Preparations of LV-Luc Wuhan, Alpha, Beta, Gamma and Delta pseudotypes (LV-Luc/Spike-C3, LV-Luc/Spike-UKC3, LV-Luc/Spike-SAC3, LV-Luc/SpikeBRC3 and LV-Luc/SpikeINd19) were titered in VERO E6 cells seeded in a 96-well plate (View plate, PerkinElmer) at a density of 20000 cells/well. After 48 h, luciferase expression was determined by the britelite plus Reporter Gene Assay System (PerkinElmer) and measured with a Varioskan luminometer (Thermo Fisher). Dilutions providing 150000-200000 relative luminescence units (RLU) were used in the neutralization assay. Briefly, mAb serial 2-fold dilutions starting from 20 µg/ml were incubated in duplicate with the Wuhan, Alpha, Beta, Gamma and Delta LV-Luc pseudoviruses for 30 min at 37°C in 96-deep well plates (Resnova, Genzano di Roma, Italy), and thereafter added to VERO E6 cells seeded in a 96-well View plate at a density of 20000 cells/well. Virus-only and cell-only controls were included. After 48 h, luciferase expression was determined by the britelite plus Reporter Gene Assay System (PerkinElmer). RLU data points were converted to a percentage neutralization value, calculated relative to virus-only controls. Results are expressed as inhibitory concentration (IC) 50 corresponding to the mAb concentration giving 50% inhibition of infection (neutralization) compared to the virus control wells (16 (link)).
Britelite plus reporter gene assay system
Manufactured by PerkinElmer
Sourced in United States, Italy
The Britelite Plus Reporter Gene Assay System is a luminescent reporter gene assay used to measure gene expression levels. It provides a sensitive and quantitative method for analyzing the activity of reporter genes such as firefly luciferase and Renilla luciferase.
Automatically generated - may contain errors
Lab products found in correlation
G δg vsv, by Kerafast (3 mentions)
Ensight luminometer, by PerkinElmer (2 mentions)
Viafect transfection reagent, by Promega (2 mentions)
Hygromycin b, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Victor x2 multilabel plate reader, by PerkinElmer (2 mentions)
Lipo3000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Ensight device, by PerkinElmer (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ensight, by PerkinElmer (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Renilla luciferase assay system, by Promega (1 mentions)
Ace2 chos, by Creative Biolabs (1 mentions)
Ace2 chos, by PerkinElmer (1 mentions)
293t cell, by American Type Culture Collection (1 mentions)
Viewplate, by PerkinElmer (1 mentions)
Varioskan luminometer, by Thermo Fisher Scientific (1 mentions)
40 protocols using britelite plus reporter gene assay system
1
Pseudotyped Lentivirus Neutralization Assay
2
SARS-CoV-2 Pseudovirus Neutralization Assay
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Sera were inactivated for 30 min at 56 °C, then 4–fold serially diluted from a starting dilution fold of 1:20 with cell culture medium. 100 μL of diluted sera were incubated with 50 μL of luciferase–expressing SARS-CoV-2 pseudoviruses (1.3 × 104 CCID50/mL) for one hour at 37 °C, and then the mixtures were distributed to the plates (150 μL/well). Vero cells (2 × 104 in 100 μL) were then added and incubated at 37 °C for another 20–28 h. The cells were lysed, and the luciferase activity was measured by the Britelite Plus Reporter Gene Assay System (PerkinElmer, Waltham, MA, USA). The relative light unit (RLU) values were read using a chemiluminescence detector (EnSight, PerkinElmer, Waltham, MA, USA). The IC50 neutralizing antibody titer was calculated by the Reed–Muench method. Each serum sample was assayed at six dilutions in duplicates. Ancestral SARS-CoV-2 (WT), Delta, and Beta pseudoviruses were purchased from Beijing YunLing Biotechnology Co., Ltd., Beijing, China. Ancestral SARS-CoV-2 (WT), Delta, and Omicron subvariants (BA.1, BA.2, and BA.4/BA.5) pseudoviruses used in the Omicron vector immunogenicity study were donated by Wuhan University, Wuhan, China. The first WHO International Standard for anti–SARS-CoV-2 immunoglobulin (NIBSC, 20/268, high 20/150) was used to determine neutralizing antibody titers.
3
Inhibition of HIV-1 Infection in TZM-bl Cells
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TZM-bl cells (1
× 104 cells/well) were seeded in 96-well plates and
grown overnight to
permit adherence prior to treatment and viral infection. Cells were
next infected with HIV-1 NL4-3 (MOI 0.5) and incubated at 37 °C.
Compound2 (50–100 nM) and the reference compounds
DS (100 μg/mL, Sigma-Aldrich) and AZT (0.5 μg/mL, Sigma-Aldrich)
were added at different hours (0, 1, 2 h) pi. After 31 h, cells were
washed with PBS 1× and HIV-1 production was assessed following
the LTR-luciferase signal using the britelite plus Reporter Gene Assay
System (PerkinElmer, Waltman, MA, USA) according to the manufacturer’s
protocol.
× 104 cells/well) were seeded in 96-well plates and
grown overnight to
permit adherence prior to treatment and viral infection. Cells were
next infected with HIV-1 NL4-3 (MOI 0.5) and incubated at 37 °C.
Compound
DS (100 μg/mL, Sigma-Aldrich) and AZT (0.5 μg/mL, Sigma-Aldrich)
were added at different hours (0, 1, 2 h) pi. After 31 h, cells were
washed with PBS 1× and HIV-1 production was assessed following
the LTR-luciferase signal using the britelite plus Reporter Gene Assay
System (PerkinElmer, Waltman, MA, USA) according to the manufacturer’s
protocol.
