Lipofectamine 2000 (Life Technologies) was used to transiently co-transfect cells with the M-MITF or NEDD9 promoters and expression vectors for LEF1, β-catenin (WT and mutants) and/or ICAT (WT and mutants). The pRL-TK Renilla luciferase reporter construct was used as an internal control for transfection efficiency. Forty-eight hrs post-transfection, cells were lysed with passive lysis buffer and luciferase activities (Firefly and Renilla) were measured using the Dual Luciferase reporter Assay (Promega, Madison WI) in a TriStar luminometer (Berthold, Germany). Firefly luciferase activity was normalized to Renilla luciferase activity.
Tristar luminometer
Manufactured by Berthold Technologies
Sourced in France, Germany
The TriStar luminometer is a multi-mode microplate reader designed for luminescence measurements. It provides accurate and reliable detection of bioluminescent, chemiluminescent, and fluorescent signals in a variety of microplate formats.
Automatically generated - may contain errors
Lab products found in correlation
Passive lysis buffer (plb), by Promega (5 mentions)
Steadylite plus luciferase substrate, by PerkinElmer (2 mentions)
White 96 well plate, by Corning (2 mentions)
Dual luciferase kit, by Promega (2 mentions)
Dual luciferase reporter assay, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Adp glo kinase assay kit, by Promega (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Thermomixer, by Eppendorf (1 mentions)
One glo reagent, by Promega (1 mentions)
Retic lysate ivt kit, by Thermo Fisher Scientific (1 mentions)
Decanal, by Merck Group (1 mentions)
N decyl aldehyde, by Merck Group (1 mentions)
Nanoluc substrate, by Promega (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Jetoptimus, by Polyplus Transfection (1 mentions)
One glo luciferase assay buffer, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
D luciferin, by Merck Group (1 mentions)
17 protocols using tristar luminometer
1
Transient Co-transfection Assay for Promoter Activity
2
In vitro AKT1 Kinase Assay
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The in vitro phosphorylation assay was carried out with the ADP-Glo™ Kinase Assay kit (Promega; V6930) and the AKT1 Kinase Enzyme System Kit (V1911). The purified proteins UPF1, and UPF2 (761-1227) which includes the three phosphorylation sites (S886, S992 and S1046) identified by mass spectrometry (19 (link)) were obtained from Dr Hervé Le Hir. The UPF3X protein was purchased from CliniSciences (Recombinant Human Regulator of Nonsense Transcripts 3B—E. coli; CSB-EP883646HU). Kinase reactions were carried out at room temperature for 1 h with 50 ng enzyme, 1 μg substrate, purified protein, 250 μM ATP, 50 μM DTT, Reaction Buffer 1×. The ATP depletion reaction was carried out at room temperature for 40 min and the luminescence reaction was carried out for 30 min in a Tristar luminometer (Berthold).
3
Luciferase Assay of Transfected HeLa Cells
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Lipofectamine LTX (ThermoFisher Scientific, Waltham, MA, USA) was used according to the manufacturer’s recommendations to transfect HeLa cells with each FLuc-int-PTC construct. The cells were distributed 24 h later into white 96-well plates (Corning, Corning, NY, USA) and incubated for 20 h at 37°C and 5% CO2 with extract at 10 ng/μl or with G418 at 1 μg/μl. Luciferase activity was then measured by adding the Steadylite plus luciferase substrate (Perkin Elmer, Waltham, MA, USA) to each well and by measuring the visible light from each well for 10 sec with a Tristar luminometer (Berthold technologies, Versailles, France). The plate was read twice and the reported values are averages of the two reads.
4
In vitro translation assays of F-Luc mRNA
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In vitro translation assays were performed with the Retic Lysate IVT Kit (Invitrogen-AM1200, Villebon Sur Yvette, France) following the manufacturer’s instructions and using the F-Luc mRNA (12.5 ng/µL) as a reporter. The translation efficiency of this transcript was analyzed in the presence of either P. lividus eIF4B or R-Luc mRNAs, both at a final concentration of 12.5 ng/µL. Incubations were performed at 30 °C in an Eppendorf ThermoMixer with agitation at 300 rpm. For each time point, 10 µL of the reaction assay was mixed with 50 µL of ONE-Glo reagent (Promega-E6110, Charbonnières-les-Bains, France), and the sample luminescence was measured for 10 s on a 96-well microplate using a Tristar luminometer (Berthold).
5
NZX Modulation of BCG Bioluminescence
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MSP released NZX was collected and the concentration was determined using SDS-PAGE. NZX at final concentrations 3.2 uM, 6.3 uM or 12.5 uM was added to BCG in 96 well plates. An additional NZX solution was prepared the same day from powder and added as a control at a final concentration of 3.2 uM. Decanal (0.1% n-decyl aldehyde, Sigma) was added once a day for three days in triplicate wells for each condition. Bioluminescence was measured as relative luminescence units (RLU) with a TriStar luminometer (Berthold Technologies, Germany).
6
Luminescence Assay for Protein Expression
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HEK293T cells were cultured as described for the mN2H assay version. For each protein, 100 ng of purified plasmid DNA was transfected into HEK293T cells grown in 96-well, flat-bottom, cell culture microplates (Greiner Bio-One, #655083). The DNA/PEI ratio (mass:mass) was 1:3 for the transfection. 24 h later, 100 µL of the NanoLuc substrate (Promega, #N1110) were added to each well, plates were incubated for 3 min at room temperature, and luminescence output was measured using a TriStar luminometer (Berthold; 1 s integration time).
