RAW264.7 cells were transfected with the iNOS, and NF-kB luciferase reporters using SuperFect® Transfection Reagent (Qiagen, Hilden, Germany). After 24 h of incubation, the cells were incubated in the presence or absence of Z. mays husk extract (ZMHE) induced by LPS for 24 h. The cells were then harvested and lysed, and the supernatants were assayed for their luciferase activity using a Dual Luciferase Assay System (Promega, Madison, WI, USA), and an Infinite® 200 PRO luminometer (Tecan, AG, Männedorf, Switzerland).
Infinite 200 pro luminometer
Manufactured by Tecan
Sourced in United States
The Infinite® 200 PRO luminometer is a high-performance instrument designed for sensitive luminescence detection. It measures light emission from a variety of luminescent samples, such as bioluminescent, chemiluminescent, or flash-type luminescent assays. The Infinite® 200 PRO provides accurate and reliable results for a wide range of applications in life science research and drug discovery.
Automatically generated - may contain errors
Lab products found in correlation
Superfect transfection reagent, by Qiagen (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Dual luciferase assay system, by Promega (1 mentions)
Lactate colorimetric assay kit 2, by Merck Group (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Biolux gaussia luciferase assay kit, by New England Biolabs (1 mentions)
Luciferase assay system e1500, by Promega (1 mentions)
Dual reporter assay kit, by Promega (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Bright glo, by Promega (1 mentions)
Fugene 6, by Promega (1 mentions)
Cell lysis buffer, by Promega (1 mentions)
Plenti cmv luciferase, by Addgene (1 mentions)
Pspax2, by Addgene (1 mentions)
9 protocols using infinite 200 pro luminometer
1
Investigating iNOS and NF-kB activation by Zea mays
2
Quantifying Metabolic Activity in Tr1 Cells
3
Luciferase Assay for HDL Regulation
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Luciferase assays were carried out using cell extracts from transfected Y1-BS1 cells. Groups of cultured dishes with 60% confluent Y1-BS1 cells were transfected with 1 μg pCRE- luc plasmid or pLuc-MCS vector per well in a 24-well plate using Lipofectamine® 2000 as a transfection reagent (Life Technologies, Grand Island, NY, USA). Twenty-four hours after transfection, the cells were re-cultured in F-12K medium supplemented with 10% LPDS overnight followed by treatment with HDL3, 22(R)-diol or pregnenolone for an additional 5 h. Cells were harvested, lysed and cell extracts luciferase assayed with dual-luciferase reporter assay system (Promega) according to the manufacturer’s protocol and light signal (bioluminescence) was quantified using a Tecan Infinite® 200 Pro luminometer. Renilla luciferase was used as a normalization control. The results are expressed as relative luciferase activity (ratio of Firefly/Renilla luciferase activity), and data shown are the mean (±SD) of triplicate values obtained from a representative experiment that was independently repeated for at least three times.
4
HaCaT Cell Transfection and Luciferase Assay
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The HaCaT cells were transfected with the 11β-HSD1 luciferase reporters (GeneCopoeia, Rockville, MD, USA) using SuperFect® Transfection Reagent (Qiagen, Hilden, Germany). After 24 h of incubation, the cells were incubated with PVE induced by cortisone for 24 h. Subsequently, the cells were harvested and lysed, and the supernatants were measured for their luciferase activity using a BioLux® Gaussia Luciferase Assay Kit (New England Biolabs, Ipswich, MA, USA), and an Infinite® 200 PRO luminometer (Tecan, AG, Männedorf, Switzerland).
5
Luciferase Transfection Efficiency Assay
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The cells were seeded in 6-well plates (3×105 cells/well) and cultured overnight. The medium was subsequently replaced with 2 mL fresh serum-free medium and cells were treated with 100 µL nanocomplex of different formulations containing 500 ng pGL3-control pDNA. After a 4 hours transfection, the serum-free medium was replaced with fresh complete medium and the cells were incubated at 37°C for an additional 24 hours. Subsequently, cells were rinsed twice with PBS. The luciferase activity was measured using a Luciferase Assay System (E1500, Promega, WI) and an Infinite 200 Pro luminometer (Tecan, Switzerland).
6
Split-Luciferase and Dual-Luciferase Assays in Tobacco Plants
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Tobacco (Nicotiana benthamiana) plants were grown in the greenhouse with a 16-h-light/8-h-dark cycle, at 22°C. 4- to 5-week-old tobacco were used for experiments. Fully expanded leaves from N. benthamiana were used for infiltration using a needleless syringe.
The split-luciferase assays were conducted as reported (Chen et al., 2008 (link)). The Agrobacterium suspension carrying CLuc and NLuc with corresponding coding regions were co-injected into N. benthamiana leaf epidermal cells. 1.5–2 days later, substrate solution (1 mM Luciferin, 20% Triton X-100) was covered onto the epidermis of leaves, and the images were captured using a Lumazone imaging system equipped with a 2,048B CCD camera (Roper).
The dual-luciferase assays were performed as reported (Hellens et al., 2005 (link)). The reporter and effector constructs were co-expressed in N. benthamiana leaf epidermal cells by Agrobacterium-mediated transformation. 1.5–2 days later, the luciferase activities were measured using Dual-Luciferase Reporter Assay System (Promega) and Tecan Infinite 200 PRO luminometer.
