For the PPARα or FGF21 luciferase assay, 1.5 × 104 Ac2F cells/well were grown in a 96-well plate in DMEM supplemented with 10% FBS. The peroxisome proliferator response element (PPRE)-X3-TK-LUC plasmid (0.1 μg) (a kind gift from Dr. Christoper K. Glass, University of California, San Diego, CA, USA) and 0.01 μg of PPARα expression vector or pGL3B-Fgf21-LUC were transfected with Lipofectamine 3000 (0.1 μL) and P3000 (0.2 μL, Invitrogen) complexes in Opti-MEM (Invitrogen) according to the manufacturer’s instructions. The pcDNA empty vector was added to obtain an equal amount of plasmid DNA per transfection. After 24 h of transfection, cells were treated with vitamin C. Luciferase activity was tested using the ONE-Glo Luciferase Assay System (Promega, Madison, WI, USA) and a luminescence plate reader (Berthold Technologies GmbH & Co., Bad Wildbad, Germany).
Luminescence plate reader
Manufactured by Berthold Technologies
Sourced in United States, Germany
The Luminescence plate reader is a versatile laboratory instrument used for the detection and quantification of luminescent signals in multi-well microplates. It is designed to measure various luminescence-based assays, including bioluminescence, chemiluminescence, and fluorescence-based assays. The core function of the Luminescence plate reader is to provide accurate and sensitive measurements of luminescent signals generated in the sample wells.
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Lab products found in correlation
One glo luciferase assay system, by Promega (5 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (3 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
P3000, by Thermo Fisher Scientific (2 mentions)
Opti mem medium, by Thermo Fisher Scientific (2 mentions)
Bright glo luciferase assay system, by Promega (2 mentions)
Chir99021, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Winglow software mikrowin v4, by Berthold Technologies (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Lipofectamine transfection reagent, by Thermo Fisher Scientific (1 mentions)
One glo luciferase reporter assay system, by Promega (1 mentions)
Bio glo reagent, by Promega (1 mentions)
Celltiter glo assay kit, by Promega (1 mentions)
10 protocols using luminescence plate reader
1
PPARα and FGF21 Luciferase Assay
2
NF-κB Transcriptional Activity Assay
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Luciferase assays were performed to determine the transcriptional activity of NF-κB in NRK52E cells. After transfection with the NF-κB promoter-LUC plasmid, the luciferase activities were measured using the One-Glo Luciferase Assay System (Promega, Madison, WI, USA) and a luminescence plate reader (Berthold Technologies GmbH & Co., Germany).
3
PPRE-Driven Luciferase Assay for PPARα
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For the peroxisome proliferator response element (PPRE)-driven luciferase assay, 1.5 × 104 HepG2 cells per well were cultured in a 96-well cell culture plate in 100 μL of DMEM supplemented with 10% FBS. The PPRE-X3-TK-LUC plasmid (0.1 μg) (Dr. Christoper K. Glass, University of California, San Diego, CA, USA) and 0.01 μg PPARα expression vector (Dr. Han Geuk Seo, Konkuk University, Seoul, South Korea) were transfected with 0.1 μL of Lipofectamine 3000 reagent and 0.2 μL P3000 (Invitrogen, Carlsbad, CA, USA) complexes in Opti-MEM (Invitrogen), according to the manufacturer's instructions. After 24 h of transfection, cells were treated with MHY553, WY14643, or fenofibrate for 5 h. Luciferase activity was measured using the One-Glo Luciferase Assay System (Promega, Madison, WI, USA) and a luminescence plate reader (Berthold Technologies GmbH & Co., Bad Wildbad, Germany).
4
Luciferase Assay for PPARα Activity
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For luciferase assays, HepG2 cells were seeded at a density of 1 × 104 cells in a 96-well plate. Cells were transfected with lipofectamine transfection reagent (Thermo Fisher Scientific, Rockford, IL, USA), and plasmids were used for transfection with the PPRE-X3-TK-LUC plasmid (Dr. Christoper K. Glass, University of California, San Diego, CA, USA) and PPARα expression vectors (Dr. Han Geuk Seo, Konkuk University, Seoul, South Korea). After transfection for 24 h, cells were treated with WY14643 (a PPARα agonist) [71 (link)] or SSC for 6 h. Luciferase activity was measured using the One-Glo Luciferase Reporter Assay System (Promega, Madison, WI, USA) and a luminescence plate reader (Berthold Technologies GmbH & Co., Bad Wildbad, Germany.
5
Transcriptional Activity of PPAR and NF-κB
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Luciferase assays were performed to determine the transcriptional activity of the PPAR transcription factors in the Ac2F cell. Briefly, Ac2F cells were transfected with the PPRE-X3-TK-LUC plasmid (0.2 µg) with PPAR-α, PPAR-δ, or PPAR-γ expression vectors (0.1 µg) using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA). The cells were further treated with 1 –3 or WY14643 (a known PPAR-α agonist), GW501516 (a known PPAR-δ agonist), and rosiglitazone (a known PPAR-γ agonist), respectively. The luciferase activity was measured using the One-Glo Luciferase Assay System (Promega, Madison, WI, USA). After adding the luciferase substrate, the luminescence was measured using a luminescence plate reader (Berthold Technologies GmbH & Co., Bad Wildbad, Germany).
