As described previously, ELISA detected the specific anti-RBD of SARS-CoV-2 wild-type binding antibody in the serum from each mouse [40 (link)]. The recombinant RBD of wild-type SARS-CoV-2 expressed in HEK293 cells (SinoBiological, Beijing, China) was used as coating antigen, whose final concentration was 1 μg/mL. Sera were three-fold serially diluted starting at 1:200, using PBST containing 3% BSA. The antibody titer was defined as the reciprocal of the largest serum dilution for which the OD450nm value was greater than 2.1-fold of the negative sera, according to the previous method [39 (link)].
Hek293 cells
Manufactured by Sino Biological
Sourced in China
HEK293 cells are a widely used human embryonic kidney cell line. They are adherent cells that can be cultured in vitro and are commonly used as a model system for various applications, including protein expression, virus production, and cellular studies.
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Lab products found in correlation
Trizol, by Thermo Fisher Scientific (2 mentions)
Camostat, by Bio-Techne (1 mentions)
Decanoyl rvkr cmk, by R&D Systems (1 mentions)
Hace2 protein, by Sino Biological (1 mentions)
Pd0325901, by Bio-Techne (1 mentions)
Bay 11 7821, by Bio-Techne (1 mentions)
Dasatinib, by LC Laboratories (1 mentions)
Mk 2206, by Apexbio (1 mentions)
Spectramax id3 plate reader, by Molecular Devices (1 mentions)
Lipo8000, by Beyotime (1 mentions)
Sigmaplot 10, by Grafiti LLC (1 mentions)
5 protocols using hek293 cells
1
ELISA for SARS-CoV-2 RBD Antibody Detection
2
SARS-CoV-2 Infection Inhibition Assay
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The reduction of luciferase gene expression was detected to evaluate the infection inhibition effect of hACE2 protein (expressed in HEK293 cells, Sino Biological), protease inhibitor Decanoyl-RVKR-CMK (Furin inhibitor, R&D Systems), protease inhibitor Camostat (TMPRSS2 inhibitor, Tocris Bioscience), protease inhibitor E64D (Cathepsin L inhibitor, APExBIO), monoclonal antibody, and sera. As described previously19 (link),20 (link), the tested samples were diluted (starting with 30 times dilution and three times gradient, a total of eight gradients), after which the virus solution was added. On each 96-well plate, eight virus control wells and eight cell control wells were arranged. In the virus control wells, no test sample but only virus solution was added; in the cell control wells, no virus solution but only a complete culture medium was added. The 96-well plates were incubated at 37 °C for 1 h, after which trypsinized Huh7 cells (2 × 104/100 μL) were added to each well. After incubation for 24 h in an atmosphere comprising 5% CO2 at 37 °C, luminescence was measured as described above. The EC50 value of each sample was calculated using the Reed–Muench method.
3
Modulation of Cell Signaling Pathways by hRNase 1
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All cell lines used in this study were obtained from American Type Culture Collection (ATCC). Cell lines have been validated by STR DNA fingerprinting at MD Anderson Cancer Center and routinely tested for mycoplasma contamination. All cell lines were cultured in the medium recommended by ATCC. Treatment with recombinant hRNase 1 protein purified from HEK293 cells (Sino Biological Inc. #13468-H08H-100) was carried out at a concentration of 1 µg/ml for 30 min or the indicated time after serum-free starvation for 3 h. The concentrations of inhibitors used were QNZ (30 nM, Selleck Chemical, #S4902), Bay 11-7821 (0.2 μM, Tocris Bioscience, #1744/10), GSK1120212 (1 nM, Apexbio Technology, #A301850), PD-0325901 (0.2 or 2 μM, Tocris Bioscience, #4192/10), MK-2206 (20 nM, Apexbio Technology, #A301010), and Dasatinib (2 nM, LC Laboratories, #D-3307).
4
Measuring S1P-induced GPCR signaling
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HEK293T cells were transiently cotransfected with the expression construct pcDNA6/S1PR2 and a NanoLuc reporter vector pNL1.2/CRE using the transfection reagent Lipo8000 (Beyotime Technology). Next day, the transfected cells were trypsinized and seeded into a white opaque (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.23.581834 doi: bioRxiv preprint 96-well plate. After cultured in complete DMEM medium for ~24 h to ~90% confluence, the medium was removed and the activation solution (serum-free DMEM plus 1% BSA) containing varied concentrations of S1P (MedChemExpress) or recombinant MYDGF produced either from E. coli (our present work) or HEK293 cells (from Sino Biological) was added (50 μl/well). After continuously cultured at 37 °C for 4 h, bioluminescence was measured on a SpectraMax iD3 plate reader (Molecular Devices) after addition of the diluted NanoLuc substrate (10 μl/well, 30-fold dilution of the Promega substrate stock using the activation solution). The measured bioluminescence data were expressed as mean ± standard deviate (SD, n = 3) and plotted using SigmaPlot10.0 software (SYSTAT software).
The copyright holder for this preprint this version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.23.581834 doi: bioRxiv preprint 96-well plate. After cultured in complete DMEM medium for ~24 h to ~90% confluence, the medium was removed and the activation solution (serum-free DMEM plus 1% BSA) containing varied concentrations of S1P (MedChemExpress) or recombinant MYDGF produced either from E. coli (our present work) or HEK293 cells (from Sino Biological) was added (50 μl/well). After continuously cultured at 37 °C for 4 h, bioluminescence was measured on a SpectraMax iD3 plate reader (Molecular Devices) after addition of the diluted NanoLuc substrate (10 μl/well, 30-fold dilution of the Promega substrate stock using the activation solution). The measured bioluminescence data were expressed as mean ± standard deviate (SD, n = 3) and plotted using SigmaPlot10.0 software (SYSTAT software).
5
SARS-CoV-2 Spike Protein Treatment of THP-1 Cells
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THP-1 monocyte-like cells were seeded in presence of phorbol 12-myristate 13-acetate (PMA, 100ng/mL) and allowed to differentiate as described above. Cells were treated with 8nM (0.6μg/mL) of recombinant soluble SARS-CoV-2 Spike S1 protein purified from HEK293 cells (SinoBiological; catalog no. 40591-V08H-B) or an equivalent volume of vehicle control for specified times. Cell culture supernatants were collected for ELISA assays and RNA was extracted from infected cells using Trizol (ThermoFisher; catalog no. 15596026) following manufacturer’s instructions.
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