ca2+](i) when cultures were examined under routine imaging conditions. This effect was a function of the cumulative fluorescence illumination and could be minimized by attenuating light intensity or duration. Ratio imaging after exposure of neuronal cultures to 1-50 μM ionomycin revealed distinct sensitivity ranges for BTC and Fura-2. BTC reported graded neuronal [Ca2+](i) responses to glutamate receptor stimulation with N-methyl-D-aspartate in the range 10-50 μM, whereas Fura-2 did not distinguish between these stimuli. Under appropriate loading and illumination conditions, bath-loaded BTC/AM may be well suited for measurement of moderate to high calcium concentrations in cultured neurons.

AB - BTC is a low affinity calcium indicator (K(d) ~ 7-26 μM) featuring many desirable properties for cellular calcium imaging, including long excitation wavelengths (400/485 nm), low sensitivity to Mg2+, and accuracy of ratiometric measurement. To assess the usefulness of this indicator in cultured neurons, we examined properties of BTC and its acetoxymethyl ester, BTC/AM. BTC/AM had substantial calcium-independent fluorescence at all excitation wavelengths. BTC/AM was readily loaded into neurons and was rapidly hydrolysed. There was little dye compartmentalization, as assessed by digitonin lysis, Co2+ quenching of BTC fluorescence and by confocal microscopy. Despite adequate loading, BTC gradually became unresponsive to [Ca2+](i) when cultures were examined under routine imaging conditions. This effect was a function of the cumulative fluorescence illumination and could be minimized by attenuating light intensity or duration. Ratio imaging after exposure of neuronal cultures to 1-50 μM ionomycin revealed distinct sensitivity ranges for BTC and Fura-2. BTC reported graded neuronal [Ca2+](i) responses to glutamate receptor stimulation with N-methyl-D-aspartate in the range 10-50 μM, whereas Fura-2 did not distinguish between these stimuli. Under appropriate loading and illumination conditions, bath-loaded BTC/AM may be well suited for measurement of moderate to high calcium concentrations in cultured neurons.

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Neuronal free calcium measurement using BTC/AM, a low affinity calcium indicator

Abstract

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