Research Cell Calcium (1998) 24(3), 165-175 0 Harcourt Brace 8 Co. Ltd 1998 Neuronal free calcium measurement using BTC/AM, a low affinity calcium indicator Krzysztof L. Hyrc, Joanna M. Bownik, Mark P. Goldberg Center for the Study of Nervous System Injury, Department of Neurology, Washington University School of Medicine, St Louis, Missouri, USA Summary BTC is a low affinity calcium indicator (K, -7-26 pM) featuring many desirable properties for cellular calcium imaging, including long excitation wavelengths (400/485 nm), low sensitivity to Mg2+, and accuracy of ratiometric measurement [latridou H., Foukaraki E., Kuhn M.A., Marcus E.M., Haugland BP., Katerinopoulos H.E. The development of a new family of intracellular calcium probes. Cell Calcium 1994; 15: 190-1981. 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. BTWAM was readily loaded into neurons and was rapidly hydrolysed. There was little dye compartmentalization, as assessed by digitonin lysis, Co’+ quenching of BTC fluorescence and by confocal microscopy. Despite adequate loading, BTC gradually became unresponsive to [Can+], 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 l-50 PM ionomycin revealed distinct sensitivity ranges for BTC and Fura-2. BTC reported graded neuronal [Ca’+], responses to glutamate receptor stimulation with N-methyl-D-aspartate in the range lo-50 PM, whereas Fura- 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. INTRODUCTION Intracellular free calcium concentrations ([Ca2+]) can reach micromolar levels in many cell types [l-6], well above the sensitivity range of standard high affinity Ca2+ indicators such as Fura- [7]. However, the utility of many lower affinity calcium indicators is limited either by their sensitivity to Mg2+ (e.g. mag-Fura- [8] or mag-Fura- [9]) or by their single wavelength nature (e.g. Calcium Green Received 6 February 1998 Revised 8 July 1998 Accepted 10 July 1998 Correspondence to: Mark P. Goldberg MD, Department of Neurology, Box 8111, 660 S. Euclid Avenue, St Louis, MO 63110, USA Tel: +I 314 362 3258: Fax: +I 314 362 9462 E-mail: goldberg@neuro.wustl.edu 5N[lO]). Several of these concerns are addressed by N-[3- (2-benzothiazolyl)-6-[2-[2-[bis(carboxymethyl)amino]-5- methylpheno~]ethoxy]-2-oxo-2H-l-benzopyran-7-yl]-N-( carboxymethyl)-glycine (BTC), a coumarin benzothiazole- based tetra-carboxylic calcium indicator developed by Iatridou and colleagues [ 111. BTC is a ratiometric dye, and it can be excited by visible light (400 and 485 mn). It combines moderate Ca2+ affinity (Kd -7-26 PM [11,12] with low sensitivity to Mg2+ [ 121. Studies using BTC to measure [Ca2+],in many cell types [ 12-171 have demonstrated levels well above the sensitivity range of Fura- (K, - 224 r&I [7]). In particular, the use of BTC revealed a linear relationship between the intensity of stimulation and intracellular free calcium levels in frog skeletal muscle fibers [12] and cerebellar granule cell presynaptic terminals [ 131. BTC was also used to identify micromolar calcium spikes necessary to induce exocytosis in pancreatic acinar cells [ 14-161. Using BTC but not Fura-2, it was possible to distinguish [Ca*+], levels 165