Electrophysiologic analyses were performed in an Ussing chamber (Physiologic Instruments). Epithelia on Transwell inserts were mounted in an Ussing chamber and bathed in a modified Ringer’s solution (120 mM NaCl, 10 mM d -glucose, 3.3 mM KH2PO4, 0.83 mM K2HPO4, 1.2 mM MgCl2, 1.2 mM CaCl2, 25 mM NaHCO3, pH 7.4), maintained at 37°C and gassed with 5% CO2/95% O2. Epithelia were analyzed under short-circuit conditions with intermittent pulsing (200 ms pulses at ±5 mV). Cultures were treated acutely in the Ussing chamber with subsequent additions of apical amiloride (100 μM, Alfa Aesar), apical and basolateral forskolin (20 μM, Tocris) and IBMX (100 μM, Sigma) (F/I), apical CFTR(inh)-172 (10 μM, CFTR Chemical Compound Distribution Program), and apical ATP (100 μM, Sigma). Inhibition of epithelial sodium channel–dependent current with amiloride enabled normalization of basal currents between epithelial donors.
Ussing chamber
Manufactured by Thermo Fisher Scientific
The Ussing chamber is a specialized laboratory equipment used to study the transport of ions, molecules, and other substances across biological membranes. It consists of two chambers separated by the membrane of interest, allowing for the measurement of parameters such as electrical resistance and ion fluxes.
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Lab products found in correlation
Ussing chamber, by Physiologic Instruments (3 mentions)
Amiloride, by Thermo Fisher Scientific (2 mentions)
Apical atp, by Merck Group (2 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (2 mentions)
Cftrinh 172, by Merck Group (1 mentions)
3 isobutyl 1 methylxanthine ibmx, by Merck Group (1 mentions)
Vx 770, by Selleck Chemicals (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Forskolin, by Thermo Fisher Scientific (1 mentions)
Acquire and analyze software, by Physiologic Instruments (1 mentions)
Vcc mc8, by Physiologic Instruments (1 mentions)
4 protocols using ussing chamber
1
Ussing Chamber Electrophysiology of Epithelia
2
Electrophysiological Assessment of CFTR Function
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Electrophysiological measurements were performed in an Ussing chamber (Physiological Instruments) under open-circuit conditions with continuous measurement of transepithelial potential difference (TEPD). Monolayers were intermittently voltage-clamped to 0 mV for measurement of short-circuit current (Isc). During periods of short-circuiting, epithelia were pulsed (200 ms of ±5 mV) and measurements of transepithelial electrical resistance (TEER) were obtained. Cells were placed between apical and basolateral compartments filled with Ringer’s solution (120 mM NaCl, 10 mM D-glucose, 3.3 mM KH2PO4, 0.83 mM K2HPO4, 1.2 mM MgCl2, 1.2 mM CaCl2, saturated with 95% O2/5% CO2, pH 7.4 at 37°C). After mounting in the Ussing chamber, solutions were continuously gassed with 5% CO2 and 95% O2. Acute treatments of the monolayers in the Ussing chamber consisted of apical 100 µM amiloride (Alfa Aesar), apical/basal 20 µM forskolin (Fsk; Tocris)/100 µM 3-isobutyl-1-methylxanthine (IBMX; Sigma), apical 1 µM VX-770 (Selleck Chemicals), apical 10 µM CFTR(inh)-172 (CFTR Chemical Compound Distribution Program) and apical 100 µM ATP (Sigma). For non-CF cultures, CFTR was potentiated with VX-770 then activated by Fsk/IBMX. For CF cultures, CFTR was activated by Fsk/IBMX then potentiated with VX-770. After Ussing chamber analysis, monolayers were immediately frozen and stored at −80°C.
3
Ussing Chamber Measurements of Epithelial Cells
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Confluent cells grown on Snapwells were mounted in Ussing chambers (Physiologic Instruments, San Diego, California, USA) to measure short circuit current (ISC) and transepithelial electrical resistance (TER). Cells were bathed in Krebs buffer solution (pH 7.4) consisting of 115 mM NaCl, 2 mM KH2PO4, 2.4 mM MgCl2*6H2O, 25 mM NaHCO3, 8 mM KCl, 1.3 mM CaCl2, and supplemented 10 mM glucose and 10 mM mannitol on the basolateral and apical sides, respectively. Experiments with TBCA were performed in Krebs buffer without MgCl2. Tissue was voltage clamped to 0 V and unclamped every 20 seconds and a 5 mV potential difference applied using a voltage-clamp apparatus (VCC MC8, Physiologic Instruments). Change in current was measured using a digital data acquisition system (BioPac, Goleta, CA) and TER calculated using Acquire and Analyze software (Physiologic Instruments). Cell viability was determined at the end of each Ussing chamber experiment through measurement of the change in short circuit current (Isc) induced by 10 μM forskolin (Alfa Aesar, Ward Hill, MA).
4
Electrophysiological Analysis of Nasal Epithelial Cells
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Electrophysiological analyses were performed in an Ussing Chamber (Physiologic Instruments, San Diego, CA). Nasal epithelial cell monolayers on inserts were mounted in an Ussing Chamber and bathed in a modified Ringer’s solution (120 mM NaCl, 10 mM D-Glucose, 3.3 mM KH2PO4, 0.83 mM K2HPO4, 1.2 mM MgCl2, 1.2 mM CaCl2, 25 mM NaHCO3, pH 7.4), maintained at 37ºC and gassed with 5% CO2/95% O2. Open-circuit conditions with intermittent short-circuiting and voltage pulsing (200 ms pulses at +/− 5 mV) were utilized. With the use of intermittent short circuiting/pulsing, peak values may not have been captured for current and conductance, and values of “0” were utilized when changes in the expected direction were not captured. Cultures were treated acutely in the Ussing Chamber with subsequent additions of apical amiloride (100 µM, Alfa Aesar), apical and basolateral forskolin (20 µM, Tocris) and IBMX (100 µM, Sigma) (F/I), apical VX-770 (1 µM, Selleck Chemicals), apical CFTR(inh)-172 (10 µM, CFTR Chemical Compound Distribution Program) and apical ATP (100 µM, Sigma).
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