Neuronal free calcium measurement using BTC/AM, a low affinity calcium indicator.
BTC is a low affinity calcium indicator (Kd approximately 7-26 microM) featuring many desirable properties for cellular calcium imaging, including long excitation wavelengths (400/485 nm), low sensitivity to Mg2+, and accuracy of ratiometric measurement [Iatridou H., Foukaraki E., Kuhn M.A., Marcus E.M., Haugland R.P., Katerinopoulos H.E. The development of a new family of intracellular calcium probes. Cell Calcium 1994; 15: 190-198]. 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 microM 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 microM, 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.
Hyrc
K L
KL
Department of Neurology, Washington University School of Medicine, St Louis, Missouri, USA.
Bownik
J M
JM
Goldberg
M P
MP
D000818:Animals; D052160:Benzothiazoles; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D004396:Coloring Agents / Q000032:analysis / Q000737:chemistry*; D003374:Coumarins / Q000032:analysis / Q000737:chemistry*; D016257:Fura-2 / Q000032:analysis; D005998:Glycine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D007202:Indicators and Reagents / Q000032:analysis / Q000737:chemistry; D008027:Light; D051379:Mice; D009474:Neurons / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*
D052160:Benzothiazoles; D004396:Coloring Agents; D003374:Coumarins; D007202:Indicators and Reagents; C116748:N-(3-(2-benzothiazolyl)-6-(2-(2-(bis(carboxymethyl)amino)-5-methylphenoxy)ethoxy)-2-oxo-2H-1-benzopyran-7-yl)-N-(carboxymethyl)glycine; D002118:Calcium; D005998:Glycine; D016257:Fura-2