Neutralization was measured by using single-round replication-defective HIV Env-pseudoviruses and TZM-bl target cells (Montefiori, 2005 , Seaman et al., 2010 (link)). 25ul of 3-fold serially diluted mAbs or serum samples were pre-incubated at 37°C for 1h with 25ul of tissue culture infective dose-50 (TCID50) Env-pseudotyped virus in a half-area 96-well tissue culture plate. TZM-bl target cells (5,000 cells/well) in 50μl of DMEM were added and the plates were allowed to grow in humidified incubator at 37°C and 5% Co2. The luciferase activity of the lysed cells was read on instrument (Biotek) after 2-3 days, by adding lysis buffer followed by Brightglow (Promega). The 50% inhibitory concentration (IC50) or 50% inhibitory doses (ID50) was reported as the antibody concentration or serum dilution required to reduce infection by half.
Bright glow
Manufactured by Promega
Sourced in United States
Bright Glow is a luminescent product designed for use in various laboratory applications. It emits a bright glow that can be detected and measured. The core function of Bright Glow is to provide a luminescent signal for various assays and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Merck Group (2 mentions)
Fetal calf serum (fcs), by Merck Group (2 mentions)
Sodium pyruvate, by Merck Group (2 mentions)
Minimum essential medium (mem), by Merck Group (2 mentions)
Glutamine, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Pnifty, by InvivoGen (1 mentions)
Nf κb luciferase reporter construct, by InvivoGen (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Lipopolysaccharides (lps), by InvivoGen (1 mentions)
Superfect, by Qiagen (1 mentions)
Bright glotm luciferase assay kit, by Promega (1 mentions)
Glomax 20 20 luminometer, by Promega (1 mentions)
Prism 8, by GraphPad (1 mentions)
7 protocols using bright glow
1
HIV Env-Pseudovirus Neutralization Assay
2
Evaluating TLR-mediated NF-κB activation
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Human embryonic kidney cells [56 ] (Invivogen) were transformed with different human Toll like receptors (TLR1/2 [293-mtlr1/2], TLR2 [293-mtlr2] and TLR4 [293-mtlr4]) and a NF-κB luciferase reporter construct (Invivogen). Approximately 6 × 104 cells of each reporter line in DMEM medium supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma Aldrich) and 10% fetal bovine serum (Invitrogen) were seeded into wells of a black, clear bottom 96-well plate (Invitrogen) and incubated overnight at 37 °C in a humidified atmosphere containing 5% CO2. The cells were subsequently stimulated with different concentrations of EPS isolated from Lb. mucosae DPC 6426, PCSK (5 μg/ml, Invitrogen), or different concentrations of LPS (Invivogen) as positive controls or with medium alone (negative control) and incubated for 4 h at 37 °C in a CO2 incubator. After this incubation period, half of the medium was replaced with ‘Bright Glow’ (Promega), the plate was then vortexed for 5 min and the luminescence measured using a Spectramax M5 (Molecular Devices). HEK293 cells not expressing any of the TLR receptors but harbouring pNIFTY, a luciferase reporter construct for NF-κB activation (Invivogen, Toulouse, France) were used to control for TLR-independent activation of NF-κB.
3
PPARγ Activity Assessment via Luciferase Assay
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Luciferase assay for PPARγ activity was conducted with PPARγ luciferase construct (a kind gift of Dr. B. Spiegelman, Addgene #1015) using BrightGlow (Promega, WI, USA) and measured with a GLOMAX 20/20 Luminometer. Transfections were carried out on MC3T3 E1 cells (control) or cells with stable Rb1 knockdown using Superfect (Qiagen) according to manufacturer recommendations. Forty-eight hours after transfection, cells were trypsinized, pelleted, and then resuspended in serum free media. An aliquot was used to count cells for normalization, the rest was assayed using Promega’s Bright-GloTM Luciferase Assay Kit (Promega, cat. no. E2610). Equal volumes of the resuspended cells and Bright-GloTM were mixed and incubated for 2 min. Samples were read for 10 s, each after a 2 s delay. Luciferase readings for each of the samples were then normalized according to the number of cells per milliliters. All measurements were carried out on triplicate samples, and experiments were repeated at least thrice.
