Macrophages (RAW264.7 or BMDM) were plated at a density of 1 × 106 cells/well in 6-well plates in 2.5 mL of medium. After 24 hours, media was either replaced with tumor conditioned media (TCM), or was supplemented with IL-4, IL-13, lactic acid, tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), or lipopolysaccharide (LPS). Unless otherwise noted, activation was performed with 25 ng/mL IL-4 or IL-13, 100 ng/mL TNFα or IFNγ, or 200 ng/mL LPS from Escherichia coli serotype O55:B5 (Sigma-Aldrich, St. Louis, MO). “High” and “low” TCM were generated by culturing 2 × 106 or 3 × 106 CT26 cells respectively in 2.5 mL media per well in a 6-well plate for 24 hours. For Gluc activation experiments with BMDMs, “high” and “low” TCM were generated with 2.5 × 105 or 1 × 105 CT26 cells respectively due to BMDM toxicity at the higher cell numbers. Conditioned media was centrifuged for 10 min at 300 × g to eliminate debris prior to use. After 3, 6, 12, or 24 hours, macrophages were either harvested for RNA extraction or 20 μL of culture media was collected to assay for Glue using a BioLux Gaussia Luciferase Assay Kit (New England BioLabs, Ipswich, MA) according to manufacturer’s instructions. Luminescence measurements were performed on a TD 20/20 luminometer (Turner Designs, San Jose, CA) with 10 seconds of integration and luminescence expressed in relative light units (RLU).
Biolux gaussia luciferase assay kit
Manufactured by New England Biolabs
Sourced in United States, Germany, Canada, United Kingdom, Australia
The BioLux Gaussia Luciferase Assay Kit is a tool used to detect and quantify the Gaussia luciferase reporter protein. It provides a sensitive and reliable method for measuring luciferase activity in various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (7 mentions)
Lipofectamine, by Thermo Fisher Scientific (3 mentions)
Luciferase assay system, by Promega (2 mentions)
Seap reporter assay kit, by InvivoGen (2 mentions)
Lumat lb 9507 luminometer, by Berthold Technologies (2 mentions)
Ab23750, by Abcam (2 mentions)
Gaussia luciferase, by GeneCopoeia (2 mentions)
Hpa002926, by Merck Group (2 mentions)
Accel sirnas, by Horizon Discovery (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Veritas microplate luminometer, by Promega (2 mentions)
Spectramax m3 microplate reader, by Molecular Devices (2 mentions)
Fluostar omega microplate reader, by BMG Labtech (2 mentions)
Lumitrac 600 plates, by Greiner (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Glomax discover microplate reader, by Promega (2 mentions)
Steady glo luciferase assay system, by Promega (2 mentions)
Biolux cypridinia, by New England Biolabs (2 mentions)
Escherichia coli serotype o55 b5, by Merck Group (1 mentions)
Td 20 20 luminometer, by Turner Designs (1 mentions)
93 protocols using biolux gaussia luciferase assay kit
1
Macrophage Activation and Secretion Assay
2
Gaussia Luciferase Reporter Assay
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Cells cultured in a 24-well plate were transfected with 100 ng reporter vector in combination with 200 ng shRNA and 100 ng each of the CRX and NRL expression vectors unless specifically stated using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Empty pcDNA3 plasmid was used to adjust the plasmid level so all transfections were done with 600ng total plasmid DNA. Twenty uLs of culture medium containing the secreted Gaussia luciferase was collected 1 and 2 days post-transfection and luciferase activity was assayed using the BioLux Gaussia luciferase assay kit (NEB). The rho-Fluc reporter was assayed in cell lysates collected 2 days post-transfection using the Luciferase Assay System (Promega). Luminescence from both reporters was measured using the FLUOstar OPTIMA (BMG Labtech, Cary, NC) plate reader and reported as relative light units (RLU). The number of cells expressing the rho-mRFP reporter and the signal intensity of the expressed fluorescent protein was measured by flow cytometry using the Accuri C6 flow cytometer (BD Biosciences, San Jose, CA) as described below (see Flow cytometry section).
3
Measuring Tumor Cell-Stromal Cell Interaction
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GLuc-expressing MDA-MB-231 or A431 cells were seeded in 24-well plates (5 × 103 per well) in triplicate with or without the same number of indicated MEFs or CAFs. For the separate culture, MEFs were seeded on transwell inserts with 0.4 μm pore size filters (BD Biosciences). Forty 8 h after seeding, culture media were replaced by fresh media and cells were cultured for 6 h. Luciferase activity in conditioned medium was measured in a GloMax 20/20 luminometer (Promega, Madison, WI, USA) using the BioLux Gaussia Luciferase Assay Kit (New England Biolabs).
