The luciferase reporter plasmids pGL-NF-κB-Luc (10 μg) and pGL-TK-Luc (10 μg) (Promega) were electroporated (250 V, 950 μF) into Jurkat cells (5 × 106). After 48 h, cells were collected and stimulated with murine recombinant TNFα (20 ng/ml, R&D Systems) or anti-CD3ε/CD28 antibodies (1 μg/ml, BioLegend) for the indicated times. Next, the cells were lysed and ligand-dependent NF-κB activity was measured (in terms of luciferase activity) using the Dual-Luciferase Reporter or Bright-Glo luciferase assay system (Promega), according to the manufacturer’s protocol. Luminescence was detected in a Lumat luminometer (Berthold).
Lumat luminometer
Manufactured by Berthold Technologies
Sourced in Germany
The Lumat luminometer is a high-performance instrument designed for the detection and measurement of luminescence signals. It is a versatile tool suitable for a wide range of applications in life science research and analytical laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (5 mentions)
Hilymax, by Dojindo Laboratories (2 mentions)
Passive lysis buffer (plb), by Promega (2 mentions)
Dual luciferase reporter (dlr), by Promega (1 mentions)
Pgl nf κb luc, by Promega (1 mentions)
Recombinant murine tnfα, by R&D Systems (1 mentions)
Bright glo luciferase assay system, by Promega (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay, by Promega (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Pgl3 basic, by Promega (1 mentions)
Thiazolyl blue tetrazolium bromide, by Merck Group (1 mentions)
Caspase glo 3 7 reagent, by Promega (1 mentions)
Cell culture lysis reagent, by Promega (1 mentions)
Lipofectin, by Thermo Fisher Scientific (1 mentions)
T7 ribomax express large scale rna production system, by Promega (1 mentions)
Luciferase assay system, by Promega (1 mentions)
Golgiplug, by BD (1 mentions)
Pgl4.74 hrluc tk, by Promega (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
11 protocols using lumat luminometer
1
NF-κB Transcriptional Activity Assay
2
Mapping GLI1 Binding Sites via Luciferase Assay
3
Deletion Analysis of 2kb Promoter
4
Assessing AGE-Induced Cell Death
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To examine whether AGE treatment induces cell death and/or apoptosis, we treated NRCMs with 1 mg·mL−1 AGE for 24 h and performed MTT and caspase 3/7 assays. For the MTT assay, NRCMs were plated in 24‐well plates at a density of 3 × 105 cells per well. Following 24‐h treatment with AGE, 0.45 mg·mL−1 thiazolyl blue tetrazolium bromide (Sigma) was added to each well and cells were incubated for 1 h at 37 °C. Formazan crystals were then dissolved by adding 100 μL of a solubilization solution containing 0.1N HCl in isopropanol, and cell viability was analysed by measuring the absorbance at 570 nm on a spectrophotometer.
For analysing caspase 3/7 activity, cells were plated at a density of 3 × 105 and treated with AGE for 24 h. Following treatment, cells were lysed for 30 min through the addition of 100 μL of cell culture lysis reagent (Promega, Madison, WI, USA). To assess caspase activity, 20 μL of lysate was mixed with an equal volume of caspase‐Glo 3/7 reagent (Promega) and incubated for 1 h at room temperature. Luminescence was measured using a Lumat luminometer (Berthold Technologies, Bad Wildbad, Germany).
For analysing caspase 3/7 activity, cells were plated at a density of 3 × 105 and treated with AGE for 24 h. Following treatment, cells were lysed for 30 min through the addition of 100 μL of cell culture lysis reagent (Promega, Madison, WI, USA). To assess caspase activity, 20 μL of lysate was mixed with an equal volume of caspase‐Glo 3/7 reagent (Promega) and incubated for 1 h at room temperature. Luminescence was measured using a Lumat luminometer (Berthold Technologies, Bad Wildbad, Germany).
