To investigate the NF-κB-dependent transcriptional activity, RAW 264.7 cells (2 × 105) were seeded onto 24-well plates and transfected using Lipofectamine 2000 reagent (Invitrogen). For the transfections, 2 µg p6κB-LUC (kindly provided by Dr Patrick Baeuerle) and 80 ng pRL-CMV (Promega) were used. Cells transfected with p6κB-LUC plasmids were infected and treated with LPS (1 µg ml−1, Sigma). Subsequent to infection and treatment, the cells were washed with PBS, lysed according to Dual Luciferase System protocol (Promega) and analysed in a TD-20/20 Luminometer (Turner Designs).
Dual luciferase system protocol
Manufactured by Promega
Sourced in United States
The Dual Luciferase System protocol is a laboratory tool that allows for the simultaneous measurement of two different luciferase reporter genes within the same sample. The protocol provides a method for normalizing experimental data by using a constitutively expressed control reporter to account for differences in transfection efficiency or cell number between samples.
Automatically generated - may contain errors
Lab products found in correlation
Prl cmv, by Promega (5 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (4 mentions)
Td 20 20 luminometer, by Turner Designs (3 mentions)
Glomax multi detection system, by Promega (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Igf 1, by R&D Systems (1 mentions)
Anti igf1r, by R&D Systems (1 mentions)
Luciferase assay system, by Promega (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
Glomax luminometer, by Promega (1 mentions)
Nucleofector technology, by Lonza (1 mentions)
Prl cmv plasmid, by Promega (1 mentions)
Dl sulforaphane sfn, by Merck Group (1 mentions)
6 protocols using dual luciferase system protocol
1
Analyzing NF-κB Transcriptional Activity
2
Investigating iNOS Promoter Activity
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As previously described, to investigate the activity of the iNOS promoter, RAW 264.7 macrophages (105 cells per well) were plated in 48-well polystyrene plates55 and transfected via Lipofectamine 2000 reagent (Invitrogen) by way of the manufacturer’s instructions. Cells were transfected with 1 μg pTK-3XNS or pTK-3XS (kindly provided by Dr. David Geller) plus plasmid pRL-CMV (Promega) that was the co-reporter for the normalization of experimental variations. Cells transfected with pTK-3XNS or pTK-3XS and pRL-CMV were treated with IGF-I (50 ng/ml; R&D Systems), IFN-γ (10 ng/ml; R&D Systems), or anti-IGF-1R (5 μg/ml; R&D Systems). Unstimulated cultures were included as controls. After these treatments, cells were washed with PBS, lysed according to the Dual Luciferase System Protocol (Promega), and analyzed in the TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA, USA). Luciferase activity was measured using the Luciferase Assay System (Promega), according to the manufacturer’s protocol. Each transfection was performed in triplicate, and three independent experiments were conducted.
3
Measuring ATF4 and Nrf2 Activity
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To analyze the ATF4-dependent transcriptional activity, shSCR or shATF4 transduced cells were plated at concentrations of 105 cells/mL in a 48 well-plate and transfected with Lipofectamine 2000 (Thermo Scientific). For transfections, 1 μg of pGL23xARE-LUC, 1 μg of pGL2NRF2-LUC and 40 ng pRL-CMV (PROMEGA) were used.
Transfected cells were incubated for 24 hours and then infected with L. amazonensis (10:1) for an additional 24 hours. After infection, cells were washed with PBS, lysed according to the Dual Luciferase System protocol (PROMEGA) and analyzed using a Glomax luminometer (PROMEGA). Sulforaphane treatment was used as a positive control of Nrf2- Luciferase assyas.
Transfected cells were incubated for 24 hours and then infected with L. amazonensis (10:1) for an additional 24 hours. After infection, cells were washed with PBS, lysed according to the Dual Luciferase System protocol (PROMEGA) and analyzed using a Glomax luminometer (PROMEGA). Sulforaphane treatment was used as a positive control of Nrf2- Luciferase assyas.
4
Promoter Activity Assay in RAW-264.7 and THP-1 Cells
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To investigate the promoter activity, RAW-264.7 cells (1 × 105 cells per well) was plated in 48-well polystyrene plates and transfected with 1 µg of reporter plasmids using LIPOFECTAMINE 2000 reagent (Invitrogen, Carlsbad, CA, USA). THP-1 cells (2 × 106) were transfected with 0.5 µg of luciferase reporter plasmids using Nucleofector™ Technology (Lonza, Basel, Switzerland) according to the manufacturer’s instructions. The following plasmids were employed in the assays: Sod1-basal, Sod1-ΔARE, Sod1-WT, 3xARE, and Nrf2-WT. For normalization of the luciferase readout, the plasmid pRL-CMV (Promega) was used. After infection and treatment, the cells were washed with PBS, lysed according to the Dual Luciferase System protocol (Promega), and analyzed using the GloMax®-Multi detection system (Promega Corp., Madison, WI, USA).
5
NF-κB Transcriptional Activity Assay
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RAW cells (2×105/well) were transfected with 1 μg of the plasmid 6KB (6 consensus binding sites for NF-κB; kindly provided by Dr. P. Bauerle, Ludwig-Maximilians-Universität, Munich, Germany; ref. 12 (link)). The cells were cotransfected with pRL-CMV (80 ng), which expresses Renilla constitutively (Promega, Fitchburg, WI, USA) for luciferase activity normalization. The cells were infected overnight in RPMI-FCS, washed with PBS, and lysed according to the dual-luciferase system protocol (Promega), and the lysates were analyzed in a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA, USA).
6
Investigating Promoter Activity in RAW-264.7 Cells
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To investigate the promoter activity, RAW-264.7 cells were plated in 24-well polystyrene plates (1 × 105 cells per well) and transfected with 1μg of reporter plasmids using LIPOFECTAMINE 2000 reagent (Invitrogen, Carlsbad, CA, USA). The following plasmids were employed in the assays: 3xARE and Nrf2-WT. Luciferase activity was normalized using 40 ng of pRL-CMV plasmid (Promega Corp., Madison, WI, USA), 24 h after transfection the cells were treated. After treatment, cells rested for 24 h and were washed with PBS and lysed according to the Dual Luciferase System protocol (Promega Corp.), and analyzed using the GloMax®;-Multi detection system (Promega Corp., Madison, WI, USA). Positive controls consisting of cells stimulated with 10 mM DL-sulforaphane (SFN) (Sigma-Aldrich) were used to induce the activation of ARE and Nrf-2 gene expression.
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