Liquid ion exchange cocktail

The SIET was used to measure K⁺ activities at the skin and ionocyte surfaces of larvae. Glass capillary tubes (no. TW 150-4, World Precision Instruments, Sarasota, FL) were pulled on a Sutter P-97 Flaming Brown pipette puller (Sutter Instruments, San Rafael, CA) into micropipettes with tip diameters of 3–4 μm. These were then baked at 120 °C overnight and coated with dimethyl chlorosilane (Sigma-Aldrich) for 3 h. The micropipettes were backfilled with a 1-cm column of electrolytes and frontloaded with a 50-μm column of liquid ion-exchange cocktail (Sigma-Aldrich) to create an ion-selective microelectrode (probe). The following ionophore cocktails (and electrolytes) were used: potassium ionophore I - cocktail B (100 mM KCl) and NH₄⁺ ionophore I cocktail B (100 mM NH₄Cl). To calibrate the ion-selective probe, the Nernstian property of each microelectrode was measured by placing the microelectrode in a series of standard solutions (0.1, 1, 10, and 100 mM KCl for the K⁺ probe; 0.1, 1, and 10 mM NH₄Cl for the NH₄⁺ probe). By plotting the voltage output of the probe against log[K⁺] and log[NH₄⁺]values, a linear regression yielded a Nernstian slope of 59.1 ± 0.5 (n = 10) for K⁺ and 58.6 ± 0.8 (n = 10) for NH₄⁺.

In the present study, we used the SIET technique to detect H⁺ flux at the surface of zebrafish larvae. The method was performed largely as described previously (Shih et al., 2008 (link), 2012 (link)). In brief, micropipettes with tip diameters of 3–4 μm were pulled by a Sutter P-97 Flaming Brown pipette puller (Sutter Instruments, San Rafael, CA). Micropipettes were then baked at 120°C overnight and coated with dimethyl chlorosilane (Sigma-Aldrich) for 30 min. To make an ion-selective microelectrode (probe), micropipettes were backfilled with a 1-cm column of electrolytes and frontloaded with a 20–30-μm column of liquid ion-exchange cocktail (Sigma-Aldrich). The following ionophore cocktail and electrolytes were used: H⁺ ionophore I cocktail B (40 mM KH₂PO₄ and 15 mM K₂HPO₄; pH 7). To calibrate the ion-selective probe, the Nernstian property of each microelectrode was measured by placing the microelectrode in a series of standard solutions (pH 6, 7, and 8 for the H⁺ probe). By plotting the voltage output of the probe against [H⁺] values, a linear regression yielded a Nernstian slope of 58.6 ± 0.8 (n = 10) for H⁺.

SIET was used to measure Na⁺ flux activity at the epithelium surface of medaka larva. Glass capillary tubes (no. TW 150 – 4; World Precision Instruments, Sarasota, FL) were pulled on a Sutter P-97 Flaming Brown pipette puller (Sutter Instruments, San Rafael, CA) into micropipettes with tip diameters of 3–4 μm. The micropipettes were then baked at 120 °C overnight and coated by incubation with dimethyl chlorosilane (Sigma-Aldrich) for 30 min. The micropipettes were backfilled with a 1-cm column of electrolytes and frontloaded with a 20–30 μm column of liquid ion-exchange cocktail (Sigma-Aldrich) to create an ion-selective microelectrode (probe). The ionophore cocktail (and electrolytes) was Na⁺ ionophore II cocktail A (100 mM NaCl). To calibrate the ion-selective probe, the Nernstian response of each microelectrode was evaluated by placing it in a series of standard solutions (0.1, 1, and 10 mM NaCl dissolved in distilled water). By plotting the voltage output of the probe against log [Na⁺] value, linear regression yielded a Nernstian slope of 56.7 ± 0.5 (N = 10). In preliminary tests, the selectivity of the Fluka Na⁺ ionophore II cocktail A was 10–16 times more selective to Na⁺ than to NH₄⁺ (measured in 1–10 mM Na⁺ solution).