Cells were transfected with 200 ng of CLIC5-pMirTarget or pMiRTarget CTRL (Origene, Rockville, MD, USA) with Promofectin Transfection Reagent (PromoCell GmbH, Heidelberg, Germany) and the luciferase activity measured after 48 h using the Dual Glo Assay system (Promega, Madison, WI, USA) according to the manufacturer’s protocol in a multiwell plate luminometer (Perkin-Elmer, Seer Green, Beaconsfield, UK). Luciferase activity was normalized to that of renilla activity for each transfected well.
Dual glo assay system
Manufactured by Promega
Sourced in United States
The Dual-Glo assay system is a luminescent reporter assay designed to measure firefly and Renilla luciferase activities in a single sample. The system allows for the sequential detection of the two luciferase activities, providing a convenient way to normalize the experimental reporter data.
Automatically generated - may contain errors
Lab products found in correlation
X tremegene hp, by Roche (3 mentions)
Psuper retro puro, by Oligoengine (2 mentions)
Puromycin, by Merck Group (2 mentions)
Azd6244, by Selleck Chemicals (2 mentions)
Pcmv renilla luciferase, by Promega (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Prl tk, by Promega (1 mentions)
Bmp 2, by R&D Systems (1 mentions)
Mia paca 2, by American Type Culture Collection (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Panc 1, by American Type Culture Collection (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Mmessage sp6 in vitro transcription kit, by Thermo Fisher Scientific (1 mentions)
Envision plate reader, by PerkinElmer (1 mentions)
Hemin, by Merck Group (1 mentions)
L4151, by Promega (1 mentions)
Pcdna3 vector, by Thermo Fisher Scientific (1 mentions)
Quikchange lightning multi site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Phtn halotag cmv neo, by Promega (1 mentions)
19 protocols using dual glo assay system
1
Luciferase Assay for CLIC5 Expression
2
Establishing Stable Cell Lines for Luciferase Assay
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Cells were transfected with the appropriate plasmids using Fugene 6 (Roche) according to the manufacturer’s instructions. Luciferase reporter assays were performed as previously described, using the Dual-Glo assay system (Promega) following the manufacturer's instructions (Levy et al., 2007 (link)).
HaCaT SMAD4 KO lines stably expressing either EGFP or EGFP-SMAD4 WT or mutants were generated by transfecting the cells with the appropriate plasmids. Transfected cells were selected with 500 µg/ml of G418 (Invitrogen), then FACS sorted for EGFP-positive cells, and expanded. EGFP expression was confirmed by microscopy. To generate stable HEK293T cell lines expressing either CAGA12-Luciferase or BRE-Luciferase together with TK-Renilla, cells were transfected with the appropriate plasmids together with a plasmid carrying the puromycin resistance gene (pSUPER-retro-puro; OligoEngine). Cells were then selected with 2 μg/ml puromycin (Sigma).
HaCaT SMAD4 KO lines stably expressing either EGFP or EGFP-SMAD4 WT or mutants were generated by transfecting the cells with the appropriate plasmids. Transfected cells were selected with 500 µg/ml of G418 (Invitrogen), then FACS sorted for EGFP-positive cells, and expanded. EGFP expression was confirmed by microscopy. To generate stable HEK293T cell lines expressing either CAGA12-Luciferase or BRE-Luciferase together with TK-Renilla, cells were transfected with the appropriate plasmids together with a plasmid carrying the puromycin resistance gene (pSUPER-retro-puro; OligoEngine). Cells were then selected with 2 μg/ml puromycin (Sigma).
3
Quantifying CIAR-Driven RAS/MAPK Activation
4
Luciferase Assay in MEF Cells
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At 72 h after transfection, MEF cells were lysed and substrates for Renilla and Firefly Luciferase were added, using the Dual Glo Assay System according to the manufacturer´s recommendations (Promega). Luminescence emission was detected on a Tecan Infinite M200 Pro.
5
Endosomal Release Enhances Lipid Transfection
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The stably transfected 15b cell line was a gift from miRagen Therapeutics, Inc. Cells were seeded in DMEM medium containing 10% FBS at 5000 cells per well in 96-well plates. Cells were then incubated at 37 °C for 24 h before transfection. For endosomal release, cells were pretreated with chloroquine (CQ; 100 μM, 100 μl per well) for 3 h in reduced serum medium without antibiotics (DMEM with 2% FBS, 1× GlutaMAX, 1× non-essential amino acids and 1× sodium pyruvate) before lipid (0.1 μM ODN, 100 μl per well) or passive transfection (1 μM ODN, 100 μl per well) in reduced serum DMEM medium without antibiotics (above). After 24 h of lipid transfection or 72 h of passive transfection, cells were collected for luciferase assays. At this time, the medium was removed and the cells were washed with 1×D-PBS before completing a luciferase assay. The Promega Dual-Glo assay system (E2940, Madison, WI, USA) was used to measure firefly and renilla luciferase.
6
Assessing RIG-I-Mediated IFN-β Response
7
Luciferase Assay for BMP2 Signaling
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Luciferase reporter plasmids, along with Zfp423 or Satb2 expression plasmids, were cotransfected into HEK293 cells transfected using XtremeGene HP (Roche), and dual luciferase activity was assessed using a Dual-Glo assay system (Promega). Renilla luciferase (pRL-TK) (Promega) was used as an internal control for transfection efficiency. To examine BMP2 responses, cells were maintained in 0.5% FBS and treated with 100 ng/ml BMP2 (R&D Systems) or vehicle 16 h prior to assay.
