Primary nasal cells were grown on transwells and studied in a non-perfused Ussing chamber (Physiologic Instruments, San Diego, CA, USA). The buffer solutions were as follows: (in mM) Krebs bicarbonate: 126 NaCl, 0.38 KH2PO4, 2.13 K2HPO4, 1 MgCl2, 1 CaCl2, 24 NaHCO3, 10 glucose. Zero chloride buffer: 116.2 Na gluconate, 2.4 KH2PO4, 1.24 K2HPO4, 1 MgSO4, 1 Ca gluconate, 25 NaHCO3, 10 glucose. zero bicarbonate buffer: 145 NaCl, 3.3 K2HPO4, 10 HEPES, 1.2 MgCl2, 1.2 CaCl2, 10 glucose, pH adjusted with acetic acid. Bath solutions were maintained at pH 7.4 and 37 °C and continuously gassed with a 5% CO2/95% O2 mixture or air in the case of bicarbonate free buffer. The transepithelial potential (Vte) was recorded in open-circuit mode and the baseline resistance (Rte) was measured following repeated, brief short-circuit current pulses (1 µA every 30 s). The results are presented as equivalent transepithelial current (Ieq), which was calculated using Ohm’s law. CFTR function was determined after inhibition of the epithelial sodium channel (ENaC) with amiloride (30 µM, Spectrum Chemical, Gardena, CA, USA) and following cAMP activation with forskolin (10 µM, Sigma-Aldrich, St. Louis, MO, USA). CFTR activity was confirmed as Ieq difference following CFTR inhibition with CFTRInh-172 (10 µM, EMD Millipore Corp, Burlington, MA, USA) [22 (link)].
Cftrinh 172
Manufactured by Merck Group
Sourced in United States, Germany, France
CFTRinh-172 is a laboratory reagent that functions as a selective inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. It is used in research applications to study the role of CFTR in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Forskolin, by Merck Group (16 mentions)
Amiloride, by Merck Group (10 mentions)
Ussing chamber, by Physiologic Instruments (8 mentions)
Genistein, by Merck Group (6 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (6 mentions)
Amiloride, by Spectrum Chemical (5 mentions)
Vx 770, by Selleck Chemicals (5 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (4 mentions)
Vx 809, by Selleck Chemicals (4 mentions)
Ouabain, by Merck Group (3 mentions)
U2500 self contained ussing chambers, by Warner Instruments (3 mentions)
T16ainh a01, by Merck Group (3 mentions)
Ivacaftor, by Selleck Chemicals (3 mentions)
Vx 770, by Merck Group (2 mentions)
Mannitol, by Merck Group (2 mentions)
Capsazepine, by Merck Group (2 mentions)
Calcein green, by Thermo Fisher Scientific (2 mentions)
Adenosine triphosphate (atp), by Merck Group (2 mentions)
Monna, by Merck Group (2 mentions)
Tannic acid, by Merck Group (2 mentions)
68 protocols using cftrinh 172
1
Nasal Epithelial Cell CFTR Function Assay
2
Evaluating CFTR Modulators in Nasal Epithelial Cells
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Primary nasal epithelial cultures were studied in a non-perfused Ussing chamber (Physiologic Instruments, San Diego, CA, USA) after 48 h treatment with 0.1% DMSO, 3 µM VX-809, 0.5 µM AC1 + 3 µM AC2-1, 0.5 µM AC1 + 3 µM AC2-2 or 3 µM VX-661 + 3 µM VX-445. The buffer solution (126 mM NaCl, 24 mM NaHCO3, 2.13 mM K2HPO4, 0.38 mM KH2PO4, 1 mM MgSO4, 1 mM CaCl2 and 10 mM glucose) was maintained at pH 7.4 and 37 °C and continuously gassed with a 5% CO2/95% O2 mixture. The transepithelial potential (Vte) was recorded in open-circuit mode and the baseline resistance (Rte) was measured following repeated, brief short-circuit current pulses (1 µA every 30 s). The results are presented as equivalent transepithelial current (Ieq), which was calculated using Ohm’s law. Following the epithelial sodium channel (ENaC) inhibition with 30 µM of Amiloride (Spectrum Chemical, Gardena, CA, USA), CFTR was stimulated with 10 µM forskolin and 1 µM VX-770 for cells treated with VX-809 or VX-661 + VX-445, or 1.5 µM for AP2 for cells treated with AC1 + AC2-2. Then, CFTR was inhibited with 5 µM CFTRInh-172 (EMD Millipore Corp., Billerica, MA, USA) [15 (link),24 (link)].
