For FXR and GP BAR1 mediated transactivations, HepG2 cells and HEK293T cells were transfected as described previously25 (link). At 24 h post-transfection, cells were stimulated 18 h with 10 μM CDCA (1 ), TLCA (2 ), 6-ECDCA (3 ) and compounds 7–19 . After treatments, 20 μL of cellular lysates were read using Dual Luciferase Reporter Assay System (Promega Italia s.r.l., Milan, Italy) according manufacturer specifications using the Glomax 20/20 luminometer (Promega Italia s.r.l., Milan, Italy). To evaluate GPBAR1 mediated transactivation, HEK-293T cells were transfected with 200 ng of human pGL4.29 (Promega), a reporter vector containing a cAMP response element (CRE) that drives the transcription of the luciferase reporter gene luc2P, with 100 ng of pCMVSPORT6-human GPBAR1, and with 100 ng of pGL4.70. Dose-response curves were performed in HepG2 and HEK-293T cells transfected as described above and then treated with increasing concentrations of compounds 7 (1– 10 μM), 13 , 14 and 19 (100 nM–25 μM). At 18 h post stimulations, cellular lysates were assayed for luciferase and Renilla activities using the Dual-Luciferase Reporter assay system (E1980, Promega). Luminescence was measured using Glomax 20/20 luminometer (Promega). Luciferase activities were normalized with Renilla activities.
Glomax 20 20 luminometer
Manufactured by Promega
Sourced in United States, China, Italy, Germany, United Kingdom, Japan, Canada
The GloMax 20/20 luminometer is a compact and versatile instrument designed for the detection and quantification of luminescent signals. It is capable of measuring a wide range of luminescent assays, including those involving luciferase-based reporter systems. The GloMax 20/20 provides accurate and reliable data for various applications in life science research and molecular biology.
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Lab products found in correlation
Dual luciferase reporter assay system, by Promega (261 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (148 mentions)
Passive lysis buffer (plb), by Promega (68 mentions)
Dual luciferase reporter assay kit, by Promega (55 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (39 mentions)
Dual glo luciferase assay system, by Promega (33 mentions)
Luciferase assay system, by Promega (28 mentions)
Dual luciferase assay kit, by Promega (26 mentions)
Dual luciferase reporter assay, by Promega (25 mentions)
Pgl3 basic vector, by Promega (19 mentions)
Prl tk, by Promega (18 mentions)
Pmir reporter, by Huayueyang (13 mentions)
Pmirglo vector, by Promega (13 mentions)
Pmirglo dual luciferase mirna target expression vector, by Promega (12 mentions)
Pmir report luciferase reporter plasmid, by Thermo Fisher Scientific (12 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (11 mentions)
Dual luciferase assay system, by Promega (11 mentions)
Reporter lysis buffer, by Promega (11 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (11 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (11 mentions)
1 046 protocols using glomax 20 20 luminometer
1
FXR and GPBAR1 Transcriptional Assays
2
PPARγ Activation and Transcriptional Regulation
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The plasmids encoding PPARγ and PPARγ-Luc were kindly provided by Dr. Jae Bum Kim of Seoul National University. The control plasmid encoding Renilla luciferase and the Dual-luciferase assay kit were purchased from Promega (www.promega.com ). Because CHO cells do not express PPARγ, a mixture containing the PPARγ, RXRα, PPARγ-Luc, and Renilla-Luc plasmids were co-transfected into the CHO cells using the Lipofectamine 2000 reagent (Invitrogen, USA) according to the manufacturer's instruction. After transfection, the cells were treated with 100 µM rosiglitazone (PPARγ agonist) in the absence or presence of CCC for 24 h. The post-transfection luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI) using a GloMax20/20 luminometer (Turner Biosystems, Sunnyvale, CA). The results were normalized to the Renilla luciferase activity.
