SARS-CoV-2 spike protein-pseudotyped lentiviral stocks were produced by the cotransfection of 293T cells with pMDL, pLenti.GFP-NLuc, pcCoV2.S-Δ19 (or variants thereof), and pRSV.Rev as previously described (41 (link)). Virus stocks were normalized by real-time PCR reverse transcriptase (RT) activity (51 (link)). Pseudotyped virus infections were done with 1 × 104 cells/well in 96-well tissue culture dishes at a multiplicity of infection (MOI) of 0.2 as previously described (41 (link)). Luciferase activity was measured after 2 days using the Nano-Glo luciferase substrate (Promega), and plates were read in an Envision 2103 microplate luminometer (PerkinElmer). To quantify antibody neutralization, sera were serially diluted 2-fold and incubated for 30 min at room temperature with pseudotyped virus (corresponding to approximately 2.5 × 107 cps luciferase) in a volume of 50 μl. The mixture was added to 1 × 104 ACE2.293T cells (corresponding to an MOI of 0.2) in a volume of 50 μl in a 96-well culture dish. After 2 days, the medium was removed, and the Nano-Glo luciferase substrate (Nanolight) was added to wells. Luminescence was read in an Envision 2103 microplate luminometer (PerkinElmer).
Envision 2103 microplate luminometer
Manufactured by PerkinElmer
Sourced in United States
The Envision 2103 microplate luminometer is a compact and versatile laboratory instrument designed for luminescence-based assays. It provides accurate and sensitive detection of luminescent signals emitted from microplates, enabling researchers to perform a variety of luminescence-based experiments and analyses.
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10 protocols using envision 2103 microplate luminometer
1
SARS-CoV-2 Spike Protein Pseudotyped Virus Neutralization
2
Pseudotyped Virus Neutralization Assay
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Serially diluted recombinant sACE2 protein prepared from transfected CHO cells was incubated with pseudotyped virus for 30 minutes at room temperature and added to 1 X 104 ACE2.293T cells. After 2 days, luciferase activity was measured using Nano-Glo luciferase substrate (Nanolight) in an Envision 2103 microplate luminometer (PerkinElmer).
3
Neutralizing Antibody Titer Quantification
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To measure neutralizing titer, sera or mAbs were serially diluted (2-fold for sera and 5-fold for mAbs) and incubated for 30 min at room temperature with pseudotyped virus. The mixture was added on 1 × 104 of ACE2.293T cells (MOI of 0.2). After 1 day of infection, medium was removed and infectivity was measured with Nano-Glo luciferase substrate (Nanolight). Luminescence was read in an Envision 2103 microplate luminometer (PerkinElmer). D614G virus (original Wuhan virus carrying the D614G spike mutation that was selected early during the pandemic) was used as reference.
4
SARS-CoV-2 Pseudotyped Virus Neutralization Assay
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For the serum neutralizing assay, 50 μL of serially diluted sera (2-fold diluted in DMEM containing 10% FBS) were incubated with 50 μL of pseudotyped lentiviruses (MOI = 0.2). For the monoclonal antibody neutralizing assay, 50 μL of serially diluted monoclonal antibodies (5-fold diluted in DMEM containing 10% FBS) and incubated with 50 μL of pseudotyped lentiviruses (MOI = 0.2). After 30 min, the mixture (100 μL) was added to ACE2.293T target cells (1 × 104 cells) in a 96 well culture dish. After 2 days, supernatant was removed and luciferase activity was measured with Nano Glo substrate (Promega) and luminescence was read in an Envision 2103 microplate luminometer (PerkinElmer) [12 (link),24 (link)].
5
SARS-CoV-2 Variant Pseudotyped Lentivirus Production
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SARS-CoV-2 variant S protein pseudotyped lentiviral stocks were generated by cotransfection of 293T cells (4 × 106) with pMDL, pLenti.GFP-NLuc, pcCoV2.S.Δ19, and pRSV.Rev (4:3:4:1 mass ratio) by calcium phosphate coprecipitation as previously described [58 (link)]. VSV-G pseudotyped lentivirus was generated substituting the S protein vector for pcVSV-G. Two days posttransfection, supernatant was harvested and passed through a 0.45-μm filter and ultracentrifuged over a 20% sucrose cushion at 30,000 rpm for 90 minutes. The virus pellet was resuspended to 1/10 the initial volume in DMEM with 10% FBS, and virus titers were normalized by real-time PCR reverse transcriptase activity. Pseudotyped virus infectivity assay was done with HI and LI monolayers at an MOI of 0.2. After 72 hours of infection, luciferase activity was measured by Nano-Glo luciferase substrate (Promega, Madison, WI, USA) with an Envision 2103 microplate luminometer (PerkinElmer).
