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Bright glo luciferase reagent

Manufactured by Promega
Sourced in United States

Bright-Glo luciferase reagent is a luminescent assay solution designed for the detection and quantification of firefly luciferase reporter gene activity. It provides a rapid, sensitive, and quantitative method for measuring gene expression levels in cell-based assays.

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69 protocols using bright glo luciferase reagent

1

Neutralization Titers of Monoclonal Antibodies

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Neutralization titers of monoclonal Abs were determined using a luciferase-based assay in TZM.bl cells as previously described.50 (link), 51 (link) Briefly, mAb samples were tested using a primary concentration of 25 μg/mL with 5-fold serial dilutions against a panel of 10 HIV-1 Env pseudoviruses that were selected for being either 3BNC117 sensitive/PGT121 resistant or 3BNC117 resistant/PGT121 sensitive. This allowed for the measurement of neutralizing activity of single Abs in samples containing a mixture of both 3BNC117 and PGT121. Antibody titrations were incubated with HIV-1 Env pseudoviruses for 1 h at 37°C, and TZM.bl cells were then added in growth media containing DEAE-dextran at a final concentration of 11 μg/mL. Assay plates were incubated for 48 h at 37°C, 5% CO2, and luciferase reporter gene expression was measured using Bright-Glo luciferase reagent (Promega) and a Victor 3 luminometer (Perkin Elmer). Neutralization titers (50% and 80% inhibitory concentrations, IC50 and IC80, respectively) were calculated as the mAb concentration at which relative luciferase units (RLU) were reduced by 50% or 80% compared to RLU in virus control wells after subtraction of background RLU in cell control wells. All assays were performed in a laboratory meeting good current laboratory practice standards.
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2

Serum Stability Assay for Peptides

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To determine serum stability, peptides were incubated with fresh mouse serum at 37 °C. The incubations were initiated with the final peptide concentration of 10 µM. Aliquots of 20 µl mixture were taken at 0 min, 10 min, 30 min, 1 h, 8 h, 24 h and 48 h, and the amount of intact peptides was analyzed by the CRE reporter assay. Briefly, HEK293 cells expressing the GLP-1R were seeded overnight in a white 384-well plate at 5,000 cells per well in 20 µl DMEM medium supplemented with 10% FBS in a humidified 37 °C, 5% CO2 incubator. Then, 5 µl peptide (prepared as 5× solution in OPTI-MEM) were added to each well and incubated at 37 °C. The following day, 10 µl/well Bright-Glo Luciferase reagent (Promega) were added and incubation was continued at room temperature for 10 min before luminescence acquisition using a luminometer (Envision). The measurements were performed in duplicate and the average concentrations were plotted using GraphPad Prism 6 software (GraphPad, San Diego, CA).
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3

Viral Replication Assay in Macrophages

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To assess viral replication, macrophages (2.5 × 105 cells/well in 24-well plates) were incubated with VSV-G-pseudotyped HIV-luciferase reporter virus at a multiplicity of infection (MOI) of 0.1 for 4 h at 37°C. Cells were washed four to five times with PBS to remove unbound virus, and then cultured in growth medium. Following 48 h of culture, cells were treated with TLR ligands or vehicle, as indicated in the text and figure legends. After 18 h, the cells were washed twice with PBS and lysed in PBS-0.02% Triton X-100. Luciferase activity was measured using BrightGlo luciferase reagent (Promega) and an MSII luminometer.
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4

Adcy3 Regulation of Luciferase Activity

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Full-length mouse Adcy3 cDNA was obtained from Origene. A cAMP-responsive element-luciferase reporter gene was obtained from the Montminy lab [31] (link). Transient transfections of human hepatoma HuH7 cells were performed as follows. Exponentially growing cells were seeded onto a 96-well plate. The following day, cells were transiently transfected with a cocktail of the CRE-luciferase reporter and a pcDNA6-2 × FLAG-tagged expression vector (empty vector, wild-type Adcy3, or Adcy3Jll) using Lipofectamine 2000 (Life Technologies). The next day, all wells were switched to 100 µl per well fresh DMEM/10% FBS, plus either DMSO or forskolin (0.2 µM, 1 µM, 5 µM final concentrations). Forskolin was serially diluted in DMSO to 100× final concentration, and the same volume of DMSO or DMSO + forskolin was added to each well. After a five-hour incubation in this media, all wells were harvested and luminescence assessed using BrightGlo luciferase reagent (Promega) and an Acquest luminometer (Molecular Devices).
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5

