The pGL3-Basic plasmid with the endogenous promoter was co-transfected with the pRL-TK plasmid expressing hRluc (Renilla reniformis) luciferase into the P. xylostella cells. On the third day after transfection, we added 100 μL of lysate to each well, lysed for 20 minutes, then collected the lysate, and added 10 μL of lysate per well to a 96-well plate. Luciferase Assay Reagent II from Promega ONE-GloTM Luciferase Assay System (Promega, Madison, WI, USA) was mixed in the 96-well plate in a way of testing 8 wells at a time, and detected the fluorescence intensity with GloMax 96 (Promega, Madison, WI, USA). After all samples were added with Luciferase Assay Reagent II and tested, the Stop & Glo® Reagent was added in the same way and detected fluorescence intensity. We normalized the expression data by dividing the fluorescence intensity value of the first measurement by the magnitude of the second, and performed one-way ANOVA with GraphPad Prism 6.01 to analyze the differences between groups (Duncan, p < 0.05).
One glotm luciferase assay system
Manufactured by Promega
Sourced in United States
The ONE-Glo™ Luciferase Assay System is a luminescent reporter assay designed to quantify firefly luciferase activity in cell-based experiments. The assay reagent provides a fast, sensitive, and stable luminescent signal that can be measured using a luminometer.
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Lab products found in correlation
Luciferase assay reagent 2, by Promega (1 mentions)
Glomax 96, by Promega (1 mentions)
Prism 6, by GraphPad (1 mentions)
Mini maxtm 300 imaging cytometer, by Molecular Devices (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Clariostar plus microplate reader, by BMG Labtech (1 mentions)
Synergy 2, by Agilent Technologies (1 mentions)
Cignal lenti vdre reporter luc kit, by Qiagen (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Accuri c6, by BD (1 mentions)
Hek293 cells, by Keygen Biotech (1 mentions)
Viafect transfection reagent, by Promega (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Opti mem culture medium, by Thermo Fisher Scientific (1 mentions)
Turbofect transfection reagent, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Penicillin streptomycin amphotericin antibiotic solution ab, by Merck Group (1 mentions)
Recombinant human wnt3a, by R&D Systems (1 mentions)
20 protocols using one glotm luciferase assay system
1
Dual-Luciferase Reporter Assay in Plutella xylostella
2
Quantification of Firefly Luciferase Activity
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The firefly luciferase reporter was detected by applying the ONE-GLOTM Luciferase Assay System (E6120, Promega, Madison, WI, USA). The Relative Luminescence Units (RLU) were measured using the SpectraMax i3x Luminescence Glow cartridge (Lum 384) (0200-7015POS, Molecular Devices) and normalized to the cell count or area, which were determined using the Mini MaxTM 300 Imaging Cytometer (5024062, Molecular Devices). The treatments were performed in sextuplicate, and the fold changes of the different treatments, relative to the untreated control sample, were calculated.
3
SARS-CoV-2 Pseudotype Entry Inhibition
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A luciferase reporter assay was used to assess if C1q, ghA, ghB, ghC, or C4BP treatment could affect SARS-CoV-2 pseudotype particle cell entry. Briefly, A549-hACE2 + TMPRSS2 cells (20,000 cells/well) were seeded in a 96-well plate and incubated in complete growth media overnight at 37 °C. The cells were then challenged with C1q, ghA, ghB, ghC, MBP or C4BP treated SARS-CoV-2 lentiviral pseudoparticles in incomplete growth medium–DMEM with Glutamax (Gibco) supplemented with 100 U/mL penicillin (Gibco) and 100 μg/mL streptomycin (Gibco), and incubated at 37 °C for 24 h. Next, the cells were washed twice in PBS, and a complete fresh medium was added and incubated for another 48 h at 37 °C. Subsequently, luciferase activity (RLU) was measured using the ONE-GloTM Luciferase Assay System (Promega) and read on the Clariostar Plus Microplate Reader (BMG Labtech, Cary, NC, USA).
4
In Vitro Luciferase Production Assay
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When producing luciferase as a reporter protein from the plasmid pK7Luc, 15 μl iSAT reactions were performed in 1.5 ml microtubes and incubated in heat blocks within an incubator for 4 h. Microtubes were then placed on ice to stop the reactions. Luciferase concentration in each reaction was determined by mixing 1–10 μl of sample with 30 μl ONE-GloTM Luciferase Assay System (Promega) in a white half-area 96-well plate. Resulting luminescence was read at 26°C in a BioTek Synergy2 plate reader over 20 min. The maximum relative luminescence units were converted to molar concentrations using a standard curve generated from a dilution series of QuantiLum® recombinant luciferase (Promega). To control for background expression, reactions were performed without additional rRNA or rRNA genes and assessed for active luciferase production, and this value (∼1 nmol/l at 4 h) was subtracted from experimental expression values.
5
Secosteroid Transcriptional Activity Assay
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Caco-2, HaCaT and Jurkat cells were cultured as described previously1 (link),4 (link),23 (link),32 (link), and were transduced with lentiviral VDRE luciferase using a Cignal Lenti VDRE Reporter (luc) Kit according to the manufacturer’s protocol (QIAGEN, Valencia, CA, USA). After one week selection by puromycin (1 µg/mL), cells were seeded in a 96-well plate (10,000 cells/well with a volume of 100 µL/well) using FBS-free medium and synchronized for 24 h. DMSO solutions (1 µL) of secosteroids to be tested were added to cells, which were then incubated for another 24 h. The luciferase signal was then measured according to the manufacturer’s procedure for the ONE-GloTM Luciferase Assay System (Promega, Madison, WI, USA). The final concentration of DMSO was 0.1% and 0.1% DMSO was used as the vehicle control. All concentrations were tested in triplicate.
