U2500 self contained ussing chambers

Manufactured by Warner Instruments

The U2500 Self-contained Ussing chambers are a piece of lab equipment designed for the study of epithelial tissue transport. The chambers allow for the measurement of various parameters, such as transepithelial electrical resistance, short-circuit current, and ion fluxes, across a sample of epithelial tissue.

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Lab products found in correlation

Cftrinh 172, by Merck Group (3 mentions) Forskolin, by Abcam (2 mentions) Ab120058, by Abcam (1 mentions) Vcc mc6, by Physiologic Instruments (1 mentions) Vx 770, by Selleck Chemicals (1 mentions)

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Ussing chambers (2 citations)

3 protocols using u2500 self contained ussing chambers

Assessing ENaC and CFTR Ion Channel Function

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Ion channel function of ENaC and CFTR was assessed with and without VX-661. Before channel measurements were conducted, VX-661 (5μM) was added to CF-HBE cultures for 48 hours (basally and apically). For apical treatment, VX-661 was added for a few hours and removed to keep cultures at air-liquid interface until being assessed. Control experiments included addition of vehicle (DMSO). Inserts were mounted into Ussing chambers (system VCC MC6 from Physiologic Instruments, Inc. San Diego, CA) with U2500 Self-contained Ussing chambers (Warner Instruments, Hamden CT) as previously described by our group (21 , 38 (link)). Ringer’s solution was placed on the apical and basal side of the transwell. Once current stabilized, a low chloride solution was exchanged on the apical side with Ringer’s maintained on the basal side. Following this, 100μM amiloride was added apically to inhibit ENaC. Forskolin (10μM; Abcam, Cambridge MA) was later added apically to activate CFTR channels via activation of adenylyl cyclase and then inhibited with CFTR inh-172 (10μM) (Sigma Aldrich, St. Louis MO).

Rayner R.E., Wellmerling J., Osman W., Honesty S., Alfaro M., Peeples M.E, & Cormet-Boyaka E. (2020). In Vitro 3D culture lung model from expanded primary cystic fibrosis human airway cells. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society, 19(5), 752-761.

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Ussing Chamber Analysis of CFTR Function

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Four weeks after cells were plated in the transwell plates, ion channel function of CFTR was assessed with the Ussing chamber system (VCC MC6 from Physiologic Instruments Inc. San Diego, CA) with the U2500 Self-contained Ussing chambers (Warner Instruments, Hamden CT). To measure Cl⁻ secretion, the fully-differentiated epithelial sheets were placed with mucosal solution on apical side [120mM NaGluconate, 25mM NaHCO₃, 3.3mM KH₂PO₄, 0.8mM K₂HPO₄, 4mM Ca(Gluconate)₂, 1.2mM Mg(Gluconate)₂, 10mM mannitol] and serosal solution on basal side [120mM NaCl, 25mM NaHCO₃, 3.3mM KH₂PO₄, 0.8mM K₂HPO₄, 1.2mM CaCl₂, 1.2mM MgCl₂, 10mM Glucose]. Following this, ion channel activators and inhibitors were added as follows: 10μM amiloride apically to inhibit Epithelial Sodium Channel (ENaC), 10μM forskolin bilaterally (Abcam #ab120058) to activate CFTR channels via activation of adenylyl cylase concentrations, 10μM CFTR potentiator drug VX-770 (SelleckChem, #S1144) apically, 10μM CFTR_inh-172 (Sigma Millipore, #C2992) apically to inhibit CFTR channel, and 100μM uridine-5’-triphosphate trisodium (UTP) salt (Thermo Scientific Chemicals #AAJ63427MC) apically.

Kanke K.L., Rayner R.E., Abel E., Venugopalan A., Suu M., Stack J.T., Nouri R., Guo G., Vetter T.A., Cormet-Boyaka E., Hester M.E, & Vaidyanathan S. (2024). Single-Stranded DNA with Internal Base Modifications Mediates Highly Efficient Gene Insertion in Primary Cells. bioRxiv.

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Evaluating Epithelial Ion Channel Function

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To assess ion channel function, specifically Epithelial Sodium Channel (ENaC) and Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel function, NHBE cells were mounted into Ussing chambers (system VCC MC6 from Physiologic Instruments, Inc. San Diego, CA) with U2500 Self-contained Ussing chambers (Warner Instruments, Hamden CT) as previously described by our group^{35 (link)}. Ringer’s solution was placed on the apical and basal side of the transwell. Once current was stabilized, a low chloride solution was exchanged on the apical side with Ringer’s maintained on the basal side. Following this, 100 μM amiloride was added apically to inhibit ENaC function. Forskolin (10 μM; Abcam, Cambridge MA) was later added apically to activate CFTR channels via activation of adenylyl cyclase and then inhibited with CFTR inh-172 (10 μM) (Sigma Aldrich, St. Louis MO).

Rayner R.E., Makena P., Prasad G.L, & Cormet-Boyaka E. (2019). Optimization of Normal Human Bronchial Epithelial (NHBE) Cell 3D Cultures for in vitro Lung Model Studies. Scientific Reports, 9, 500.

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