4
Luciferase Assay in Transfected 293T Cells
5
Neutralizing Antibody Assay for SARS-CoV-2 Variants
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SARS-CoV-2 pseudovirus stocks encoding for the wildtype, Alpha, Gamma, Beta, Delta, or Omicron Spike proteins were produced as previously described [10] (link), [36] . To assess the extent of which neutralizing antibodies are present in the sera, CHO cells stably expressing ACE2 (ACE2-CHOs – Creative Biolabs) were used as target cells at 7,000 cells/well. Sera was heat inactivated and serially diluted prior to incubation with the different SARS-CoV-2 variant pseudoviruses. After a 90-minute incubation, sera-pseudovirus mixture was added to ACE2-CHOs, then 72 h later, cells were lysed using Britelite plus Reporter Gene Assay System (PerkinElmer) and RLU was measured using an automated luminometer. Neutralization titers (ID50) were calculated using GraphPad Prism 9 and defined as the reciprocal serum dilution that is reduced by 50% compared to the signal in the infected control wells.
6
Neutralization Activity of Broadly-Neutralizing mAbs
7
Production and Titration of Pseudotyped SARS-CoV-2 Variants
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The Omicron variant gene of SARS-CoV-2 spike protein (GISAID: EPI_ISL_6590782.2) was optimized using mammalian codon and synthesized, then cloned into pcDNA3.1 vector as described previously [11 (link)]. The plasmid expressing the S protein of 614G, Alpha, Beta, Gamma, Delta, Lambda, and Mu SARS-CoV-2 variants were previously constructed (Figures S1). The VSV-based pseudotyped SARS-CoV-2 variants was produced by transfecting 293T cells (American Type Culture Collection, CRL-3216) with S protein expression plasmids and simultaneously infected with G*ΔG-VSV (Kerafast, Boston, MA). The titres of pseudotyped viruses were evaluated using Huh 7 (Japanese Collection of Research Bioresources, 0403) cells by 3-fold serial dilutions. Chemiluminescence signals were detected 24 h after the incubation of cells and virus at 37°C with 5% CO2. The Britelite plus reporter gene assay system (PerkinElmer, Waltham, MA) and PerkinElmer Ensight luminometer were used for signal collection. The detailed procedure was described in our previous paper [12 (link)].
8
Antiviral Susceptibility Assay for HIV-1 Mutants
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Susceptibility assays for WT and mutant HIV-1 (NLdE-luc) with NNRTIs DAP, DOR, EFV, ETR, and RPV as well as the NRTI FTC were performed in TZM-bl cells as previously described (49 (link)). Briefly, TZM-bl cells (2 × 104 cells/well of a 96-well plate) were incubated with or without dilutions of drugs and infected with each virus at a multiplicity of infection (MOI) of0.02 in phenol red-free medium. After 48h, luciferase was measured using the Britelite Plus Reporter Gene Assay System (PerkinElmer). Infectivity was measured by luciferase expression in the cells, with uninfected cells set as background and infected cells in the absence of inhibitor set as 100%. Experiments were performed in triplicate in 3 or 4 independent experiments. The 50% effective concentration (EC50) was calculated in Prism (GraphPad) using nonlinear regression for each experiment.
9
HEK293 Cell Line-based Receptor Assay
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The human embryonic kidney 293 (HEK293) cell line (CRL-1573™) was obtained from the American Type Culture Collection (Manassas, VA, USA). RPMI1640, fetal bovine serum (FBS), Geneticin (G418), and hygromycin B were purchased from Gibco (Grand Island, NY, USA). Human growth hormone receptor (hGHR) plasmid was obtained from OriGene Technologies (Beijing, China). ViaFect™ transfection reagent was purchased from Promega (Madison, WI, USA). The Britelite Plus Reporter Gene Assay System was obtained from PerkinElmer (Waltham, MA, USA). The SG-luciferase reporter plasmid (including SIE and GAS response elements) [19 (link)], an in-house reference of rhGH-Fc, rhGH-Fc, rhGH, Fc-fusion recombinant human erythropoietin (rhEPO-Fc), Fc-fusion vascular endothelial growth factor receptor (VEGFR-Fc), Fc-fusion recombinant human interleukin 15 (rhIL15-Fc), and Fc fusion recombinant human GLP1 (rhGLP-Fc) were stored at 4 °C or –80 °C in our laboratory. The 96-well flat clear bottom white polystyrene TC-treated microplates were purchased from Corning (New York, NY, USA).
10
Luciferase Reporter Assay for ICP0 and ICP27
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Vectors pGL4.10-ICP0 and pGL4.10-ICP27 (150 ng each) were transfected in 1.2 × 105 U2-OS cells per well onto 12-well plates, using Lipo3000 transfection reagent (Invitrogen, Life Technologies Italia, Monza, Italy). B19 was added to the cell medium 1 h after transfection at increasing concentrations (5–20 µM), to avoid interference, if any, with transfection. Expression of firefly luciferase was determined 24 h after transfection using the Britelite plus Reporter Gene Assay System (PerkinElmer Inc., Milan, Italy) at a Victor X2 multilabel plate reader (PerkinElmer Inc., Milan, Italy), according to the manufacturer’s instructions. Cells were lysed in 0.1% Triton-X100-PBS and protein concentration was determined by BCA assay (Thermo Scientific Pierce, Monza, Italy). Luciferase signals were subsequently normalized to total protein content, according to the manufacturer’s protocol (http://ita.promega.com/~/pdf/resources/pubhub/cellnotes/normalizing-genetic-reporter-assays/ ). Each assay was performed in duplicate and each set of experiments was repeated at least three times.
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