3
Measuring CFTR-Dependent Intestinal Ion Transport
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The method of measuring the CFTR‐dependent intestinal epithelial ion transport properties in rectal organoid‐derived monolayers have been described previously (Li et al, 2004 (link); Molinski et al, 2017 (link); Cao et al, 2018 (link); Zomer‐van Ommen et al, 2018 (link)). Briefly, upon dissociation of the 3D intestinal organoid cultures, the cells were grown into the 2D monolayer on PureCol®‐coated Transwell (Costar™ 3470, Corning, Tewksbury, US). They were studied in a non‐perfused Ussing chamber (Physiologic Instruments, San Diego, CA). The buffer was maintained at pH 7.4 and 37°C and continuously gassed with 5% CO2/95% O2 mix. The transepithelial potential (Vte) was recorded and the baseline resistance (Rte) was measured following repeated, brief short‐circuit current pulses (1 μA every 30 s). The results are presented as equivalent transepithelial current (Ieq), which was calculated using Ohm’s law. CFTR function was determined after inhibition of the epithelial sodium channel (ENaC) with amiloride in the apical bath (100 μM, Spectrum Chemical, Gardena, CA) and cAMP activation with forskolin (10 μM, Sigma‐Aldrich, US). CFTR activity was quantified as Ieq difference following forskolin stimulation, CFTRInh‐172 (10 μM, EMD Millipore Corp. US) was applied to inhibit CFTR activity.
4
Inhibition of CFTR and PAF signaling
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CFTRinh-172 (Thiazolidinone CFTR inhibitor, 3-[(3-Trifluoromethyl) phenyl]-5-[(4-carboxyphenyl) methylene]-2-thioxo-4-thiazolidinone) was purchased from EMD Millipore (Billerica, MA). MalH-2 (di-sulfonate Glycine Hydrozide), a water soluble CFTR inhibitor, was provided by Verkman AS (UCSF). SB203580 and Wortmannin were purchased from Calbiochem (San Diego, CA). LPS (E. coli 0111:B4) was from Sigma-Aldrich (St. Louis, MO). Lipoxin A4 analog, Methyl Arachidonyl Fluorophosphonate (MAFP), and PAF acetylhydrolase assay kit were from Cayman Chemical (Ann Arbor, MI). Purified NA/LE Rat Anti-Mouse CD162, Rat (LEW) IgG1, κ; and purified NA/LE rat IgG1, κ isotype control antibodies; PE-conjugated PSGL-1 antibody or PE-conjugated isotype control antibody were purchased from BD Pharmingen (San Diego, CA). WEB2086 (a platelet activating factor receptor antagonist) was from Tocris Bioscience (Ellisville, MO). PAF C16 was purchased from Avanti Polar Lipids Inc (Alabaster, AL).
5
Evaluating CFTR Function in Nasal Epithelial Cells
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Nasal epithelial cells were studied in a non-perfused Ussing chamber (Physiologic Instruments, San Diego, CA). The buffer (126 mM NaCl, 24 mM NaHCO3, 2.13 mM K2HPO4, 0.38 mM KH2PO4, 1 mM MgSO4, 1 mM CaCl2, and 10 mM glucose) was maintained at pH 7.4 and 37 °C and continuously gassed with 5% CO2/95% O2 mix.59 (link) The transepithelial potential (Vte) was recorded and the baseline resistance (Rte) was measured following repeated, brief short-circuit current pulses (1 µA every 30 sec). The results are presented as equivalent transepithelial current (Ieq), which was calculated using Ohm’s law. CFTR function was determined after inhibition of the epithelial sodium channel (ENaC) with amiloride (100 µM, Spectrum Chemical, Gardena, CA) and cAMP activation with FSK (10 µM, Sigma-Aldrich, USA). CFTR activity was calculated as Ieq difference following CFTR inhibition with CFTRinh-172 (5 µM, EMD Millipore Corp., USA). For drug rescue experiments nasal cell were treated with the corrector VX-809 (6 µM) for 48 h and acutely with the potentiator VX-770 (1 µM).
6
Inhibiting CFTR Ion Channel Activity
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CFTRinh-172 and 4,4′-diisothiocyanatostilbene-2,2 ´-disulfonic acid disodium salt hydrate (DIDS) were purchased from Merck (Darmstadt, Germany). GlyH-101 was obtained from R&D Systems (Minneapolis, MN, USA). Piperine (purity > 98%) was prepared from black peppers as previously described [20 ].
