The identified KLF2-binding site in the Cxcr5 promotor (GAGGCAAGCA) was cloned into the pGL3 minimal promotor luciferase plasmid (Promega). HEK cells were cotransfected with pGL3, pcDNA3-Klf2, and pRL-TK Renilla luciferase plasmid by calcium phosphate precipitation. Luciferase activity was measured on an Orion L Microplate Luminometer (Berthold) after 24 h using the dual luciferase assay system (Promega). Data were normalized to the activity of Renilla luciferase.
Orion l microplate luminometer
Manufactured by Berthold Technologies
Sourced in Germany
The Orion L Microplate Luminometer is a laboratory instrument used for the measurement of luminescence in microplate samples. It is designed to detect and quantify the light emitted from various luminescence-based assays, such as those involving luciferase, aequorin, and other bioluminescent reporters. The Orion L provides precise and sensitive luminescence detection capabilities for a wide range of applications in life science research, drug discovery, and cellular analysis.
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Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Ros glo h2o2 assay kit, by Promega (2 mentions)
Bioluminescent somatic cell assay kit, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Dual luciferase assay system, by Promega (1 mentions)
Turbofect reagent, by Thermo Fisher Scientific (1 mentions)
Luciferase assay kit, by Berthold Technologies (1 mentions)
Cell culture lysis 5x reagent, by Promega (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay kit, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Luminol, by Merck Group (1 mentions)
Secrete pair dual luminescence assay kit, by GeneCopoeia (1 mentions)
Chenodeoxycholic acid cdca, by Merck Group (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Adp glo kinase assay kit, by Promega (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Luciferase assay system kit, by Promega (1 mentions)
Ros glo assay kit, by Promega (1 mentions)
14 protocols using orion l microplate luminometer
1
Assessing KLF2-Mediated Cxcr5 Regulation
2
Quantification of Abcb1 gene expression
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pGL3-Abcb1a-Luc and pGL3-Abcb1b-Luc reporter plasmids were transfected into D12, col500 and col1000 cells using TurboFect reagent (Fermentas, Maryland, USA) according to the manufacturer’s instructions. After a 24 h incubation, the media was removed and the cells were washed and harvested. Cells were resuspended in 1x lysis buffer (Promega, Cell Culture lysis Reagent 5x) and incubated on ice for 30 min. Next, the samples were centrifugated at 13,000 rpm at 4°C for 5 min, and the supernatants were collected and used to determine protein concentrations by the Bradford method and to perform luciferase enzyme assays. Luciferase activity in the samples was determined using the Promega luciferase assay kit and the Orion L Microplate Luminometer (Berthold Detection System, Simplicity 4.2 software). To determine transfection efficiency, pEGFP-N3 was co-transfected, and the number of GFP-positive cells were determined by FACS (FACSCalibur) using an FL-1 filter.
3
Investigating miR-27a's Impact on ESCC Proliferation
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To investigate the effect of miR-27a on cell proliferation, a comparison of the growth rates of ESCC cells transduced with miR-27a or miRNA-NC was performed. Cell growth was determined using a CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit (Promega Corporation). A total of ~5,000 cells were seeded in a 96-well plate 48 h post-transfection and incubated at 37°C for three days. Cell growth was then detected using a 3-(4,5-dimethylthiazol-2-yl) 5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) reduction cell proliferation assay kit every 24 h (0, 24, 48 and 72 h). Absorbance at a wavelength of 490 nm was determined using a microplate reader (OrionL Microplate Luminometer; Titertek-Berthold, Pforzheim, Germany). To investigate whether miR-27a suppresses tumor progression in vivo, TE-1 cells were transfected with pEGFP-miR-27a, and G418 was added to the medium and the cells were cultured for one month. The resultant TE-1 cells, which stably expressed miR-27a, were subcutaneously implanted into nude mice to generate tumor xenograft models. Four days after implantation, all of the animals in the control group developed palpable tumors, compared with the mice overexpressing miR-27a, which lacked any detectable tumors.
4
Hydrogen Peroxide ROS Assay in Cells
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Determination of ROS in cultured cells was performed by analysis of hydrogen peroxide (H2O2) formation. H2O2 production was measured with a homogenous bioluminescence ROS-Glo™H2O2 Assay Kit according to the manufacturer’s protocol (Promega). Briefly, cells were seeded in 96-well plates (5.000 cells/ well). ROS levels were determined at day 0 (two hrs after after the first CO exposure) and at day 6 in vitro (two days after the second CO exposure). The ROS-Glo™H2O2 Substrate was added during treatment (final concentration 25 μM), and the cells were incubated for an additional hours (37°C, CO2 incubator). After incubation, 50 μl medium from each well was transferred to 96-well plates. ROS-Glo™H2O2 Detection Solution was added (incubation for 20 min) before luminescence was determined using an Orion L Microplate Luminometer (Titertek Berthold). Luminescence signals were normalized to protein concentrations determined by the BCA Protein Assay Kit (Thermo Fisher Scientific).
