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 journalArticle

12 Citations (Scopus)

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 languageEnglish (US)
Pages (from-to)165-175
Number of pages11
JournalCell Calcium
Volume24
Issue number3
StatePublished - Sep 1998

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Calcium
Fura-2
Lighting
Neurons
Fluorescence
Digitonin
Ionomycin
Glutamate Receptors
N-Methylaspartate
Baths
Fluorescence Microscopy
Confocal Microscopy
Esters
Coloring Agents
Light

ASJC Scopus subject areas

  • Cell Biology
  • Endocrinology

Cite this

Neuronal free calcium measurement using BTC/AM, a low affinity calcium indicator. / Hyrc, Krzysztof L.; Bownik, Joanna M.; Goldberg, Mark P.

In: Cell Calcium, Vol. 24, No. 3, 09.1998, p. 165-175.

Research output: Contribution to journalArticle

Hyrc, Krzysztof L. ; Bownik, Joanna M. ; Goldberg, Mark P. / Neuronal free calcium measurement using BTC/AM, a low affinity calcium indicator. In: Cell Calcium. 1998 ; Vol. 24, No. 3. pp. 165-175.
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