7
Nasal Epithelial Cells Ussing Chamber
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Nasal epithelial cells were studied in a nonperfused Ussing chamber (Physiologic Instruments, San Diego, CA). The buffer (126 mmol/l NaCl, 24 mmol/l NaHCO3, 2.13 mmol/l K2HPO4, 0.38 mmol/l KH2PO4, 1 mmol/l MgSO4, 1 mmol/l CaCl2, and 10 mmol/l glucose) was maintained at pH 7.4 and 37 °C and continuously gassed with a 5% CO2/95% O2 mixture. The transepithelial potential (Vte) was recorded and the baseline resistance (Rte) was measured following repeated, brief short-circuit current pulses (1 µA every 30 seconds). The results are presented as equivalent transepithelial current (Ieq), which was calculated using Ohm’s law. CFTR function was determined after inhibition of the epithelial sodium channel (ENaC) with amiloride (100 µmol/l, Spectrum Chemical, Gardena, CA) and cAMP activation with forskolin (10 µmol/l, Sigma-Aldrich). CFTR activity was calculated as Ieq difference following CFTR inhibition with CFTRInh-172 (5 µmol/l, EMD Millipore, Billerica, MA).
8
CFTR Function in Nasal Epithelial Cells
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Primary nasal epithelial cells were grown on transwells and studied in a nonperfused Ussing chamber (Physiologic Instruments, San Diego, CA, USA). Cells expressing mutant CFTR were transduced as described earlier. Where indicated, cells were treated with either 0.1% dimethyl sulfoxide (DMSO) or the CFTR modulator 3 µM VX-809, 48 h before the experiments at 37°C. The buffer solution (126 mM NaCl, 24 mM NaHCO 3 , 2.13 mM K 2 HPO 4 , 0.38 mM KH 2 PO 4 , 1 mM MgSO 4 , 1 mM CaCl 2 and 10 mM glucose) was maintained at pH 7.4 and 37°C and continuously gassed with a 5% CO 2 /95% O 2 mixture. The transepithelial potential was recorded in open-circuit mode and the baseline resistance was measured following repeated, brief short-circuit current pulses (1 µA every 30 s). The results are presented as equivalent transepithelial current (I eq ), which was calculated using Ohm's law. CFTR function was determined after inhibition of the epithelial sodium channel (ENaC) with amiloride (30 µM, Spectrum Chemical, Gardena, CA, USA) and following cAMP activation with forskolin (10 µM, Sigma-Aldrich, St Louis, MO, USA) and when indicated 1 µM VX-770. CFTR activity was confirmed as I eq difference following CFTR inhibition with CFTR inhibitor 172 (CFTR Inh172 ) (10 µM, EMD Millipore Corp., Billerica, MA, USA) [28, 29] .
9
Whole-Cell Patch-Clamp Recordings of CFTR Activity
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Patch-clamp experiments were performed with an automatic electrophysiology workstation (Port-a-Patch, Nanion Technologies GmbH, Hambourg, Germany) coupled to an external amplifier unit HEKA EPC-10 [37 (link),38 (link)]. Whole-cell recordings were performed with treated or non-treated cells with the MBTP1 inhibitor. All measurements were obtained at room temperature. The voltage clamp protocol was carried out between −80 and +80 mV (10 mV per steps) with a holding membrane potential of −80 mV. The following buffers were used to suspend the cells: 140 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM Hepes (pH 7.4), 5 mM D-glucose monohydrate, 298 mOsm. The internal buffers were 50 mM CsCl, 10 mM NaCl, 60 mM Cs-Fluoride, 20 mM EGTA, 10 mM Hepes/CsOH, 5 mM Mg-ATP; pH 7.2; 285 mOsmol. CFTR’s activators (forskolin, 10 µM and genistein, 30 µM; Sigma Aldrich, Saint-Quentin Fallavier, France) and inhibitor (CFTRinh172, 10 µM; Sigma Aldrich) were added to activate or inhibit its activity, respectively.
10
Inhibitor Assays for Cell Signaling
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The antibody against pMLC20 was purchased from AbCam (ab2480). CFTR (Inh)-172 was purchased from Sigma and stored as frozen aliquots of 20 mM in DMSO prior to being used at a final concentration of 20 µM. Blebbistatin enantiomers were purchased from Santa Cruz Biotechnology, Inc., reconstituted in DMSO to a stock of 40 mM, and used at a final concentration of 40 µM. Latrunculin A was purchased from Sigma; stocks were made up in DMSO to 2 mM and used at a final concentration of 5 µM. All inhibitor assays included DMSO in media used at a final concentration of 0.1%. Mannitol and NaCl were also purchased from Sigma. TNF-α was purchased from Shenandoah Biotechnology Inc., stored as a stock of 100 µg/ml in double-distilled water (ddH2O) with 0.1% bovine serum albumin (BSA), and used at a final concentration of 30 ng/ml. Actinomycin D was purchased from Sigma; stocks were made at 5 mg/ml in DMSO and used at a final concentration of 5 µg/ml. All drugs were divided into aliquots and stored in −20°C or −80°C according to the manufacturer’s instructions.
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