The split-luciferase assays were conducted as reported (Chen et al., 2008 (link)). The Agrobacterium suspension carrying CLuc and NLuc with corresponding coding regions were co-injected into N. benthamiana leaf epidermal cells. 1.5–2 days later, substrate solution (1 mM Luciferin, 20% Triton X-100) was covered onto the epidermis of leaves, and the images were captured using a Lumazone imaging system equipped with a 2,048B CCD camera (Roper).
The dual-luciferase assays were performed as reported (Hellens et al., 2005 (link)). The reporter and effector constructs were co-expressed in N. benthamiana leaf epidermal cells by Agrobacterium-mediated transformation. 1.5–2 days later, the luciferase activities were measured using Dual-Luciferase Reporter Assay System (Promega) and Tecan Infinite 200 PRO luminometer.
7
Quantifying GLI2 transcriptional activity
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For luciferase assays, the dual luciferase reporter assay kit (Promega) was used. HEK293T or pluripotent iPSC cells were seeded at 1.3 x 106 cells in 12-well plates and transiently co-transfected in duplicates with 0.5 μg of wild-type or mutant GLI2 pCS2-MT plasmids together with 0.5 μg of GLI-responsive Firefly luciferase reporter construct (8 × 3’Gli- BSδ51LucII) and 0.05 μg of a constitutive Renilla luciferase reporter (pRL-SV40) construct. Protein lysates were prepared 48 hrs after transfection. Firefly and Renilla Luciferase activities were quantified using the dual reporter assay kit (Promega) according to the manufacturer’s instructions on an Infinite 200 Pro-luminometer (TECAN). Luciferase assay experiments were repeated three times on independent samples. As negative controls, the mutated version of the GLI-luciferase reporter construct (8xm3’Gli-BSδ51LucII) was used, as well as a GFP-expressing construct. Firefly/Renilla activity ratio was then calculated for each sample.
8
SARS-CoV-2 Pseudotype Virus Neutralization Assay
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Twenty-four h prior to running the assay, HEK293T/17 cells were transfected with pCAGGS-ACE2 and pCAGGS-TMPRSS2 at a ratio of 1:3 using Fugene6 (Promega, Madison, WI, USA). In a 96-well plate, sera was initially diluted 1:40, and then, a 2-fold serial dilution was undertaken across the plate. Then, 100 TCID50 of the SARS-CoV-2 pseudotype virus that resulted in an output of 1 × 104 relative light units was added to the test sera and incubated at 37 °C for 1 h before 2 × 104 transfected HEK293T/17 cells were added to the sera/pseudotype virus mix. Following incubation for 48 h, the media was removed and replaced with 50 μL serum free DMEM, 50 μL of Bright Glo (Promega, Madison, WI, USA) reagent was also added at this stage. Luciferase activity was measured 2.5 min later using an Infinite 200 PRO luminometer (TECAN, Männedorf, Switzerland) plate reader. IC50 values were determined as described by Ferrara and Temperton [30 (link)].
9
SARS-CoV-2 Pseudovirus Neutralization Assay
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The mouse serum samples were detected for neutralizing antibody activity against infection of SARS-CoV-2 prototypic strain and mutant pseudoviruses using our established pseudovirus neutralization assay with some modifications.7 (link)
,26 Briefly, a plasmid encoding the S protein of SARS-CoV-2 prototypic strain or each of the variants containing single or multiple amino acid mutations was transfected into 293T cells in the presence of 2 additional plasmids (psPAX2 and pLenti-CMV-luciferase encoding luciferase protein) (Addgene, Watertown, MA). Pseudovirus-containing culture supernatants were collected at 72 hours after transfection, incubated with serially diluted mouse sera for 1 hour at 37°C, and the virus-serum mixture was added into 293T cells expressing SARS-CoV-2 receptor human ACE2 (hACE2/293T). After being cultured at 37°C for 72 hours, the cells were lysed using cell lysis buffer (Promega, Madison, WI), and detected for relative luciferase activity using Infinite 200 PRO Luminometer (Tecan, Morrisville, NC). Neutralizing activity of serum antibodies against SARS-CoV-2 was calculated using the CalcuSyn computer program and expressed as 50% pseudovirus neutralizing antibody titer (NT50).
,26 Briefly, a plasmid encoding the S protein of SARS-CoV-2 prototypic strain or each of the variants containing single or multiple amino acid mutations was transfected into 293T cells in the presence of 2 additional plasmids (psPAX2 and pLenti-CMV-luciferase encoding luciferase protein) (Addgene, Watertown, MA). Pseudovirus-containing culture supernatants were collected at 72 hours after transfection, incubated with serially diluted mouse sera for 1 hour at 37°C, and the virus-serum mixture was added into 293T cells expressing SARS-CoV-2 receptor human ACE2 (hACE2/293T). After being cultured at 37°C for 72 hours, the cells were lysed using cell lysis buffer (Promega, Madison, WI), and detected for relative luciferase activity using Infinite 200 PRO Luminometer (Tecan, Morrisville, NC). Neutralizing activity of serum antibodies against SARS-CoV-2 was calculated using the CalcuSyn computer program and expressed as 50% pseudovirus neutralizing antibody titer (NT50).
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