To measure luciferase activity, cells were lysed in Galacto-Star lysis buffer (Tropix), incubated with ONE-Glo substrate (Promega), and measured on a GloMax 96 microplate luminometer (Promega).
One glo substrate
Manufactured by Promega
ONE-Glo substrate is a luminescent reagent used for the detection and quantitation of ATP in cell-based assays. It provides a rapid and sensitive way to measure ATP levels in various cell types and experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Glomax 96 microplate luminometer, by Promega (2 mentions)
Glomax, by Promega (1 mentions)
Dmem 10 fbs, by Thermo Fisher Scientific (1 mentions)
Black opaque 96 well plate, by Corning (1 mentions)
Prism software, by GraphPad (1 mentions)
Glo lysis buffer, by Promega (1 mentions)
One glo luciferase assay system, by Promega (1 mentions)
Omni bead ruptor 24, by Omni International (1 mentions)
Synergy h1 plate reader, by Agilent Technologies (1 mentions)
6 protocols using one glo substrate
1
Quantifying Luciferase Activity in Cells
2
SARS-CoV-2 Spike Pseudovirus Neutralization
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For the virus neutralization assay, sera were incubated for 30 min at 56°C in order to inactivate complement factors. A propagation-incompetent VSV∗ΔG(FLuc) pseudovirus system bearing the SARS-CoV-2 spike protein in the envelope was incubated with quadruplicates of twofold serial dilutions of immune sera in 96-well plate prior to infections of Vero E6 cells (1x104 cells / well) in DMEM + 10% FBS (Life Technologies). At 18 hours post infection, firefly luciferase (FLuc) reporter activity was determined after addition of 25 μL of firefly luciferase ONE-Glo™ substrate (Promega) using a GloMax® plate reader (Promega) and the reciprocal antibody dilution causing 50% inhibition of the luciferase reporter calculated (PVND50).
3
Cytotoxicity Evaluation of Compounds in A549-hACE2 Cells
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The cytotoxicity of compounds was determined in A549-hACE2 cells in the following manner. A549-hACE2 cells (12,000 cells per well in medium containing 2% FBS) were plated into a black opaque 96-well plate (Corning). The next day, 3-fold serial dilutions of compounds were prepared in DMSO in a manner to normalize the DMSO concentration among all cell culture wells. The compounds were further diluted 500-fold in the culture medium containing 2% FBS. Spent medium from overnight incubation was aspirated, 100 μl of diluted compound solutions were added to each well, and cultures were returned to 37°C with 5% CO2. After 48 h, 100 μl OneGlo substrate (Promega) was added to each well. Luciferase signals were measured using an Envision microplate reader. The relative luciferase signals were calculated by normalizing the luciferase signals of the compound-treated groups to that of the DMSO-treated groups (set as 100%). The relative luciferase signal (y axis) versus the compound concentration (x axis) was plotted in software GraphPad Prism 8 (version 8). The compound concentration for reducing 50% of luciferase signal as a measure of cell viability (CC50) values were calculated using a nonlinear 4-parameter regression model. Three independent experiments were performed with technical duplicates.
4
TZM-bl Virus Infection Assay
5
Luciferase Assay for Inducible Gene Expression
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The luciferase assay was performed using the ONE-Glo luciferase assay system (Promega). Flies were exposed to induction at 29°C for 5 or 15 days. Three adult flies per replicate were transferred into 150 μL Glo lysis buffer (Promega, #E2661) and homogenized in a bead homogenizer (OMNI bead ruptor 24, OMNI International) at 3.25 m/s for 2 min. After centrifugation at 3000 × g for 3 min, 50 μl of the sample was mixed with 50 μl of ONE-Glo substrate (Promega, #E6110). Luciferase luminescence was measured with a plate reader (Synergy H1 Plate Reader, Biotek Instruments Inc.,).
6
Luciferase and β-Galactosidase Assays in mESCs
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To measure luciferase activity, cells were lyzed in Galacto-Star Lysis buffer (Tropix), incubated with ONE-Glo substrate (Promega), and measured on a GloMax 96 Microplate Luminometer (Promega). For measuring ß-galactosidase (encoded by LacZ) activity, cells were fixed for 10 min in 0.8% glutaraldehyde in fixation buffer (50 mL NaPi, 2 mM MgCl2, 1 µM EGTA pH 7.3) and washed for 10 min in X-gal wash buffer (2 mM MgCl2, 0.01% sodium deoxycholate, 0.1% NP40 in 20 mM Tris pH 7.3). Subsequently, the cells were stained in X-gal staining solution (0.5 mg/ml X-gal, 2.5 mM potassium ferrocyanide, and 2.5 mM potassium ferricyanide in LacZ wash buffer) for 1 h at 37 °C, protected from light. For each mESC line, 12 puromycin-resistant colonies were picked, expanded and screened by X-gal staining.
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