5
Mapping GLI1 Binding Sites via Luciferase Assay
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Full-length human GLI1 intron (chr12:57,460,202-57,463,664; assembly hg38 from Dec. 2013) was cloned from HeLa cell lysate and ligated into pGL3 luciferase expression vectors using Gibson Assembly (NEB). Potential GLI binding sites (8/9 consensus bases) were identified using MacVector and deleted individually or in groups via inverse PCR. For luciferase assays, plasmids were cotransfected into HeLa cell culture using HilyMax (Dojindo) reagent. 30 h post transfection, cells were lysed and relative luciferase and renilla signal was detected using the Dual-Luciferase Reporter Assay system (Promega) with a Lumat luminometer (Berthold). After subtracting baseline noise and normalizing signal against renilla activity, statistical significance was determined by ANOVA (R).
Deletion primers:
Deletion primers:
6
Replicon RNA Transfection Assay
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Replicon RNA, an in vitro transcript from full-length AiV cDNA in which the capsid-coding region is replaced by a firefly luciferase gene, was synthesized using T7 RiboMAX express large scale RNA production system (Promega) as described previously [39 (link),40 (link)]. Cells were all prepared in 48 well-plates and 125 ng/well of replicon RNA was transfected using Lipofectin (Invitrogen) according to the manufacturer’s protocol. At the indicated time points after transfection, cell lysates in 1× Passive lysis buffer (Promega) were harvested, and luciferase activity was measured using a Luciferase assay system (Promega) with Lumat luminometer (Berthold).
7
Evaluating Transfection Efficiency in Cells
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HEK293T and 786-O cells (ATCC, Manassas, VA, USA) were seeded overnight in a 24-well plate at a density of 1 × 104 cells/well and transfected with liposomes (8 nM lipids/µg DNA) with 1 µg of total DNA/well. After 24 h incubation, the cells were washed with DMEM medium, trypsinized and centrifuged for 5 min at 1500 rpm. The pellets were resuspended in PBS and analyzed for GFP expression by flow cytometry (Guava easycyte). In vivo assays were done by intraperitoneal inoculation of 10 µg pcDNA3-GFP (8 nM lipids per µg DNA). After 24 h, the peritoneal region from euthanized mice was washed with PBS and the cells were recovered and analyzed by flow cytometry. The samples checked for luciferase expression were treated with Brefeldin A (BD GolgiPlug™), according to manufacturer’s instructions, 6–8 h before every analysis. The cells were pelleted at 14,000 rpm for 20 s and resuspended in Luciferase Assay Substrate and Buffer, according to the manufacturer’s instructions (Promega), before analysis in a Berthold Lumat luminometer.
8
PPARα Transcriptional Activity Assay
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The CV-1 cells were plated on 24-well cell culture plates at a concentration of 5 × 104 cells/well for 24 h. The cells were then transiently co-transfected with 0.2 µg of Cignal PPAR Reporter construct (QIAGEN, Hilden, Germany) and 0.4 µg of pcDNA3 WT PPARα or PPARα variant (His117Gln, Arg141Cys, Arg226Trp, Cys122Ser, Arg128Thr) constructs or empty expression plasmid. The Cignal PPAR Reporter comprised an inducible PPAR-responsive firefly luciferase reporter and a constitutively-expressed Renilla luciferase construct (40:1). At 24 h post-transfection, the cells were lysed using Passive Lysis Buffer (Promega). The firefly luciferase activity relative to the Renilla luciferase activity was examined to determine the PPARα activity using the dual-luciferase reporter assay system (Promega) and Lumat luminometer (Berthold Technologies, Bad Wildbad, Germany). All the luciferase assays were performed at least in triplicate.
9
TCF-Luciferase Assay for ROCK2 Kinase
10
Measuring NF-κB Activation in HEK293T Cells
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To measure NF-κB activation, HEK293T cells were co-transfected with pGL4.32 (Luc2p/NF-κB-RE/Hygro) and pGL4.74 (hRLuc/TK) (Promega), together with various expression plasmids. At 24 h after transfection, cells were lysed, and luciferase activity was measured by a Lumat Luminometer (Berthold) using the Dual-Luciferase reporter assay system (Promega).
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