Vero, HEK293T, and CaCo-2 cells were seeded at a density of 15.000 cells/well in flat bottom 96-well plates. Serial fourfold dilutions of antibodies were pre-incubated with the different viral strains (MOI 0.05) for 1h at 37°C. Thereafter, the antibody-virus mixture was added to the cells. Cells without antibody and without virus served as ‘cell controls’, cells without antibody but with virus as ‘virus controls’ and cells fixed with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO), washed with DPBS for five times, and infected in parallel as ‘background controls’. Control wells fixed with paraformaldehyde were plated separately to prevent evaporation and interference with non-fixed cell layers and viruses. At 2h p.i., cells were washed and antibodies were replenished, and at 48h p.i., supernatants were harvested and analyzed using SARS-CoV-2 RT-qPCR as described above. A similar set-up was used for testing the susceptibility of SARS-CoV-2 variants aloxistatin and camostat as inhibitors of endocytosis and fusion, respectively (both MedChemExpress, Monmouth Junction, NJ). Toxicity of both inhibitors at indicated concentrations was excluded using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl¬tetrazolium bromide (MTT) assay (23 (link)). The percent neutralization was calculated as 100-(viral load of inhibited sample/viral load of uninhibited virus control)*100.
Camostat
Manufactured by MedChemExpress
Sourced in United States
Camostat is a laboratory equipment product offered by MedChemExpress. It is a serine protease inhibitor that can be used for various research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Aloxistatin, by MedChemExpress (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Decanoyl rvkr cmk, by Bio-Techne (1 mentions)
Naphthofluorescein, by Merck Group (1 mentions)
Hyclone, by GE Healthcare (1 mentions)
Chloroquine, by MedChemExpress (1 mentions)
Apilimod, by MedChemExpress (1 mentions)
7 protocols using camostat
1
SARS-CoV-2 Neutralization Assay
2
Evaluating Anti-Viral Protease Inhibitors
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The serine protease inhibitor, camostat, and the cysteine protease inhibitor, E64D, were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Calu3, Caco2, or VeroE6-TMPRSS2 cells were seeded in 96-well plates and treated with DMSO, camostat, or E64D at concentration of 1, 25, and 50μM for 2h. Calu3 and Caco2 cells were challenged with viruses at 0.1 MOI or 0.5 MOI. At 24 hpi, the cell lysates were lysed in 90 μL RLT buffer and RNA were extracted for qRT-PCR quantification of virus replication. For pseudovirus transduction, the cells were incubated with different spikes of pseudovirus, followed by luciferase signal measurement at 24 hpi.
3
Huh-7 Cells Inhibitor Pretreatment
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Huh-7 cells were incubated with 10 μM decanoyl-RVKR-CMK (Tocris), 50 μM Camostat (MedChemExpress), or 50 μM E64D (Selleck) for 2 h before infection. Equal volumes of water and dimethylsulfoxide (DMSO) served as vehicle controls. After the 2-h pretreatment, the media containing inhibitors and control media were removed. The Huh-7 cells were infected with MjHKU4r-CoV-1 at a multiplicity of infection (MOI) of 10 in a 5% CO2 incubator at 34°C for 8 h and then fixed with 4% paraformaldehyde and subjected to IF staining.
4
Inhibitor Testing in VeroE6 Cells
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VeroE6 cells were cultured at 37°C in DMEM containing 10% FBS (HyClone, GE Healthcare Life Sciences) in a 5% CO2 incubator. The indicated plasmid was transfected into VeroE6 cells with Lipofectamine™ 2000 (Thermo Fisher Scientific). The inhibitors were added to the medium at the indicated concentrations 2 hr post transfection. Cells were harvested or fixed 24 hr post transfection for subsequent western blot or immunofluorescence analysis. The stock inhibitors tested in this study were all prepared in DMSO solutions, including CMK (10 mM), D6R (5 mM), SSM3 (25 mM) (Tocris Bioscience), naphthofluorescein (10 mM) (Sigma-Aldrich) and camostat (100 mM) (Medchemexpress). The concentration and percentage of DMSO (v/v) in the working solution of these inhibitors after dilution of the stock solutions are as follows: CMK 50 μM (0.5% DMSO); D6R 50 μM (1% DMSO); SSM3 25 μM (0.1% DMSO); naphthofluorescein 20 μM (0.2% DMSO); camostat 500 μM (0.5% DMSO).
5
Inhibition of SARS-CoV-2 Entry and Replication
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The serine protease inhibitor, camostat (HY-13512), and the cysteine protease inhibitor, E64D (HY-100229), were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Calu3 or VeroE6-TMPRSS2 cells were treated with DMSO, camostat, or E64D at concentrations of 1, 25, and 50 µM for 2 h before authentic virus infection. At 24 hpi, the cell lysates were harvested for qRT-PCR quantification of virus replication. For pseudovirus entry assays, VeroE6-TMPRSS2 cells were treated with DMSO, camostat, or E64D at concentrations of 1, 25, and 50 µM for 2 h before pseudoviruses transduction. The cell lysates were lysed for detection of luciferase signal 18 h post-transduction.
6
Inhibiting Endocytosis and Proteases
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For experiments involving endocytosis and protease inhibitors, 293T-hACE2 or 293T-hACE2-TMPRSS2 cells were pretreated with either endocytosis inhibitors (Chloroquine, MedChemExpress, 0-10 μM; Tetradrine, Sigma-Aldrich, 0-2 μM and Apilimod, MedChemExpress, 0-100 nM) or protease inhibitors (E64d, MedChemExpress, 0-5 μM; Camostat, MedChemExpress, 0-100 μM) for 2 h. Then the pseudovirions was added into the corresponding wells of plates. 24 h post infection, cells were lysed, and the infection efficiency of viruses was detected by measuring the luciferase activity using Luciferase Assay System.
7
Protease Inhibitors Suppress SARS-CoV-2 Omicron Infection
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The serine protease inhibitor, camostat (HY-13512), and the cysteine protease inhibitor, E64D (HY-100229), were purchased from MedChemExpress (Monmouth Junction, NJ, USA). VeroE6-TMPRSS2 cells were treated with DMSO, camostat, or E64D at concentration of 50 μM for 2 h before pseudovirus transduction. At 24 hpi, the cell lysates were lysed for detection of luciferase signal. The DMSO-treated cells transduced with pseudovirus carrying the same spike served as the control. Luciferase readings from the inhibitor-treated cells were normalized with the mock-treated controls. For protease inhibitor treatment on Calu3 and VeroE6 cells, Calu3 and VeroE6 cells were infected with the indicated Omicron sublineages at 0.5 MOI or 0.1 MOI after treatment with DMSO, camostat, or E64D at 25 μM for 2 h. At 24 hpi, virus infected cells were lysed for detection of viral sgE gene copies. The human nasal epithelial cells were infected with the indicated Omicron sublineages at 2 MOI after treatment with DMSO, camostat, or E64D at 10 μM for 2 h. At 72 hpi, supernatants were lysed for detection of viral RdRp gene copies.
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