Dual-luciferase assay was performed to evaluate the luciferase activity of the reporter constructs according to manufacturers’ instructions (Promega, USA) with some modifications. In brief, HEK293 cells were seeded in 24-well culture plates at a density of 1 × 106 cells per well, and allowed to grow until they reached 80% confluency. Cells were then transfected with TOP Flash or pmiR-GLO luciferase reporter plasmids together with linc-MCF2L-AS1 expression vectors or miRNA mimics. The pRSVb-galactoside vector was co-transfected into HEK293 cells as an internal control for normalization. The plate was placed into a PerkinElmer VictorTM X2 2030 multilabel reader (Waltham, USA) to measure the firefly luciferase activity and β-galactosidase activity. The ratio of firefly luciferase to β-galactosidase activity in each sample was used as a measurement of the normalized luciferase activity. All experiments were performed in triplicate.
Victor x2 2030
Manufactured by PerkinElmer
Sourced in United States
The Victor X2 2030 is a multimode microplate reader designed for high-throughput analysis of various assays. It can detect and quantify a range of signals, including luminescence, fluorescence, and absorbance, in 6- to 384-well microplates. The Victor X2 2030 is a versatile instrument that can be used for a variety of applications in life science research and drug discovery.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase assay reagent, by Promega (4 mentions)
Amplex red hydrogen peroxide assay kit, by Thermo Fisher Scientific (4 mentions)
Hek293 cell, by American Type Culture Collection (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Amplex red, by Thermo Fisher Scientific (1 mentions)
Dcfh da dye, by Merck Group (1 mentions)
13 protocols using victor x2 2030
1
Dual-Luciferase Assay for Reporter Activity
2
Dual-Luciferase Assay for Gene Expression
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Dual-luciferase assay was performed to evaluate the luciferase activity of the reporter constructs according to the instructions of dual-luciferase assay reagents (Promega, USA) with some modifications. In brief, HEK293 cells were seeded on a 24-well plate, and the cells were allowed to grow until 80% confluency. Cells were then transfected with TOPFlash or pmiR-GLO luciferase reporter plasmids together with linc-ROR expression vectors or miRNA mimics. The pRSV-β-galactoside (ONPG) vector was co-transfected into HEK293 cells as an internal control for normalization. The plate was placed into a PerkinElmer VictorTM X2 2030 multilabel reader (Waltham, USA) to measure the firefly luciferase activity, as well as the β-galactosidase activity. The ratio of firefly luciferase to β-galactosidase activity in each sample was revealed as a measurement of the normalized luciferase activity. All experiments were performed in triplicate.
3
High-Throughput Screening of Wnt/NF-κB Modulators
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Wnt/β-catenin response element (RE) luciferase reporter vector TOPFlash, NF-κB response element (RE) luciferase reporter vector and Renilla luciferase control reporter vectors were purchased from Upstate Cell Signaling (Charlottesville, VA, USA). The Malat1 small interfere RNAs (si-Malat1) and control siRNAs (si-NC) were synthesized by GenePharma (Shanghai, China). The human embryonic kidney 293 (HEK293) cells were purchased from ATCC (CRL-1573). BMSCs were isolated from the previous studies and stocked in our laboratory. The transient transfection was performed using Lipofectamine 3000 (Invitrogen, Waltham, MA, USA). To perform the high-throughput luciferase screening, the HEK293 cells were seeded in 96-well plates at the density of 5000 cells/well. After 80% confluency, the cells were co-transfected with TOPFlash or NF-κB RE reporter vector, together with Renilla vector, respectively. The cells were then treated with chemicals from library as mentioned before at a concentration of 10 μM for 24 h. The dual-luciferase assay was then conducted in a PerkinElmer VictorTM X2 2030 multilateral reader (Waltham, MA, USA) according to the instructions as described previously [30 (link)]. The ratio of firefly luciferase of each treatment was normalized with Renilla activity.
4
High-throughput Luciferase Screening of Small Molecules
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A high-throughput luciferase activity screening method was applied to screen out the putative natural products which regulate promoter luciferase activities. The HEK293 cells were seeded in 24-well plates at the density of 5000 cells/well. When the cells were 80% confluent, they were co-transfected with Renilla vector together with TOPFlash or NF-κB RE reporter vector, respectively. The cells were then treated with chemicals from Selleck small product chemical library at the final concentration of 10 μM. After one-day treatment, the cells were harvested and applied to dual-luciferase assay according to the instructions of dual-luciferase assay reagent (Promega, Madison, WA, USA) with some modifications. In brief, cell lysate was placed into a PerkinElmer VictorTM X2 2030 multilateral reader (PerkinElmer, Waltham, MA, USA) to measure the firefly luciferase activity as well as the renilla luciferase activity. The ratio of firefly luciferase to renilla activity in each sample was revealed as a measurement of the normalized luciferase activity. Triplicated experiments were performed.
