In situ calibration and [H⁺] sensitivity of the fluorescent Na⁺indicator SBFI

Abstract

Despite the popularity of Na+-binding benzofuran isophthalate (SBFI) to measure intracellular free Na+ concentrations ([Na+](i)), the in situ calibration techniques described to date do not favor the straightforward determination of all of the constants required by the standard equation (Grynkiewicz G, Poenie M, and Tsien RY. J Biol Chem 260: 3440-3450, 1985) to convert the ratiometric signal into [Na+]. We describe a simple method in which SBFI ratio values obtained during a "full" in situ calibration are fit by a three-parameter hyperbolic equation; the apparent dissociation constant (K(d)) of SBFI for Na+ can then be resolved by means of a three-parameter hyperbolic decay equation. We also developed and tested a "one-point" technique for calibrating SBFI ratios in which the ratio value obtained in a neuron at the end of an experiment during exposure to gramicidin D and 10 mM Na+ is used as a normalization factor for ratios obtained during the experiment; each normalized ratio is converted to [Na+](i) using a modification of the standard equation and parameters obtained from a full calibration. Finally, we extended the characterization of the pH dependence of SBFI in situ. Although the K(d) of SBFI for Na+ was relatively insensitive to changes in pH in the range 6.8-7.8, acidification resulted in an apparent decrease, and alkalinization in an apparent increase, in [Na+](i) values. The magnitudes of the apparent changes in [Na+](i) varied with absolute [Na+](i), and a method was developed for correcting [Na+](i) values measured with SBFI for changes in intracellular pH.

Sodium Influx Pathways during and after Anoxia in Rat Hippocampal Neurons

“…Primary cultures of hippocampal neurons from 2-to 4-d-old postnatal Wistar rats (Animal Care Centre, University of British Columbia) were prepared as described previously (Diarra et al, 2001). Briefly, rat pups were anesthetized and decapitated, and the hippocampi were removed.…”

“…As described previously (Diarra et al, 2001), media used to calibrate sodium-binding benzofuran isophthalate (SBFI)-derived fluorescence ratio measurements contained 0.6 mM MgCl 2 , 0.5 mM CaCl 2 , 10 mM HEPES, 130 mM [Na ϩ ] plus [K ϩ ], 100 mM gluconate, 30 mM Cl Ϫ , and 4 M gramidicin D, titrated with 10 M KOH to pH 7.35. In experiments in which seminaphthorhodafluor (SNARF) pH indicators were used, SNARF-derived fluorescence ratio measurements were calibrated with media containing the same components as the standard HEPES-buffered medium, except for the substitution of K gluconate (130.5 mM) and Na gluconate (9 mM) for NaCl and KCl and the addition of 10 M nigericin .…”

“…As described by Diarra et al (2001), fluorescence emissions (at Ͼ510 nm) were measured by a single intensified charge-coupled device camera from regions of interest placed on individual neuronal somata. Raw emission intensity data at each excitation wavelength (334 and 380 nm) were corrected for background fluorescence before the calculation of a ratio.…”

“…Ratio pairs were acquired at 2-15 sec intervals and analyzed off-line. A one-point calibration technique was used to convert background-corrected SBFI ratio values (BI 334 /BI 380 ) into [Na ϩ ] i values as described previously (Diarra et al, 2001). In brief, at the end of an experiment, neurons loaded with SBFI were exposed to a pH 7.35 medium containing 10 mM Na ϩ and 4 M gramicidin D, and the resulting background-corrected ratio value at 10 mM [Na ϩ ] i was used as a normalization factor for experimentally derived BI 334 /BI 380 ratio values.…”

Temporary Sequestration of Potassium by Mitochondria in Astrocytes

“…to the normalized, background-subtracted PBFI ratio values were fitted (r 2 Ͼ 0.99) by a three-parameter hyperbolic equation (28).…”