6
Pseudotyped Virus Neutralization Assay
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ACE.293T or ACE2.A549 cells were plated in a 96 well tissue culture dish at 1 X 104 cells/well. Cells were infected with pseudotyped virus normalized for RT activity at MOI=0.2. After 2 days, the culture medium was removed, Nano-Glo luciferase substrate (Promega) was added and plate was read in an Envision 2103 microplate luminometer (PerkinElmer). To determine neutralizing antibody titers, sera or monoclonal antibodies were serially diluted 2-fold and then incubated with pseudotyped virus (approximately 2.5 X 107 cps) for 30 minutes at room temperature and then added to target cells. After 2 days, medium was removed, same amount of fresh medium and Nano-Glo luciferase substrate (Promega) was added and transferred to a microtiter plate which was read in an Envision 2103 microplate luminometer (PerkinElmer).
7
Spike-ACE2 Interaction Assay
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HEK293T cells were transfected with variants spike expression vectors by lipofectamine 2000 (Invitrogen). 2.5 μg purified sACE2-Nuc protein prepared from transfected CHO cells was incubated with variant spikes-expressing cells for 30 minutes. Free sACE2-Nuc protein was washed away with PBS twice and luciferase substrates were added to the cells. Luciferase activity was measured using Nano-Glo luciferase substrate (Nanolight) in an Envision 2103 microplate luminometer (PerkinElmer).
8
SARS-CoV-2 Neutralization Assay
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ACE-MAP, ACE2, and ACEBINDER were tested against SARS-CoV-2 using the previously established pseudotyped lentiviral neutralization assay [62] which is employed with ACE 0.293 T cells. Cells were plated in a 96 well tissue culture dish a 1 × 104 cells/well. ACE-MAP and ACEBINDER was serially diluted and incubated with pseudotyped virus for 30 min before being added to the target cells. Cells were infected with pseudotyped SARS-Cov-2 and normalized for activity at MOI= 0.2. The next day, culture medium was removed and Nano-Glo luciferase substrate (Promega) was added before reading on an Envision 2103 microplate luminometer (PerkinElmer). Luciferase activity was normalized to the untreated cells and IC50 values were be calculated by fitting a sigmoidal nonlinear regression fit using inhibitor concentration vs. normalized response – Variable slope equation against concentration of protein using GraphPad Prism.
9
Protocol for Assaying SARS-CoV-2 Spike Protein Neutralization
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Spike protein-pseudotyped lentiviruses were generated as previously described.35 (link) Briefly, virus stocks were generated by cotransfection of 293T cells with pMDL, pLenti.GFP.nLuc, pcCoV2.S-Δ19 and pRSV.Rev using the calcium phosphate method. After 2 days, the culture supernatant was harvested and the virus was concentrated by ultracentrifugation at 4°C for 1 hour at 30,000 X g and normalized for reverse transcriptase (RT) activity. To determine the neutralizing titer of the decoy proteins, serially diluted decoys were incubated with pseudotyped virus (MOI = 0.2) for 30 minutes at room temperature and then added to ACE2.293T or ACE2.TMPRSS2.Vero E6 cells. At 2-dpi, luminescence was measured in an Envision 2103 microplate luminometer (PerkinElmer). All samples were assayed in duplicate and IC50s were calculate by Prism 8 software.
10
Spike-ACE2 Interaction Quantification
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HEK293T cells were transfected with variants spike expression vectors by lipofectamine 2000 (Invitrogen). 2.5 μg purified sACE2-Nuc protein prepared from transfected CHO cells was incubated with variant spikes-expressing cells for 30 minutes. Free sACE2-Nuc protein was washed away with PBS twice and luciferase substrates were added to the cells. Luciferase activity was measured using Nano-Glo luciferase substrate (Nanolight) in an Envision 2103 microplate luminometer (PerkinElmer).
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