Pseudoviruses were generated as described below36 (link),38 (link)–40 (link). Specifically, the plasmid encoding original or mutant S protein was co-transfected with pLenti-CMV-luciferase and PS-PAX2 plasmids (Addgene) into 293T cells using the PEI transfection method described above. Supernatants containing pseudoviruses were collected at 72 h after transfection and processed for the below pseudovirus neutralization assay. Pseudoviruses were incubated with serial dilutions of mouse sera for 1 h at 37 °C, and the virus-serum mixture was added to 96-well plates pre-seeded with 293T cells expressing hACE2 (hACE2/293T). Fresh medium was added to the cells 24 h later, which were sequentially incubated with cell lysis buffer, and luciferase substrate (Promega) 72 h later. Relative luciferase activity was measured using Cytation 7 Microplate Multi-Mode Reader and Gen5 software (BioTek Instruments). Pseudovirus neutralization was detected, based on which 50% neutralizing antibody titer (NT50) was calculated.
Plenti cmv luciferase
Manufactured by Addgene
The PLenti-CMV-luciferase is a lentiviral vector that expresses the luciferase reporter gene under the control of the constitutive CMV promoter. This vector can be used to generate stable cell lines or to transduce cells for luciferase-based assays.
Automatically generated - may contain errors
2 protocols using plenti cmv luciferase
1
SARS-CoV-2 Pseudovirus Neutralization Assay
2
SARS-CoV-2 Pseudovirus Neutralization Assay
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The mouse serum samples were detected for neutralizing antibody activity against infection of SARS-CoV-2 prototypic strain and mutant pseudoviruses using our established pseudovirus neutralization assay with some modifications.7 (link)
,26 Briefly, a plasmid encoding the S protein of SARS-CoV-2 prototypic strain or each of the variants containing single or multiple amino acid mutations was transfected into 293T cells in the presence of 2 additional plasmids (psPAX2 and pLenti-CMV-luciferase encoding luciferase protein) (Addgene, Watertown, MA). Pseudovirus-containing culture supernatants were collected at 72 hours after transfection, incubated with serially diluted mouse sera for 1 hour at 37°C, and the virus-serum mixture was added into 293T cells expressing SARS-CoV-2 receptor human ACE2 (hACE2/293T). After being cultured at 37°C for 72 hours, the cells were lysed using cell lysis buffer (Promega, Madison, WI), and detected for relative luciferase activity using Infinite 200 PRO Luminometer (Tecan, Morrisville, NC). Neutralizing activity of serum antibodies against SARS-CoV-2 was calculated using the CalcuSyn computer program and expressed as 50% pseudovirus neutralizing antibody titer (NT50).
,26 Briefly, a plasmid encoding the S protein of SARS-CoV-2 prototypic strain or each of the variants containing single or multiple amino acid mutations was transfected into 293T cells in the presence of 2 additional plasmids (psPAX2 and pLenti-CMV-luciferase encoding luciferase protein) (Addgene, Watertown, MA). Pseudovirus-containing culture supernatants were collected at 72 hours after transfection, incubated with serially diluted mouse sera for 1 hour at 37°C, and the virus-serum mixture was added into 293T cells expressing SARS-CoV-2 receptor human ACE2 (hACE2/293T). After being cultured at 37°C for 72 hours, the cells were lysed using cell lysis buffer (Promega, Madison, WI), and detected for relative luciferase activity using Infinite 200 PRO Luminometer (Tecan, Morrisville, NC). Neutralizing activity of serum antibodies against SARS-CoV-2 was calculated using the CalcuSyn computer program and expressed as 50% pseudovirus neutralizing antibody titer (NT50).
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