Costar Transwell filters (6.5 mm) were used for culturing of F508del-CFTR CF cells and transduction with SFPQ/GFP adenoviral vector or control GFP vectors was performed on the basolateral side of the cell monolayer at 37 °C for 48 h. These filters were then mounted and equilibrated in Physiologic Instruments Ussing chambers as previously described78 (link), with some modifications. In the subsequent step, 10 µM amiloride was added to the apical chamber to inhibit epithelial sodium channel (ENaC)-mediated sodium absorption. After 2 min, 10 µM forskolin was added to both the basolateral and apical chambers to activate CFTR-mediated anion excretion. Following another 2 min, CFTR-mediated anion excretion was inhibited using a CFTR Inhibitor-172 (Sigma-Aldrich) to the apical chamber. During this time, currents had achieved steady-state. Short-circuit current (Isc) and transepithelial resistance (TER) were continuously measured using a Physiologic Instruments VCC-MC8 and Physiologic Instruments Acquire and Analyze 2.3 data acquisition hardware and software. Agonist- or inhibitor-induced changes in short-circuit current (ΔIsc) were calculated from differences in the mean Isc over the 10 s period preceding reagent additions.
Acquire and analyze 2
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The Acquire and Analyze 2.3 is a data acquisition and analysis system designed for laboratory environments. It provides the core functionality of data collection and processing, without interpretation or extrapolation.
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Lab products found in correlation
4 protocols using acquire and analyze 2
1
Measuring CFTR-mediated Anion Excretion
2
Electrophysiological Measurements of Mouse Jejunum
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The full-thickness preparations of mouse jejunum with a cross-sectional area of 0.3 cm2 were mounted in 4-mL Üssing chambers (Physiologic Instruments). Transepithelial potential difference was measured using paired Ag-AgCl electrodes via 3% Agar with 3-M KCl bridges and clamped at 0 mV by another pair of Ag-AgCl electrodes. The mucosal and serosal surfaces of the tissue were bathed with 4 mL of Krebs-Ringer solution with mannitol and glucose, respectively, maintained at 37 °C during the experiments. Tissue equilibrated to attain stable basal short-circuit current (Isc) and tissue conductance (Gt) for 30 min before conducting the experiment. Hydrostatic pressure was applied using DPM-1 pneumatic transducer (Bio-Tek Instruments) in a sealed mucosal chamber. Pressure stimuli of 10-s duration were applied from rest (atmospheric pressure). To assure tissue viability, acetylcholine (100 µM) was applied to the serosal side at the end of the experiment. Tissue was not used if there was no response to acetylcholine. Data were recorded using Acquire and Analyze 2.3 (Physiologic Instruments).
3
Measuring Epithelial Ion Transport
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Electrophysiological experiments to measure the transport of ions across epithelial monolayers were accomplished using established methods [32 (link)]. Reagents were introduced into bath solution in the Ussing chamber (Physiologic Instruments, San Diego, CA, USA) in the following order: Amiloride, 100 μM (apical) to inhibit the ENaC current; forskolin, 10 µM (apical and basal) to stimulate CFTR; VX-770 10 µM (apical and basal) to further potentiate the current; and CFTRinh-172 10 µM (apical). Electrophysiological data were collected and analyzed using Acquire and Analyze 2.3 software (Physiologic Instruments, San Diego, CA, USA) [32 (link)].
4
Ussing Chamber Assay for Organoid Monolayers
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Ussing chamber assays were performed essentially as described elsewhere41 (link). In short, organoids were dissociated into single cells and seeded on a permeable support (Transwell 3470; Corning) that were coated with Matrigel (1:20 in PBS), and cultured in EM organoid medium. After the cells had formed a confluent monolayer (circa 10 days after seeding), filters were inserted in Ussing chambers (P2302T/P2300; Physiologic Instruments, San Diego, CA), and bathed in modified Meyler solution (128 mM NaCl, 4.7 mM KCl, 1.3 mM CaCl2, 1.0 mM MgCl2, 0.3 mM Na2HPO4, 0.4 mM NaH2PO4, 20 mmol/l NaHCO3, 10 mM HEPES), supplemented with glucose (10 mM), in 95% O2, 5% CO2, pH 7.3, at 37 °C. The transepithelial potential difference (PD) was clamped at 0 mV using a VCC MC8 voltage clamp module (Physiologic Instruments), and the resulting short-circuit current (Isc) was digitally recorded using an analog-to-digital signal converter and associated software (Acquire and Analyze 2.3; Physiologic Instruments). Anion secretion was stimulated by addition of the adenylyl cyclase activator forskolin (10 µmol/l, Sigma-Aldrich) or the purinergic receptor agonist UTP (50 µmol/l, Sigma-Aldrich) to the luminal bathing solution. forskolin-induced and UTP-induced secretion was inhibited by Glyh-101 (20 µmol/l, Sigma-Aldrich) and T16inh-A01 (50 µmol/l, Sigma-Aldrich), respectively.
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