3
Dual Luciferase Assay Protocol
4
Elucidating TFEB-mediated Dusp-1 Regulation
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Cells were seeded in 24-well plates at a density of 4 × 104 cells per well. After ChIP-seq analysis [59 (link)] the relative TFEB MACS peak on the promoter gene was identified in D4M cells that were transfected with a pMCS-GreenRenillaLuc_Dusp-1_full promoter, pMCS-GreenRenillaLuc_Dusp-1_promoter_dell1 (Del1) (in which the putative TFEB-binding site had been deleted), or pMCS-GreenRenillaLuc_Dusp-1_promoter _dell2 (Del2) (in which 100 bp before and after the same putative TFEB-binding site had been deleted) synthesised with GeneArt support (Thermo Fisher Scientific) using Lipofectamine Reagent according to the manufacturer’s instructions.
Luciferase activity was analysed with a Pierce Renilla Luciferase Glow Assay Kit or the Dual-Luciferase® Reporter (DLR™) Assay System using a GloMAX 20/20 luminometer (Turner Biosystems, Sunnyvale, CA, USA). The relative reporter activity was calculated by normalising the luciferase activity to the Renilla luciferase activity.
Luciferase activity was analysed with a Pierce Renilla Luciferase Glow Assay Kit or the Dual-Luciferase® Reporter (DLR™) Assay System using a GloMAX 20/20 luminometer (Turner Biosystems, Sunnyvale, CA, USA). The relative reporter activity was calculated by normalising the luciferase activity to the Renilla luciferase activity.
5
Tgif1 Promoter Luciferase Assay
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The mouse Tgif1 promoter sequence (pTGIF1-full) and the promoter with a deleted TFEB-binding region (pTGIF1-del) were cloned into the luciferase-expressing vector pGL4-luc2P-Hygro (Promega). The Tgif1 promoter was defined as a sequence (−1106; +310) from the TSS of Tgif1 transcript variant 1. The TFEB binding peak according to ChIP-seq data is situated at −438 and +187. The central 321 bp of the peak containing a possible TFEB-binding motif agcatgtgag according to the Jaspar algorithm (score 9.3) was deleted in the pTGIF1-del construct. pTGIF1-full and pTGIF1-del were electroporated into MEC-TFEBS142A cells with an Amaxa electroporator (Lonza), and the cells were selected with 0.5 mg/ml hygromycin. Cells were seeded in 96-well plates (5000 cells per well) and stimulated with doxycycline for the indicated time intervals, and luciferase activity was analyzed with a Luciferase Assay System Kit (Promega) using a Glomax 20/20 luminometer (Turner Biosystems, Sunnyvale, CA, USA). The relative reporter activity was calculated by normalizing the luciferase activity in doxycycline-treated cells to that in untreated cells.
6
Dual Luciferase Assay Protocol
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Dual measurement of FLuc and coelenterazine-utilizing luciferases was performed either with the Dual Luciferase Assay (Promega) or with homemade reagents as described13 (link). Single measurement of GLucSDEL was performed by adding one volume of sample to ten volumes of assay buffer (0.5× phosphate-buffered saline, 0.025% Nonidet P-40, 1 µg/mL coelenterazine). Luciferase intensity was measured on a GloMax 20/20 luminometer (Turner Biosystems) or a Spectramax L plate reader (Molecular Devices). Minimum signals obtained using transient and stably transfected lines were ≥ 5-fold and ≥103-fold above background, respectively, and within instrumentation linear response ranges.
7
Transcriptional Regulation of CDK4 and MYO1C
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Cells were seeded in 24‐well plates at a density of 4 × 104 cells per well. After ChIP‐seq analysis, to identified the relative TFEB MACS peak on the different promoter gene, ECs were transfected with CDK4‐luciferase full‐length promoter (from J. Modiano, Addgene plasmid #86656) and CDK4‐luciferase fragment A (from J. Modiano, Addgene plasmid #86658) in which the predicted ChIP‐seq TFEB peak near TSS on CDK4 promoter is deleted. ECs were transfected with pMCS‐CypridiLuc_AC167_MYO1C_promoter (sequences −1,500/+200 from TSS of the longest isoform of MYO1C gene according to Ensembl GRCh37, transcript ID: ENST00000359786), pMCS‐CypridiLuc_AC167_MYO1C_promoter_d1 (in which the putative TFEB binding site has been deleted), pMCS‐CypridiLuc_AC167_MYO1C_promoter_d2 (in which 100 bps around the same putative TFEB binding site has been deleted) synthesized Geneart support (Thermo Fisher Scientific) and with pMCV‐GreenReLuc using Lipofectamine® RNAiMAX Reagent according to the manufacturer's instructions.