8
Preparation and Characterization of Pancreatic Cell Lines
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Human pancreatic cancer cell lines MIAPaCa-2 and PANC-1 were obtained from ATCC. Cells were cryopreserved in liquid nitrogen upon receipt. Cells were maintained as previously described.12 (link),13 (link) The human pancreatic duct epithelial cell line (H6c7) was purchased (Kerafast) and cultured in Keratinocyte SFM with epidermal growth factor and bovine pituitary extract (Invitrogen) supplemented with 1× antibiotic–antimycotic (Gibco). Human Jurkat cells (gift from Dr. Jingfang Ju, Stony Brook University) was cultured in RPMI-1640 (Fisher Scientific) with 10% FBS and 1% P/S; and was used as PD-1 positive cell line control.
PDAC PDOs were maintained in standard fashion. L cells that produce Wnt3A (gift from Dr. Hans Clevers, Hubrecht Institute) were cultured in DMEM with 10% FBS and 1% P/S.14 (link) 293T cells that produce Rspo1 (Trevigen) were cultured in DMEM with 10% FBS and 1% P/S and changed to Advanced DMEM/F12 with 10% FBS and 1% P/S before collecting conditioning medium.15 (link) Activities of Wnt3A and Rspo1 were assessed by TopFlash assay using Dual-Glo Assay System (Promega).
PDAC PDOs were maintained in standard fashion. L cells that produce Wnt3A (gift from Dr. Hans Clevers, Hubrecht Institute) were cultured in DMEM with 10% FBS and 1% P/S.14 (link) 293T cells that produce Rspo1 (Trevigen) were cultured in DMEM with 10% FBS and 1% P/S and changed to Advanced DMEM/F12 with 10% FBS and 1% P/S before collecting conditioning medium.15 (link) Activities of Wnt3A and Rspo1 were assessed by TopFlash assay using Dual-Glo Assay System (Promega).
9
Comparative Analysis of Zebrafish PPARGC1a Isoforms
10
In vitro Translation Protocol
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In vitro translation was performed according to the protocol of Penzo et al. (32 (link)). In brief, rabbit reticulocyte lysate (Promega L4151) was depleted from ribosomes after mixing with an equal volume of cold 3.2 mM MgCl2 solution and 40%;v of 1.5 M sucrose/150 mM KCl solution and centrifugation at 140 000 g in a MLS50 rotor for 4 h (S140). The supernatant was complemented with hemin (Sigma Aldrich) (50 μM). To isolate translation-initiation factors, the pelleted ribosomes were suspended with salt-wash solution and adjusted to 0.5 M KCl by the stepwise addition of 4 M KCl and incubated on ice for 10 min. The ribosomes were pelleted by a 2 h centrifugation step at 170 000 g in a (TLA-100S/N 07U1825, Optima MAX) Rotor. The supernatant and the salt-washed fraction was aliquoted and frozen. For in vitro translation analysis, rabbit reticulocyte lysates (S140) and salt-wash fraction (SWF) were combined with ribosomes and mRNA. 300 ng RNA (WT/K > N mutant), 8.8 μl S140, 0.25 μl SWF and 2 μl TM buffer were mixed in a 1.5 mL tube. A total of 2 μl of the ribosomes were added with a concentration of 0.0625 pmol/μl, diluted with nuclease-free water. Samples were incubated at 30°C for 2.5 h on a thermomixer. Following incubation, they were transferred to a 96-well plate for luminescence measurements using Dual-Glo®Assay System (Promega).
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In vitro translation was performed according to the protocol of Penzo et al. (32 (link)). In brief, rabbit reticulocyte lysate (Promega L4151) was depleted from ribosomes after mixing with an equal volume of cold 3.2 mM MgCl2 solution and 40%;v of 1.5 M sucrose/150 mM KCl solution and centrifugation at 140 000 g in a MLS50 rotor for 4 h (S140). The supernatant was complemented with hemin (Sigma Aldrich) (50 μM). To isolate translation-initiation factors, the pelleted ribosomes were suspended with salt-wash solution and adjusted to 0.5 M KCl by the stepwise addition of 4 M KCl and incubated on ice for 10 min. The ribosomes were pelleted by a 2 h centrifugation step at 170 000 g in a (TLA-100S/N 07U1825, Optima MAX) Rotor. The supernatant and the salt-washed fraction was aliquoted and frozen. For in vitro translation analysis, rabbit reticulocyte lysates (S140) and salt-wash fraction (SWF) were combined with ribosomes and mRNA. 300 ng RNA (WT/K > N mutant), 8.8 μl S140, 0.25 μl SWF and 2 μl TM buffer were mixed in a 1.5 mL tube. A total of 2 μl of the ribosomes were added with a concentration of 0.0625 pmol/μl, diluted with nuclease-free water. Samples were incubated at 30°C for 2.5 h on a thermomixer. Following incubation, they were transferred to a 96-well plate for luminescence measurements using Dual-Glo®Assay System (Promega).
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