5
Gck Promoter Luciferase Assay in Hepatocytes
6
Quantifying Neutrophil Oxidative Burst
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Primary human neutrophils were isolated and 2 x 105 cells/well were seeded in 100 µl HBSS into a FBS-coated, white 96-well cell culture plate (Costar, #3912). HBSS stimulation mixture containing 50 µM luminol (Sigma, 123072-5G) and particles {final concentration: 0.8 µg/µl silica, 1 x 106S. cerevisiae particles, 3 µl alhydrogel, and 1 µg/µl of all other crystals used [cholesterol, t-CPPD, m-CPPD, calcium oxalate mono- and dihydrate, calcium carbonate, MSU (lot1), MSU (lot2)]} was prepared. Then, 50 µl stimulus mixture were added to the cells and ROS production was immediately detected for 60 min using the Berthold Orion L Microplate Luminometer and Simplicity software 4.20 (https://www.berthold.com/en/ ).
7
Intracellular ATP Quantification Protocol
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The intracellular ATP level was measured by the Bioluminescent Somatic Cell Assay Kit (Sigma-Aldrich) using an OrionL Microplate Luminometer (Berthold, Bad Wildbad, Germany), as previously described (3 (link)). Luminescence intensity was divided by total cell number.
8
Hepatocyte miRNA Target Assay
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We transfected hepatocytes with Plasmids encoding miTarget™ 3′UTR miRNA Target Clones (1ug/5 × 105 cells), or miRNA mimics (50 nM), using Lipofectamine 2000 (Invitrogen). 48-hr after transfection, we assayed luciferase with Secrete-Pair™ Dual Luminescence Assay Kit (GeneCopoeia) in an Orion L Microplate Luminometer (Berthold).
9
Evaluating FXR Isoform Activity in Hepatic Cells
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Twelve-well plates were seeded with human (HepG2 or Huh-7) or mouse (Hepa 1-6) hepatic cells on day 0 with 5 × 105 cells/well and co-transfected the next day with 0.5 µg/well of the plasmid expressing luciferase to be tested and 0.1 µg/well of the plasmid expressing the selected FXR isoform using lipofectamine 2000 (Thermo Fisher, Waltham, Massachusetts) at a DNA/lipofectamine ratio of 1:3. To control the transfection efficiency, cells were co-transfected with 0.1 µg/well of a plasmid expressing Renilla-luciferase from a constitutive promoter (pRL-CMV, AF025843). At 24 h post-transfection, cells were incubated with 30 µM (human cells) and 100 µM (mouse cells) chenodeoxycholic acid (CDCA, Sigma-Aldrich, St. Louis, MO), or 0.3% and 1.0% DMSO (Sigma-Aldrich) as controls, for 30 h. The amount of CDCA used to induce each specific cell line was previously optimized (data not shown). The expression of the Renilla/firefly luciferase system was determined from cell lysates using a non-commercial dual luciferase enzyme assay as described Dyer et al. [28 (link)] and the results were measured with an Orion L Microplate Luminometer (Berthold Technologies, Germany).
10
YBX1 CSD Kinase Activity Assay
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A purified YBX1 CSD domain was purchased from ProteinGene Biotech. Kinase assays for AKT/RSK and YBX1 CSD were performed using a luminometric kinase assay with ADP-Glo Kinase Assay Kit (Promega). The AKT/RSK kinase was purchased from Promega Corperation. HOTAIR RNA was added to the reaction to check whether it can enhance the Kinase reaction, with Antisense, HD234 and HD34 used as negative control. In brief, the RNA was denatured at 65°C for 5 min and cooled to room temperature in the presence of RNA structure buffer. AKT/RSK and YBX1 CSD at optimal concentrations were incubated with HOTAIR, Antisense, HD234, or HD34 of equal molar concentration, in the presence of ATP. The kinase assay was performed at room temperature for 60 min. Then ADP-Glo Reagent was added to the reaction, and incubated for 40 min at room temperature. Finally, the Kinase Detection Reagent was added and the reaction mixture was incubated for another 40 min at ambient temperature. Then luminescence was recorded on an Orion L Microplate Luminometer (Berthold Technologies).
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