Inhibition of pseudovirus entry using various protease inhibitors was carried out as described previously (18 (link)). Briefly, target cells were preincubated with 10 or 50 μm proprotein convertase inhibitor Dec-RVKR-CMK (Enzo Life Sciences), 20 or 100 μm camostat mesylate (Sigma-Aldrich), 20 or 100 nm bafilomycin A1 (Sigma-Aldrich), 10 or 50 μm E-64d (Sigma-Aldrich), 50 μm cathepsin L inhibitor Z-FY-CHO (Santa Cruz Biotechnology), or 50 μm cathepsin B inhibitor CA-074 Me (Santa Cruz Biotechnology) at 37 °C for 1 h. The cells were subsequently transduced by retroviruses pseudotyped with PEDV spike, MERS-CoV spike, or VSV envelope glycoprotein. The cells were incubated at 37 °C for 6–8 h, and then the medium was replaced with fresh DMEM. 48 h later, the cells were lysed and measured for luciferase activity.
Camostat mesylate
Manufactured by Merck Group
Sourced in United States, Germany
Camostat mesylate is a laboratory reagent used in research applications. It is a serine protease inhibitor that can be used to study the role of proteases in various biological processes. The core function of camostat mesylate is to inhibit the activity of serine proteases, which are a class of enzymes involved in a wide range of physiological and pathological processes.
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Lab products found in correlation
Bafilomycin a1, by Merck Group (8 mentions)
Chloroquine, by Merck Group (5 mentions)
Nafamostat mesylate, by Merck Group (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Ammonium chloride, by Merck Group (3 mentions)
Recombinant human ifn β beta 300 02bc, by Thermo Fisher Scientific (3 mentions)
Recombinant human ifn β beta 1a 11415 1, by PBL Assay Science (3 mentions)
Nh4cl, by Merck Group (3 mentions)
Leupeptin, by Merck Group (2 mentions)
Incyclinide, by MedChemExpress (2 mentions)
Apratastat, by MedChemExpress (2 mentions)
Batimastat, by Selleck Chemicals (2 mentions)
Human ifn λ lambda, by Thermo Fisher Scientific (2 mentions)
Human ifn λ lambda 11725 1, by PBL Assay Science (2 mentions)
Eg00229, by Selleck Chemicals (2 mentions)
Amphotericin b, by Merck Group (2 mentions)
Trypsin, by Merck Group (2 mentions)
Hd fugene transfecting reagent, by Promega (2 mentions)
Mdl28170, by Merck Group (2 mentions)
Glycyrrhizin, by Merck Group (2 mentions)
40 protocols using camostat mesylate
1
Inhibition of Pseudovirus Entry by Protease Inhibitors
2
Compound Solubilization and Characterization
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Compounds were obtained from ThermoFisher Scientific (amitriptyline hydrochloride, carbamazepine) and Sigma-Aldrich (chlorpromazine hydrochloride, chlorpromazine sulfoxide, clemastine fumarate salt, desloratadine, haloperidol, imipramine hydrochloride, loratadine, remdesivir, terfenadine, camostat mesylate and E64D). All compounds were solubilized in dimethyl sulfoxide (DMSO). The final concentration of DMSO was 0.01% for all experiments.
3
Transepithelial Fluid Transport Measurement
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Transepithelial fluid transport measurement was performed according to Aarbiou et al. [26 (link)]. Briefly, the apical surface of epithelia was washed with a saline solution containing (in mM): 137 NaCl, 2.7 KCl, 8.1 Na2HPO4, 1.5 KH2PO4, 1 CaCl2 and 0.5 MgCl2. The apical medium was removed, then 200 μl of saline solution at room temperature was added to the apical surface. Filters were rotated gently to remove the medium remaining at the walls of the cup, and then the fluid was recovered. This process was repeated three times. After washing, the apical side of the epithelium was covered with 50 μl of the same saline solution (with or without 30 μM camostat mesylate, Sigma-Aldrich), and 150 μl of mineral oil to prevent evaporation. Cells were maintained at 37°C in 5% CO2 with complete medium present in the basolateral compartment. After 24 hrs, the apical fluid was carefully removed, centrifuged to separate the mineral oil, and the residual volume of aqueous phase was measured. Fluid absorption is reported as ml/(cm2 h).
4
Characterizing SARS-CoV-2 Spike Protein Interactions
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Mouse anti-SARS-CoV-2 spike S2 antibody was obtained from Euromedex, SARS-CoV-2 nucleocapsid was detected using a rabbit polyclonal antibody (Novus), human TMPRSS2 was detected with a rabbit monoclonal antibody from Abcam (catalog no. ab92323), and mouse anti-tubulin was from Sigma. Anti-capsid of murine leukemia virus (MLV; ATCC CRL1912) was produced in vitro by using a MiniPerm apparatus (Heraeus) as recommended by the manufacturer.
E64D (2S,3S)-trans-Epoxysuccinyl-l -leucylamido-3-methylbutane ethyl ester was purchased from Merck, and camostat mesylate was purchased from Sigma. Decanoyl-RVKR-chloromethylketone (CMK; furin convertase inhibitor) was from Enzo Life Science.