Luciferase assays were also performed to determine the transcriptional activity of NF-κB in the HEK293T cells. The cells were transfected with the NF-κB promoter-Luc plasmid (0.1 µg) for 24 h, co-treated with test compounds1 –3 and LPS (1 µg/mL) for 6 h, and lysed using a One-Glo Luciferase Assay System and a luminescence plate reader. The results are presented as mean ± S.E. (n = 5), and each measurement was performed in triplicates. Statistical significance was tested using a one-way ANOVA/post hoc test.
Luciferase assays were also performed to determine the transcriptional activity of NF-κB in the HEK293T cells. The cells were transfected with the NF-κB promoter-Luc plasmid (0.1 µg) for 24 h, co-treated with test compounds
6
Quantifying Wnt/β-catenin Signaling in Cells
7
Quantifying Wnt/β-catenin Signaling in Cells
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Wnt/β-catenin signaling was measured as previously described 37 . In short, M50 Super 8x TOPflash and M51 Super 8x FOPflash plasmids 70 , containing a firefly luciferase gene under the control of TCF/LEF binding sites (TOPflash) or mutated TCF/LEF binding sites (FOPflash) were used. MLE 12 cells were plated in 48-well plates at a density of 55.000 cells per well. The following day cells were transfected with either 75 ng/well of M50 Super 8x TOPflash plasmid or the negative control M51 Super 8x FOPflash using Lipofectamine 2000 reagent (Life Technologies, Carlsbad, USA) in serum-free Opti-MEM medium (Life Technologies, Carlsbad, USA). After 6 hours of transfection, cells were stimulated for 24h with an agonistic antibody to LTβR [2 μg/ml] (clone ACH6, kindly supplied by Jeffrey Browing, Biogen Idec), recombinant murine TNF-α [1 ng/ml] (Cat. No. 315–01A, PeproTech) and CHIR99021 [1μM] (Cat. No. 4423, Tocris, Minneapolis, MN). Cells were lysed using Glo lysis buffer and luciferase activity was assayed using the Bright-Glo luciferase assay system (Promega, Madison, Wisconsin, USA). Luciferase activity was determined using a luminescence plate reader (Berthold Technologies). Measured values were analyzed with WinGlow Software (MikroWin v4.41, Berthold Technologies) and TOPflash activity was normalized to FOPflash activity and expressed relative to control conditions.
8
PPAR and NF-κB Transcriptional Regulation
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Luciferase assays were performed to determine the transcriptional activity of PPAR transcription factors in the NRK49F cells. Briefly, NRK49F cells were transfected with the PPRE-X3-TK-LUC plasmid (0.1 µg) with PPARα, PPARβ/δ, or PPARγ expression vectors (0.01 µg) using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA.). The cells were further treated with MHY2013 or WY14643 (a known PPARα agonist), GW501516 (a known PPARβ/δ agonist), and rosiglitazone (a known PPARγ agonist). The luciferase activity was measured using a One-Glo Luciferase Assay System (Promega, Madison, WI, USA). After adding the luciferase substrate, the luminescence was measured using a luminescence plate reader (Berthold Technologies GmbH & Co., Bad Wildbad, Germany). Luciferase assays were performed to determine the transcriptional activity of NF-κB in the NRK52E cells. The cells were transfected with the NF-κB promoter-LUC plasmid, and the luciferase activity was measured using a One-Glo Luciferase Assay System and a luminescence plate reader.
9
Bispeci ic Antibody-Mediated T-cell Activation
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T-cell activation assays were performed using a Promega kit, following the manufacturer’s protocol. Briefly, 3 × 104 AsPC-1 or H226 cancer cells were seeded into 96-well plates in Roswell Park Memorial Institute (RPMI)-1640 media with 10% FBS. After incubation overnight, the medium was aspirated, and bsAbs and 1 × 105 TCR/CD3 effector cells (NFAT) (Promega) were added. T-cell activation was assessed after 5 h of incubation at 37 °C in 5% CO2 atmosphere. Following mixing with Bio-Glo Reagent (Promega), luminescence was measured with a luminescence plate reader (Berthold Technologies, Bad Wildbad, Germany).
10
Measuring Cell Proliferation by CellTiter-Glo
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Cell proliferation was assessed using the CellTiter-Glo assay kit (Promega, Madison, WI, USA). Cells were seeded in 96-well plates in McCoy’s 5A complete medium, containing 10% FBS and 1% P/S, and treated with 0–100 μM DFMO or with 10 μM cisplatin for 72 h. The CellTiter-Glo reagent was added to the treated cells with a volume equal to that of the medium in each well. The cells were incubated at room temperature for 10 min, and luminescence was measured using a luminescence plate reader (Berthold, Germany).
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