4
SARS-CoV-2 Spike Pseudotyped Lentivirus Assay
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The pseudotyped virus was used according to the manufacturer´s instructions: Approximately 7500 ACE2-HEK293 cells/well were infected with 750 infectious particles (MOI = 0.1) of SARS-CoV-2 Spike pseudotyped lentivirus (Luc reporter). Virus was incubated with buffer (controls) or the tests substances for 30 minutes before infection (designated as concentration “virus contact”). For infection, 5 volumes of cell culture medium (MEM (Merck KGA, Germany) containing 10% FCS (Merck KGA, Germany), 4 mM glutamine (Merck KGA, Germany), 1 mM sodium pyruvate (Merck KGA, Germany), and 1% penicillin/streptomycin (Merck KGA, Germany) were added, resulting in 1:6 dilution of the initial concentration of the test substance. (24 hours after infection, the medium was changed to fresh cell culture medium. 48 hours after infection plates were lysed by freeze/thaw before luciferase reagent (Bright Glow, Promega, Madison, WI) was added to cells to measure the luciferase activity using a BMG Fluostar Microplate reader. Mock-infected cells and infected, mock-treated (0.5% NaCl) cells served as positive and negative control. Luciferase data were routinely corrected with metabolic data (Alamar blue) derived from a parallel plate with identical set-up.
5
SARS-CoV-2 Spike Pseudotyped Virus Neutralization
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The pseudotyped virus was used according to the manufacturer’s instructions: Approximately 7500 ACE2-HEK293 cells/well were infected with 750 infectious particles (MOI = 0.1) of SARS-CoV-2 Spike pseudotyped lentivirus (Luc reporter). The SSPL were incubated with buffer (controls) or the test substances for 30 min before infection (designated as concentration “virus contact”). For infection, five volumes of cell culture medium (MEM (Merck KGA, Germany) containing 10% FCS (Merck KGA, Germany), 4 mM glutamine (Merck KGA, Germany), 1 mM sodium pyruvate (Merck KGA, Germany), and 1% penicillin/streptomycin (Merck KGA, Germany) were added, resulting in 1:6 dilution of the initial concentration of the test substance.
At 24 h after infection, the medium was changed to fresh cell culture medium. At 48 h after infection, plates were lysed by freeze/thaw before luciferase reagent (Bright Glow, Promega, Madison, WI, USA) was added to cells to measure the luciferase activity using a BMG Fluostar Microplate reader. Mock-infected cells and infected, mock-treated (0.5% NaCl) cells served as positive and negative control. Luciferase data were routinely corrected with metabolic data (Alamar blue) derived from a parallel plate with an identical setup.
At 24 h after infection, the medium was changed to fresh cell culture medium. At 48 h after infection, plates were lysed by freeze/thaw before luciferase reagent (Bright Glow, Promega, Madison, WI, USA) was added to cells to measure the luciferase activity using a BMG Fluostar Microplate reader. Mock-infected cells and infected, mock-treated (0.5% NaCl) cells served as positive and negative control. Luciferase data were routinely corrected with metabolic data (Alamar blue) derived from a parallel plate with an identical setup.
6
HIV Neutralization Assay Protocol
7
SARS-CoV-2 Spike Protein Neutralization Assay
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Frozen plasma samples were thawed, heat-inactivated at 56 °C for 30 min, and assayed for neutralisation of a lentivirus-based viral particle carrying a luciferase reporter and pseudotyped with full-length SARS-CoV-2 spike (Accession No: YP_009724390.1)l34 (link). Briefly, neutralising antibody titres were determined by incubating serial two-fold plasma dilutions with ~105 RLU pseudotyped virus for 2 h before the addition of 104 HEK293T cells transfected with full-length human ACE2 24 h prior. After 72 h incubation at 37 °C, luciferase expression was quantified using BrightGlow (Promega Corp.), readouts were normalised, and −Log(IC50) determined via non-linear regression using GraphPad Prism 8 (GraphPad Software).
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