An 3xSTAT3/Luc fragment containing tandem repeats of double-stranded oligonucleotides spanning the STAT3 binding site (5′-CATTTCCCGTAAATC-3′ ) [30] (link) was amplified with the primers sense: 5′-CATT TCCCGTAAAT CCATTTCCCGTAAATCCATTTCCCGTAAATC-3′ and then introduced into the pGL3 promoter plasmid (Promega, Madison, WI). All transfection experiments were performed using Lipofectamine 2000 reagent (Invitrogen) in accordance with the manufacturer's instructions. For luciferase assays, the cell lysate was first mixed with luciferase substrate solution (Promega), and the resulting activity was measured using a luminometer. For each experiment, luciferase activity was determined in triplicate and normalized using β-galactosidase activity.
Luciferase substrate solution
Manufactured by Promega
Sourced in United States
Luciferase substrate solution is a reagent used in biochemical assays to detect and quantify the activity of the luciferase enzyme. The solution contains the necessary substrates and cofactors required for the luciferase-catalyzed bioluminescent reaction, which produces light that can be measured using a luminometer or other light detection device.
Automatically generated - may contain errors
Lab products found in correlation
Victor3, by Promega (3 mentions)
Victor3, by PerkinElmer (3 mentions)
Passive lysis buffer (plb), by Promega (3 mentions)
Pgl2 promoter plasmid, by Promega (2 mentions)
Lipofectamine reagent, by Thermo Fisher Scientific (2 mentions)
Wrhek293a cells, by AMS Biotechnology (2 mentions)
Pgl3 promoter plasmid, by Promega (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Lysis buffer, by Promega (1 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
Fb12 tube luminometer, by Berthold Technologies (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
8 protocols using luciferase substrate solution
1
STAT3-Responsive Luciferase Assay
2
ARE-luciferase Vector Construction
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To construct the ARE-luciferase vector, tandem repeats of double-stranded oligonucleotides spanning the Nrf2 binding site (5′-TGACTCAGCA-3′) were introduced into the restriction sites of the pGL2 promoter plasmid (Promega, Madison, WI, USA). All transfection experiments were performed by using Lipofectamine reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. For luciferase assays, the cell lysate was first mixed with the luciferase substrate solution (Promega, Madison, WI, USA), and luciferase activity was measured by using an AutoLumat LB953 luminometer (EG and G Berthold, Bad Wildbad, Germany). For each experiment, the luciferase activity was determined in triplicate and was normalized in each sample by using β-galactosidase activity.
3
Quantifying luciferase expression in vivo
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Hind limb muscles, the tattooed skin region and inguinal lymph nodes were isolated to quantify luciferase expression ex vivo 6 h and 30 h after rSFV injection. Immediately after excision, the organs were frozen in liquid N2 and kept at −80 °C. The organs were crunched to powder in a mortar on dry ice. The material was lysed in lysis buffer (Promega, Leiden, The Netherlands) by three freeze-thaw cycles with vigorous vortexing in between. Cell debris was removed by centrifugation. Immediately before measurement, lysates of the samples were mixed with luciferase substrate solution (Promega). The luciferase signal was determined in a luminometer (Synergy HT, BioTek, Winooski, VT, USA). Background intensity was subtracted from the signal of measured samples.
4
Transfection of ARPE-19 Cells for Luciferase Assay
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The ARPE-19 cells were transfected with 1 µg of NF-κB/Luc or p3xARE/Luc using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Briefly, the reporter DNA (2 µg) and β-galactosidase DNA (0.5 µg) were mixed with 5 µl Lipofectamine 3000 for 10 min at room temperature. The mixture was added to ARPE-19 cells and, 4 h later, 10% FBS DMEM/F-12 was added for 24 h. For the luciferase assays, the cell lysate was mixed with luciferase substrate solution (Promega Corporation), and then the resultant luciferase activity was measured using the FB12 Tube Luminometer (Titertek-Berthold). The luciferase activities were standardized to β-galactosidase activity.
5
Luciferase Assay for TCF/LEF Activity
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WRHEK293A cells (Amsbio, Abingdon, UK) were seeded in a black 96-well plate and cultured for 24 h. Cells were treated with various concentrations of samples and incubated for another 24 h. Cells were then lysed by adding 50 μL of 1x passive lysis buffer (Promega, Madison, WI, USA) to each well and shaken for 10 min. GFP expression (internal cell viability control) was assessed by measuring the fluorescence at a 488/510 nm wavelength using VICTOR3 (PerkinElmer, Waltham, MA, USA). A total of 50 μL of luciferase substrate solution (Promega, Madison, WI, USA) was added and luciferase activity was measured using VICTOR3. Luminescence (TCF/LEF activity) values were normalized to GFP (cell viability) values.
6
Cell Viability and Transcriptional Activity Assay
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WRHEK293A cells (Amsbio, Abingdon, UK) were seeded in black 96 well plates and cultured for 24 h. Cells were treated with various concentrations of chemicals and incubated for another 24 h. Cells were then lysed by adding 50 μL of 1× Passive Lysis Buffer (Promega, Madison, WI, USA) to each well and shaking for 10 min. The expression of GFP (internal cell viability control) was assessed by measuring the fluorescence at 488/510 nm wavelength using VICTOR3 (PerkinElmer, Waltham, MA, USA). Then, 50 μL of luciferase substrate solution (Promega, Madison, WI, USA) was added to each well and the luciferase activity was measured using VICTOR3. Luminescence (TCF/LEF activity) values were normalized to GFP (cell viability) values.
7
Constructing ARE-Luciferase Reporter Vector
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To construct the antioxidant response element (ARE)-luciferase vector, tandem repeats of double-stranded oligonucleotides spanning the Nrf2 binding site (5’-TGACTCAGCA-3’) were introduced into the restriction sites of the pGL2 promoter plasmid (Promega, Madison, WI, USA). All transfection experiments were performed using lipofectamine reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. For luciferase assays, the cell lysate was first mixed with the luciferase substrate solution (Promega) and luciferase activity was measured using a luminometer. For each experiment, luciferase activity was determined in triplicate and normalized using β-galactosidase activity for each sample.
8
Escin Modulates TCF/LEF Activity
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WRHEK293A cells (3 × 105 cells per well) were seeded in black 96-well plates and cultured for 24 h. Cells were treated with various concentrations of escin and incubated for another 24 h. Cells were then lysed by adding 50 μL of 1× Passive Lysis Buffer (Promega, Madison, WI, USA) to each well and shaking for 10 min. GFP expression levels (internal cell viability control) were assessed by measuring the fluorescence at a 488/510 nm wavelength using VICTOR3 (PerkinElmer, Waltham, MA, USA). Then, 50 μL of luciferase substrate solution (Promega, Madison, WI, USA) was added and luciferase activity was measured using VICTOR3. Luminescence (TCF/LEF activity) values were normalized to GFP (cell viability) values.
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