HEK293T cells were transiently transfected with human APN (hAPN). hAPN with a C-terminal Flag tag was cloned into pcDNA3.1+ by GenScript. HEK293T cells seeded at 16E6 cells in 100 mm dishes coated with poly-D-Lysine and incubated overnight at 37 °C with 5% CO2. The following day cells were transfected with 8 μg of DNA using 30 μL of Lipofectamine 2000 transfection kit in Opti-MEM. Post 5 hours of incubation with transfection reagent, cells were trypsinized with 0.05% Trypsin EDTA for 3 minutes and neutralized with DMEM with 10% FBS 1% penicillin–streptomycin. 40,000 cells were seeded in 96-well plates (Corning 3610) coated with poly-lysine (Sigma P4707) and allowed to incubate at 37 °C with 5% CO2. Following day, media was removed and 20 μL of DMEM was added followed by 20 μL of VSV pseudotyped with PDCoV S variants for 2 hours at 37 °C with 5% CO2. Cells were supplemented with 40 μL of DMEM containing 20% FBS and 2% PenStrep and incubated for 22 hours at 37 °C with 5% CO2. 40 μL of One-Glo-EX substrate (Promega) was added and cells were incubated at 37 °C for 10 minutes with shaking. Luminescence was read using BioTek Neo2 plate reader. Technical replicates of three were run for three different lots of pseudovirus for each variant. Luciferase units were plotted using Graphpad Prism.
One glo ex substrate
Manufactured by Promega
One-Glo-EX substrate is a luminescent reagent designed for the quantitative detection of ATP in cell-based assays. It produces a stable luminescent signal proportional to the amount of ATP present in the sample.
Automatically generated - may contain errors
4 protocols using one glo ex substrate
1
Evaluating SARS-CoV-2 Spike Variants
2
mAb Neutralization Assay for PDCoV S VSV
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For mAb neutralization assays against PDCoV S VSV, HEK293T cells were transfected with hAPN as described in the cell entry assay. 40,000 cells/well were seeded into 96-well plates coated with poly-lysine and incubated overnight at 37°C. Antibody titration series was performed in a half-area 96-well plate (Greiner) starting at a 2x concentration of 20 μg/ml and diluting 1:3 with DMEM. Equal volume of pseudovirus was added and incubated at room temperature for 30 minutes and 40 μL was transferred to cells and incubated for 2 hours at 37 °C with 5% CO2. Cells were supplemented with 40 μL of DMEM with 20% FBS and 2% PenStrep and incubated for 22 hours at 37 °C with 5% CO2. After 22 hrs, 40 μL of One-Glo-EX substrate (Promega) was added to each well and incubated on a plate shaker in the dark for 5 min before reading the relative luciferase units using a BioTek Neo2 plate reader. Relative luciferase units were used to determine (%) neutralization in Prism (GraphPad). Nonlinear regression curve fit and sigmoidal 4PL where x is the concentration was run to determine IC50 values for 3 biological replicates of pseudovirus and three technical replicates for each titration series.
3
SARS-CoV-2 Pseudovirus Neutralization Assay
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HEK293-hACE2 cells (Crawford et al., 2020 (link)) were cultured in DMEM with 10% FBS (Hyclone) and 1% PenStrep with 8% CO2 in a 37°C incubator (ThermoFisher). One day prior to infection, 40 μL of poly-lysine (Sigma) was placed into 96-well plates and incubated with rotation for 5 min. poly-lysine was removed, plates were dried for 5 min then washed 1 × with water prior to plating with 40,000 cells. The following day, cells were checked to be at 80% confluence. In an empty half-area 96-well plate a 1:3 serial dilution of sera was made in DMEM and diluted pseudovirus was then added to the serial dilution and incubated at room temperature for 30-60 min. After incubation, the sera-virus mixture was added to the cells at 37°C and 2 hours post-infection, 40 μL 20% FBS-2% PenStrep DMEM was added. After 17-20 hours (VSV) or 48 hours (HIV, MLV) 40 μL/well of One-Glo-EX substrate (Promega) was added to the cells and incubated in the dark for 5-10 min prior reading on a BioTek plate reader. Measurements were done in at least duplicate. Relative luciferase units were plotted and normalized in Prism (GraphPad). Nonlinear regression of log(inhibitor) versus normalized response was used to determine IC50 values from curve fits. Kruskal Wallis tests were used to compare two groups to determine whether they were statistically different.
4
Pseudovirus Neutralization Assay in VeroE6-TMPRSS2 Cells
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VeroE6-TMPRSS2 cells84 (link) were cultured in DMEM with 10% FBS (Hyclone), 1% PenStrep, and 8 μg/mL puromycin with 5% CO2 in a 37°C incubator (Caron-VWR). One day prior to infection, 96-well plates were plated with 20,000 cells. The following day, cells were checked to be at 80% confluence. In an empty half-area 96-well plate a 1:3 serial dilution of sera was made in DMEM and then diluted pseudovirus was added to the serial dilution and incubated at room temperature for 30-60 min. After incubation, the sera-virus mixture was added to the cells at 37°C and 2 hours post-infection, 40 μL 20% FBS-2% PenStrep DMEM was added. After 17-20 hours 40 μL/well of One-Glo-EX substrate (Promega) was added to the cells and incubated in the dark for 5-10 min prior to reading on a BioTek plate reader. Measurements were done in at least duplicate. Relative luciferase units were plotted and normalized in Prism (GraphPad). Nonlinear regression of log(inhibitor) versus normalized response was used to determine IC50 values from curve fits.
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