Luciferase activities were analyzed with Pierce Cypridina Luciferase Glow Assay Kit and Pierce Renilla Luciferase Glow Assay Kit or The Dual‐Luciferase® Reporter (DLR™) Assay System using a Glomax 20/20 luminometer (Turner Biosystems, Sunnyvale, CA, USA). The relative reporter activity was calculated by normalizing the luciferase activity with Renilla luciferase activity.
Luciferase activities were analyzed with Pierce Cypridina Luciferase Glow Assay Kit and Pierce Renilla Luciferase Glow Assay Kit or The Dual‐Luciferase® Reporter (DLR™) Assay System using a Glomax 20/20 luminometer (Turner Biosystems, Sunnyvale, CA, USA). The relative reporter activity was calculated by normalizing the luciferase activity with Renilla luciferase activity.
8
Synchronized Parasite Life Cycle Profiling
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Ring-stage parasites were tightly synchronized using 0.3 M alanine for three consecutive IDCs before induction. Synchronized parasites were induced using 0.5 μM aTc at ring-stage and allowed to go through a complete IDC before collecting ring-, trophozoite- and schizont-stage samples for luciferase assays. Firefly and RLuc measurements were made with the Dual Luciferase Assay kit (Promega) on a GloMax 20/20 luminometer (Turner Biosystems).
9
Optimized ATP Quantification Protocol
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ATP was measured using the BacTiter-GloTM Microbial Cell Viability Assay (G8231, Promega Corporation, Dübendorf, CH) and a GloMax 20/20 Luminometer (Turner BioSystems, Sunnyvale, CA, USA). We applied an optimized protocol as described by Hammes et al.15 (link), comparing with the manufacturer’s protocol for this product. Total ATP was measured as follows: 2 mL of the supernatant for each sample was transferred into a 2 mL Eppendorf tube and 50 μl of the ATP reagent was transferred to a separate sterile 1.5 mL reaction tube (Axygen). Both sample and reagent were heated for at least 1 min in a heating block at 38 °C. Then, 500 μl of the sample was transferred to the 50 μl reagent, and the mixture was incubated for a further 20 s in the heating block (38 °C). The luminescence was subsequently measured as an integral over 10 s, expressed in relative light units (RLU). The RLU values were converted to ATP concentrations using a calibration curve (R2 > 0.99), which was prepared with pure ATP standard (10 mM; Promega Corporation) diluted in ATP free, sterile water (Milli-Q) to different concentrations (10−2–104 nM). A conversion factor of 1.75 × 10−10 nmol ATP per cell was applied to calculate the bacterial cell number15 (link). All ATP measurements were done in triplicate, and all procedures were conducted under a sterile condition.
10
Quantifying Microbial Abundance in Bioelectrochemical Systems
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The scanning electron microscope (SEM) was observed by the field-emission SEM (FE-SEM), which was made by Hitachi, Japan (model: SU8020). Adenosine triphosphate (ATP) was determined by the BacTiter-GloTM Microbial Cell Viability Assay (G8231, Promega Corporation, Dübendorf, CH) and a GloMax 20/20 Luminometer (Turner BioSystems, Sunnyvale, CA, USA). We followed the description of the manufacturer's protocol for this product. We collected 2 ml of the supernatant from each reactor, and 50 µl ATP reagent to prepare to heat at 38°C for 1 min. We mixed final 500 µl sample with 50 µl ATP reagent to settle in the heating block for 20 s at 38°C. The luminescence was subsequently measured, which was expressed in relative light units (RLU). We used conversion factor of 1.75 × 10−10 nmol ATP per cell to transfer the RLU to the number of cell. Quantitative PCR (qPCR) was employed to analyse the abundances of total archaea, total bacteria, Methanobacteriales, Methanomicrobiales, Methanosarcinaceae, and Methanosaetaceae for four samples collected from cathode at low and high current respectively [22 ]. All primers and the process description were shown in the electronic supplementary material, table S1.
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