6
SARS-CoV-2 Pseudovirus Neutralization Assay
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Pseudovirus expressing the SARS-CoV-2 spike protein was obtained as a general gift from the Institute of Biological Product Control from National Institute for Food and Drug Control, China. SARS-CoV-2 pseudovirus was prepared by using VSV G pseudotyped virus (G*ΔG-VSV) that packages the expression cassette for firefly luciferase instead of VSV-G in the VSV genome, and the serum neutralization capability was determined as described recently.9 (link) Briefly, the SARS-CoV-2 pseudovirus was preincubated with serum samples at 1:20 dilution at 37°C for one hour, together with the pseudovirus control and cell control wells. Serum samples from healthy controls were served as negative control in hexaplicate. Then, the 96-well plates were seeded with 100 μg of freshly trypsinized Huh7 cells (2 × 104 cells/well). After 24 hours of incubation in a 5% CO2 environment under 37°C,the luminescence was measured using luciferase substrate (One-GloTM Luciferase assay system, Promega, E6120) and the percentage of neutralization was calculated with the following formula as: [(relative light units (RLUs) of virus control wells – RLUs in cell control wells)- (RLUs of serum incubated with virus wells- RLUs of the cell control wells)]/ (RLUs of virus control wells – RLUs of cell control wells) x100%. The percentage of neutralization over 50% was considered to have neutralization activity.
7
Huh7 GNMT Promoter-Luciferase Assay
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Huh7 GNMT promoter-luciferase (H7GPL) cells75 (link) were seeded at 96-well plates, 5 × 103 cells in each well. H7GPL cells were treated with 2.5, 5, 25, 50, 100, 250, 500 μM AAI or 0.1% DMSO (vehicle negative control) in duplicate for 16 hrs. Treated H7GPL cells were lysed for luciferase activity measurement by using the One-GloTM Luciferase Assay System (Promega, E6120) and detected in a luminometer (BioTek Ins., Synergy HT). The reporter luciferase activity were normalized by cell density measured by adding alamarBlue® reagent (Thermo Scientific/Pierce Biotechnology., Rockford, lL, USA). Values were presented as the relative luciferase activity, fold increase over vehicle control.
8
Tomato Transcription Factor Interaction Assay
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The SlMYB-ATV and Slmyb-atv entry clones were individually recombined with the GatewayTM compatible bait vector pDuEx-Ac6 (Fujikawa and Kato, 2007 (link)) containing the N-terminal half of the Renilla luciferase gene. The SlAN1 and SlJAF13 entry clones were individually recombined with the GatewayTM compatible prey vector pDuEx-Dn6 (Fujikawa and Kato, 2007 (link)) containing the C-terminal half of the Renilla luciferase gene. Tomato leaf protoplasts were transfected with mixtures of two different recombined bait and prey vectors. As a control, the vector pDuEx-Ac6 in combination with each of the two recombined SlAN1- or SlJAF13-pDuEx-Dn6 vectors was used. Luciferase activity was analyzed using the ONE-GloTM Luciferase Assay System (Promega), following the manufacturer’s instructions. Luminescence was measured with a Lumat LB 9507 Tube Luminometer (Berthold Technologies GmbH & Co., KG, Bad Wildbad, Germany). Protein content of transfected protoplasts, measured by Bradford Protein Assay (Bio-Rad Laboratories, Hercules, CA, United States), was used for normalization of transfection efficiency. In each experiment four biological replicates (independent protoplasts transformations) were used for each combination of bait and prey vectors and each split-luciferase assay was repeated twice.
9
Optimizing Transfection Efficiency in Cell Lines
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Growth medium at volume of 965 μl was added to 35 µl of cell suspension after incubation for 10 min post electroporation. Afterwards 900 μl of diluted cell suspension was transferred into a well of 24 well plate (TPP) and incubated for 24 hours. Then the cells where trypsinized, centrifuged at 200 × g and resuspended in 100 µl of PBS. Then the transfection (GFP positive cells) was evaluated using flow cytometer (BD Accuri C6, BD Biosciences, USA).
For the experiments with luciferase coding plasmid, 950 µl of growth medium was added 10 min post electroporation. Then 139 µl of treated cell suspension (~1 × 104 cells) were plated in the wells of microplate (Plastibrand, Wertheim, Germany) and allowed to grow in cell culture medium for 24 h. Luciferase protein activity was measured with the ONE-GloTM Luciferase Assay System (Promega, Madison, USA) using a luminometer (Tecan GENios Pro; MTX Lab Systems, Vienna, VA).
For the experiments with luciferase coding plasmid, 950 µl of growth medium was added 10 min post electroporation. Then 139 µl of treated cell suspension (~1 × 104 cells) were plated in the wells of microplate (Plastibrand, Wertheim, Germany) and allowed to grow in cell culture medium for 24 h. Luciferase protein activity was measured with the ONE-GloTM Luciferase Assay System (Promega, Madison, USA) using a luminometer (Tecan GENios Pro; MTX Lab Systems, Vienna, VA).
10
Dual Luciferase Assay for NF-kB Activity
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Human embryonal kidney (HEK)-293 cells were obtained from KeyGen Biotech (Nanjing, China) and grown in DMEM with 10% fetal bovine serum at 37°C in 5% CO2. Cells were plated in 96-well plates at a concentration of 2×104 cells/well, and Lipofectamine 2000 transfection reagent was used for transfection. Recombinant plasmids NF-kB and TK were cotransfected into HEK-293 cells for 6 h. Cells were cultured with various concentrations of DHA and/or 30 nM PMA for 24 h, and relative luciferase activity in cell extract was determined. Firefly luciferase activity was measured using ONE-GloTM Luciferase Assay System (Promega Corp. Madison, WI) according to manufacturer’s protocol [26 (link)].
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