5
Dual-Luciferase Reporter Assay for hMSCs
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Dual-luciferase assay was performed to evaluate the luciferase activity of the reporter constructs according to the instructions of dual-luciferase assay reagents (Promega, USA) with some modifications [3 (link)]. In brief, hMSCs were cultivated on cell culture plate, and the cells were allowed to grow until 80% confluency. Cells were then transfected with TOPFlash or pmiR-GLO luciferase reporter plasmids together with β-catenin expression vectors or miRNA mimics. The pRSV-β-galactoside (ONPG) vector was co-transfected into hMSCs as an internal control for normalization. The plate was placed into a PerkinElmer VictorTM X2 2030 multilabel reader (Waltham, USA) to measure the firefly luciferase activity, as well as the β-galactosidase activity. The ratio of firefly luciferase to β-galactosidase activity in each sample was revealed as a measurement of the normalized luciferase activity. All experiments were performed in triplicate.
6
Extracellular H2O2 Measurement
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The measurement of extracellular H2O2 level from the culture medium samples was performed using the Amplex Red assay (Thermo Fisher Scientific, Waltham, MA, USA). After incubation of 50 µL of Amplex Red (100 µM), HRP (0.2 U/mL) and 50 µL of culture medium at 21 °C for 30 min, the fluorescence of the samples was measured at 531 nm with an excitation set at 590 nm using a Victor X2 2030 fluorimeter (Perkin Elmer, Waltham, MA, USA).
7
Intracellular Redox State Measurement
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Measurement of disruptions in the intracellular redox state of IPEC-J2 cells was carried out using DCFH-DA dye (Sigma-Aldrich, Budapest, Hungary). DCFH-DA is oxidized to the highly fluorescent form of dichlorofluorescein (DCF) by the intracellular ROS [20 (link)]. Following a centrifugation process for 10 min at 10 000 rpm at 5°C, 100 μL of cell-free supernatant was collected and pipetted into a 96-well plate. Samples of supernatant were collected at 24 and 48 h after treatments. The fluorescence intensities of the supernatants were measured at 530 nm with a fluorometer using 485 nm excitation wavelength (Victor X2 2030, Perkin Elmer, Waltham, MA, USA).
8
Amplex Red Assay for Extracellular H2O2 Quantification
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The changes in the extracellular H2O2 levels in cell supernatants were detected using the Amplex Red Hydrogen Peroxide Assay Kit (Invitrogen, Molecular Probes) in a 96-well plate. In the presence of horseradish peroxidase (HRP), Amplex Red reagent reacts with H2O2 (in 1:1 stoichiometry) to produce a red fluorescent product called resorufin. Following the treatment of PHHs with inhibitor MI-1851 (100 μM) for 24 h, a 50 µL aliquot of the cell free supernatant was collected and mixed with 50 µL of Amplex Red working solution in each well, then incubated for 30 min. Fluorescence intensities were measured with a fluorimeter (Victor X2 2030, Perkin Elmer, Waltham, MA, US) using 560 nm excitation and 590 nm emission wavelengths.
9
Quantification of Hydrogen Peroxide Levels
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The quantification of H2O2 concentrations in cell supernatants was carried out using the Amplex Red Hydrogen Peroxide Assay Kit (Invitrogen, Molecular Probes, Waltham, MA). In the presence of horse radish peroxidase, Amplex Red reagent reacts with H2O2 (in 1:1 stoichiometry) to produce a red fluorescent product called resorufin. Following the exposure of HIEC-6 and PHH to MI-1900 and MI-1907 (50 µM, 24 h), the H2O2 concentrations in the medium were determined using a working solution of 100 µM Amplex Red and 0.2 U/mL HRP. After 24 h, cell free supernatants were taken from the basolateral compartment. Fifty microlitres of the collected cell free supernatants were mixed with the Amplex Red working solutions. The fluorescence intensities were measured with a fluorometer (Victor X2 2030, Perkin Elmer, Waltham, MA) using 560 nm excitation and 590 nm emission wavelengths.
10
Dual-Luciferase Assay for miRNA Target Validation
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Dual-luciferase assay was performed following the previous protocol.16 (link) HEK293 cells were transfected with pmiRGLO-Atg2a or pmiRGLO-Sox6 WT and site-mutation (mut) recombinant vectors together with miR-378-3p mimics. The renilla luciferase vector was co-transfected as normalization control. The luciferase activities were measured using PerkinElmer Victor X2 2030 multilabel reader (Waltham, USA). The renilla luciferase activity was also measured for normalization.
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