E64D (2S,3S)-trans-Epoxysuccinyl-
5
Screening Cysteine Protease Inhibitor Library
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The cysteine protease inhibitor library screened in this work has been described elsewhere (Ang et al., 2011 (link)). Briefly, the library includes ∼2100 electrophilic cysteine protease inhibitors of various chemotype (glycine nitriles, ketobenzoxazoles, ketooxadiazoles, vinylsulfones, etc.), which were synthesized during the course of industrial drug discovery programs targeting human cathepsins (Palmer et al., 1995 (link), Palmer et al., 2005 (link), Palmer et al., 2006 (link), Rydzewski et al., 2002 (link)). Camostat mesylate, leupeptin, bafilomycin A1, ammonium chloride, and chloroquine were purchased from Sigma–Aldrich.
6
Evaluating Antiviral Compounds against SARS-CoV-2
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Dulbecco’s modified Eagle’s medium (DMEM) and phosphate-buffered saline (PBS) were purchased from Life Technologies (Carlsbad, CA, USA). Fetal bovine serum (FBS) was obtained from Eurobio. Remdesivir (GS-5734) and GC376 were purchased from Selleck Chemicals (Houston, TX, USA). Camostat mesylate, tariquidar, and dimethylsulfoxide (DMSO) were obtained from Sigma (Saint Louis, MO, USA). The lichen compounds were resuspended in DMSO at 100 mM.
7
Modulation of Viral Infection Pathways
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Camostat mesylate, E-64, and nanchangmycin were purchased from SIGMA and reconstituted following the manufacturer’s instructions. Adherent cells were treated with 25 μM camostat, 100 μM E-64, 100 nM nanchangmycin or DMSO for 2 hr at 37°C. After treatment, the medium was removed, and the cells were infected with indicated pseudoviruses. Luciferase activity was measured at 48 hpi. MnCl2 tetrahydrate was purchased from SIGMA and was diluted in ddH2O. Cell lines and primary cells were treated with 1mM MnCl2 for 2 hr, and subjected to viral infections, VIAs and western blot. Recombinant human IL-4 (InvivoGen) was reconstituted in ddH2O and used at different concentrations to stimulate U2OS cells for 48 hr prior to viral infections, VIAs and western blot.
8
SARS-CoV-2 Variant Interferon Sensitivity
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Recombinant human IFN-β (beta) (300-02BC) and human IFN-λ (lambda) (300-02 L) were obtained from PeproTech and were used to test IFN sensitivity of full-length, authentic SARS-CoV-2 variants (IFN was added 18 h prior to inoculation and removed prior to virus addition). Recombinant human IFN-β (beta) 1a (11415-1) and human IFN-λ (lambda) (11725-1) were obtained from PBL Assay Science and were used to test IFN sensitivity of VSV-based pseudoviruses (IFN was added 18 h prior to inoculation and removed prior to virus addition). The following inhibitors were reconstituted in DMSO: E64d (Sigma; E8640), camostat mesylate (Sigma; SML0057), incyclinide (MedChemExpress; HY-13648), apratastat (MedChemExpress; HY-119307), batimastat (SelleckChem: S7155), EG00229 (SelleckChem; E1119). Bafilomycin A1 (Sigma; SML1661) was received as a ready-made solution in DMSO, and amphotericin B (Sigma; A2942) was received as a ready-made solution in deionized water.
9
SARS-CoV-2 Spike Pseudovirus Infection Assay
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A6-001 (ChemDiv; Cat# F575-0482, N-(4-fluorobenzyl)-3-((6-phenylpyridazin-3-yl)amino)benzamide), A6-004 (ChemDiv; Cat# F474-2423, ethyl 8-methyl-4-((4-(methylthio)benzyl)amino)-2-oxo-1,2-dihydroquinoline-3-carboxylate), abamectin (Cayman Chemical, Ann Arbor, MI, USA), ivermectin (Sigma-Aldrich, St. Louis, MO, USA), camostat mesylate (Sigma-Aldrich, #SML0057), BAPTA-AM (Sigma-Aldrich, #A1076), amphotericin B solution (Sigma-Aldrich, #A2942), and trypsin (Sigma-Aldrich, #T6567) were purchased commercially. Lentifect™ SARS-CoV-2 Spike-pseudotyped Lentivirus used in the single-round infection (GFP reporter) was commercially acquired from Genecopoeia (cat# SP001). The following antibodies were acquired commercially: anti-SARS-CoV-2 S2 (GeneTex #GTX632604), anti-aldolase A (Santa Cruz Biotechnology #SC-390733), and anti-HIV-p24 (GeneTex #GTX64128).
10
Alveolar Cell Infection and Lung Culture
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Alveolar spheres were trypsinized to generate single cells then seeded on 1% Matrigel coated 48 well tissue culture plates for infection. Attached cells were incubated with 100 μl of virus containing medium for 1 day then changed back with alveolar cell medium. For small molecule test, medium containing 20 μM or 50 μM Camostat Mesylate (SigmaAldrich) or E64-D (SigmaAldrich), and 2 μM or 10 μM Ampilimod (MedChemexpress) was added to cells 1 day before infection, and was kept in the medium during infection. Engineered lungs cultured for 26 days were transferred from original incubator for extended organ culture to a new small incubator which runs with a minimum of 25 mL medium (Fig. 5 b). Medium containing WT or D614G pseudotyped viruses were added into the incubator and perfused at 1 mL/min for 24 h before transferring back to the normal organ culture incubators. For small molecule inhibition assay on the regenerated lung, medium containing 50 μM Camostat Mesylate was used for lung perfusion starting from Day 25, 24 h before virus infection.
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