Luciferase Activity Quantification Protocol

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For analysis of Luciferase activity, infected cells were lysed in 100 μL BrightGlo Luciferase reagent (Promega #E2610) and read on a LUMIstar Omega Luminometer.
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6

Pseudovirion Infection Assay in SupT1 Cells

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Viruses were propagated in 293T cells reverse-transfected with pLAIΔenvLuc2Δvif, A3H plasmid, and L-VSV-G for pseudotyping. Virus-containing supernatants were harvested 48 h after transfection, transferred to a V-bottom plate, and clarified of cells and debris by centrifugation at 1000× g for 3 min at 25 °C. An amount of 10 µL of supernatant was added to flat-bottom 96-well plates containing SupT1 cells (3.75 × 104 and 90 µL/well) pretreated with 20 µg/mL DEAE/Dextran and mixed by repipetting. An amount of 5 µL of supernatant was saved for quantification of reverse transcriptase (RT) activity, as described previously [29 (link)]. Infected SupT1 cells were incubated for 48 h and lysed in 100 µL of Bright-Glo Luciferase Reagent (Promega, E2610, Madison, WI, USA). Infection was assessed by luciferase activity using a LUMIstar Omega microplate luminometer (BMG Labtech, Ortenberg, Germany), and raw luciferase values were normalized to 2000 mU RT activity.
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7

Angiotensin II-Induced NFAT Luciferase Assay

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Chinese Hamster Overy (CHO) cells stably expressing human angiotensin AT1 and containing luciferase under an NFAT‐responsive promoter (NFAT‐hAT1‐CHO) were used. Briefly, cells are incubated with 30 nmol/L angiotensin II for 18 hours, in the presence of test compound. Luciferase activity was quantified using Bright‐Glo™ Luciferase reagent (Promega), measured on the Lumistar Galaxy (BMG Labtech).
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8

Evaluating Antiviral Activity of PE-TS and Luteolin

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RD cells (5 × 103 cells/well) were seeded in 96-well plates, cultured to 90% confluence, treated with 250 μg/mL PE-TS or 8.59 μg/ml luteolin (positive control) in DMEM-2% FBS, and infected with EV71-luciferase (MOI = 0.5). Each concentration was set with three parallel wells and incubated at 37°C with 5% CO2. After infection for 1 h, the inoculum was replaced with the drugs at corresponding concentrations in DMEM-2% FBS. The supernatant was discarded at 16 hpi, and 100 μl of Bright-Glo™ luciferase reagent (Promega, United States) was added to each well. Luciferase activity was determined with the VICTOR X2 Multilabel Plate Reader (PerkinElmer, United States).
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9

Neutralizing Antibody Assay for HIV

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Patient plasma samples were heat-inactivated (56°C for 1 hour) and tested for neutralizing activity using a luciferase-based assay in TZM.bl cells as previously described [35 (link)]. Briefly, three-fold dilutions of plasma samples starting at a 1:20 dilution were performed in duplicate. Env-pseudotyped viruses were added to the plasma dilutions at a pre-determined titre to produce measurable infection and incubated for one hour at 37°C. TZM.bl cells were then added at 1×104/well and plates incubated for 48 hours. Cells were lysed for two minutes with Bright-Glo luciferase reagent (Promega, Madison, WI, USA), and supernatant measured for luciferase activity using a Victor 3 luminometer (Perkin Elmer, Waltham, MA, USA). The 50% inhibitory dose (ID50) was calculated as the plasma dilution that resulted in a 50% reduction in relative luminescence units compared with virus control wells. All plasma samples were assayed against a standard reference panel of 11 clade B Tier 2/3 Env pseudoviruses [36 (link)]. Plasma neutralizing activity against each HIV Env pseudovirus was scored as positive, when ID50 titres were at least three-fold above Murine Leukaemia Virus negative control as previously described [37 ], and is summarized in Supplementary file 2.
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10

Pseudotyped Virus Particle Entry Assay

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The Vesicular Stomatitis Virus (VSV)-based coronavirus spike pseudotyped virus particle entry assays were performed as previously described with minor adjustments (4 (link), 7 (link), 10 (link), 13 (link)). In brief, target cells were seeded in a 96-well plate and washed with phosphate-buffered saline (PBS) once before inoculating with equivalent volumes of pseudotyped virus particle stocks in the presence or absence of trypsin. Inoculated plates were centrifuged as described above. Entry efficiency was quantified 18–20 hours post-transduction by measuring the luciferase activity using Bright-Glo luciferase reagent (Promega), following manufacturer’s instructions. Relative entry was calculated as the fold-entry in relative luciferase unit over the no spike control. All experiments were performed at least three times in triplicate.
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