7
Smad1/5 Activation by Plasmin-Cleaved AMH
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EXAMPLE 2
Methods:
SMAT-1 cells seeded in 24-wells plates at 7.5×104 cells per well. 24 h later, cells were co-transfected using lipofectamine Plus reagent with either Gal4-Smad1 or Gal4-Smad5 (250 ng) and Gal4-luc (250 ng) plasmids, and 12.5 ng of pRLTK as a control for transfection efficiency. After the transfection, cells were treated with either control medium, plasmin-cleaved AMH (0.5 or 1 μg/ml), or plasmin-cleaved AMH pre-incubated 1 h in presence of a 50 fold excess of Mab 22A2 (25 or 50 μg/ml). 24 h later, cells were washed twice with PBS, and lysed for 20 min under rocking in 125 μl of passive lysis buffer (Promega, Madison, Wis.) per well. Twenty μ1 were analysed for Firefly and Renilla luciferase activity according to the manufacturer (Dual Luciferase kit, Promega) using a TriStar luminometer (Berthold). Results were expressed as a ratio of Firefly to Renilla luciferase activity. Data are a mean±SEM of 1 experiment, done in quadriplate.
Results:
The functional activation of Smad1 pathway was assessed by a reporter system composed of Gal4-Smad1 or a Gal4-Smad5 fusion proteins and a Gal4-luc reporter construct (Clarke et al., 2001). Both concentrations of plasmin-cleaved AMH induce a 10-fold stimulation of Smad1 and Smad5 reporter systems, which is abolished by a 50-fold excess of 22A2.
8
Pathway Activity Analysis in Melanoma Cells
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SuperTopFlash, pcDNA3.1-GLuci-CRE/-AP1/-NFAT as well as pLucTKS3 (Stat3) reporter were transiently transfected for the analysis of specific pathway activity. 2.5 × 105 melanoma cells were seeded into 6 well plate cavities 24 h prior to transfection. Reporter plasmids (2 μg/well) and CMV renilla plasmids (120 ng/well) were co-transfected using ScreenFect A Transfection Kits (Genaxxon bioscience) or Lipofectamine 3000 Transfection Reagent (Life Technologies) according to the manufacturer's protocols for 24 h. Then 1 × 104 transfected cells were seeded into 96 well plate cavities at minimum in quintuplicates and optionally pre-treated with 15 mM lithium chloride for 6 h. Additional treatments were PLX4032 (up to 10 μM), Stattic (up to 2 μM) or Wnt5a and Wnt3a (100 ng/ml) for further 24 h. Cells were lysed in 50 μl passive lysis buffer (Promega). 10 μl of lysates or 10 μl of supernatant of the culture medium (Gaussia reporters) was used for the measurement of the firefly and renilla luciferase activity in a Tristar luminometer (Berthold). Luciferase activity was measured using D-luciferin or renilla and gaussia substrate coelentarazine as previously described (Albert and Silvia, 2012 ).
9
Smad5 Activation by C-terminal AMH
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EXAMPLE 3
Methods:
SMAT-1 cells seeded in 24-wells plates at 7.5×104 cells per well. 24 h later, cells were co-transfected using lipofectamine Plus reagent with Gal4-Smad5 (250 ng) and Gal4-luc (250 ng) plasmids, and 12.5 ng of pRLTK as a control for transfection efficiency. After the transfection, cells were treated with either control medium, C-terminal AMH (0.2 to 0.05 μg/ml), or C-terminal AMH (0.2 μg/ml) pre-incubated 1 h in presence of a 50-fold or 10-fold excess of Mab 22A2, or a 50-fold excess of Mab 10.6. 24 h later, cells were washed twice with PBS, and lysed for 20 min under rocking in 125 μl of passive lysis buffer (Promega, Madison, Wis.) per well. Twenty μ1 were analysed for Firefly and Renilla luciferase activity according to the manufacturer (Dual Luciferase kit, Promega) using a TriStar luminometer (Berthold). Results were expressed as a ratio of Firefly to Renilla luciferase activity. Data are a mean±SEM of 1 experiment, done in quadriplate.
Results:
The functional activation of Smad1, 5, 8 pathway was assessed by a reporter system composed of a Gal4-Smad5 fusion proteins and a Gal4-luc reporter construct (Clarke et al., 2001). C-terminal AMH induce a 10-fold stimulation of Smad5 reporter system, which is abolished by both a 50-fold and a 10-fold excess of 22A2, but not by a 50-fold excess of 10.6.
10
Measuring NMD Efficacy with One-Plasmid Screen
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The measurement of NMD efficacy with the two-plasmid screen has been described previously in detail [21 (link)]. For the one-plasmid screening system, 180ng of a vector carrying the cDNA coding for firefly luciferase and two copies of the same intron located in the coding sequence and in the 3′UTR, are introduced into 8 million U2OS cells with JetOptimus (PolyPlus). After 24 h, the cells were distributed into 96-well white clear-bottom plates and treated with cell culture before adding the molecules for an additional 24 h. Then, the luciferase substrate Steadylite Plus (Revvity, Bussy Saint Martin, France) was added to each well before reading the luciferase activity with a Tristar luminometer (Berthold, Thoiry, France).
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