4
Bioluminescence Split Complementation Assay for α-Synuclein
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Bioluminescence split complementation assay with the different mutants was performed using α-SYN-hGLuc1 (S1) and α-SYN-hGLuc2 (S2) constructs based on the principle reported in the publication [59 (link), 64 (link)]. Briefly, equal numbers of α-SYN knock out HEK293T cells were seeded in 24-well plate in triplicates. For each well, 500 ng of the individual α-SYN containing split luciferase constructs (S1 + S2) were transfected along with 10 ng of the WT or mutant α-SYN constructs in a 1:100 ratio. At each time point, the media was assayed for luciferase activity using BioLux® Gaussia Luciferase Assay Kit (NEB# E3300S) following the manufacturer’s protocol.
5
Evaluation of GSDMC Promoter Activity
6
Luciferase Activity Quantification
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Gaussia luciferase activity was measured with the BioLux Gaussia luciferase assay kit (New England BioLabs). A 50-μl volume of cleared culture supernatant was added into a 96-well white plate, and 20 μl of GLuc assay solution was prepared and thoroughly mixed with the sample immediately before luminescence detection with a Synergy H1 reader (Biotek). NanoLuc activity was measured with Nano-Glo luciferase assay system (Promega). A 25-μl volume of cleared culture supernatant was added into a 96-well white plate, and 25 μl of NanoLuc assay solution was prepared and thoroughly mixed with the sample before luminescence detection with a Synergy H1 reader.
7
Evaluating GBF1 Mutant Effects on Secreted Luciferase
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HeLa cells were cotransfected in 96-well plates with plasmids encoding Gaussia luciferase (GLuc) and the GBF1 mutants. An empty vector was used as a negative control. The next day, the medium was removed, and the cells were washed to remove secreted luciferase and incubated in 25 μl of fresh medium supplemented with the indicated amount of BFA. After 4 h of incubation, the medium was transferred into another 96-well plate, and the amount of secreted luciferase was measured with a BioLux Gaussia Luciferase Assay Kit (New England BioLabs, Ipswich, MA) according to the manufacturer’s recommendations.
8
Gaussia Luciferase Assay Protocol
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Luciferase assays were performed with a BioLux Gaussia Luciferase Assay Kit (NEB, Ipswich, MA, USA) according to the manufacturer’s instructions. In cell culture, 50 μL of culture medium was removed and assayed with 50 μL of luciferase substrate. For animal samples, 20 μL of lung homogenate (appropriate dilution adopted) was added to 50 μL of luciferase substrate, and the relative lighting unit was detected.
9
Gaussia Luciferase Assay and Cell Characterization
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For the Gaussia luciferase (gLuc) assay, the BioLux Gaussia Luciferase Assay kit (New England BioLabs) was utilized. OVCAR4 and PEO1 cells were grown in a 96-well plate, starting with 2000 cells per well. Media were collected every 24 h and stored in at −20°. For the assay and luminometer readings, the media were thawed and placed in a new 96-well plate. The assay was performed following the manufacturer’s protocol and the relative light units were obtained using luminometry (GloMax) and charted with Prism software. Colony formation assays were performed in parallel using crystal violet staining. Briefly, cells were fixed (10% methanol/10% acetic acid/PBS), stained with crystal violet (0.4%) and washed with de-ionized water. Crystal violet was dissolved and absorbance was measured using a spectrophotometer (SpectraMaxM2e, Molecular Devices, San Jose, CA) at 590 nm and SoftMaxPro software. For the spheroid assay, 4000 cells were plated from a single cell suspension onto growth factor reduced Matrigel (Corning, Corning, NY) and allowed to incubate for 12 days. Microscopic images were obtained and the diameter of each spheroid was measured in ImageJ (NIH). At least 50 spheroids were measured for each cell type and the diameters were averaged and graphed using Prism software.
10
Gaussia Luciferase Assay for IAV Infection
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Cells were aliquoted in 24 well plates for 24 hr prior to IAV infection. Human primary and cell lines were infected with 0.01 MOI PR8-GLuc and MEF were infected with 0.1MOI. After 1 hr the viral inoculum was aspirated, cells were washed twice and incubated at 37°C with 0.2% BSA-DMEM without serum. Cells were lysed 12–16 hr p.i. and the luciferase assay was performed using BioLux Gaussia Luciferase Assay Kit (NEB, Ipswich, MA).
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