The cells were harvested at indicated timepoints after transfection and/or stimulation and lysed with 25 ul of 1 × Passive Lysis buffer (Promega). Firefly luciferase and Renilla luciferase expression were measured using the dual luciferase assay (Promega) on an Orion II microplate reader (Berthold Technologies). Relative luciferase units (RLU) were calculated by normalizing each sample’s firefly luciferase activity to the constitutive Renilla luciferase activity determined in the same sample.
Orion 2
Manufactured by Berthold Technologies
Sourced in Germany
The Orion II is a multipurpose laboratory instrument designed for a variety of analytical applications. It features advanced electronics and a digital display for precise measurements and data handling.
Automatically generated - may contain errors
Lab products found in correlation
Passive lysis buffer (plb), by Promega (5 mentions)
Dual luciferase assay, by Promega (3 mentions)
Dual luciferase reporter assay system, by Promega (3 mentions)
Prism 8, by GraphPad (2 mentions)
Metafectene pro, by Biontex (2 mentions)
96 well opaque white plate, by Thermo Fisher Scientific (2 mentions)
Celltiter glo luminescent cell viability assay, by Promega (2 mentions)
Reporter lysis buffer, by Promega (2 mentions)
Methanol, by Merck Group (1 mentions)
Atp determination kit, by Thermo Fisher Scientific (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Atp assay kit, by Beyotime (1 mentions)
Adp atp assay kit, by Merck Group (1 mentions)
β gal reporter gene assay, by Roche (1 mentions)
Mithras lb 940, by Berthold Technologies (1 mentions)
Opaque 96 well plate, by Corning (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Native coelenterazine, by Merck Group (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Costar white 96 well plates, by Corning (1 mentions)
21 protocols using orion 2
1
Dual Luciferase Assay Protocol
2
Quantifying ATP/ADP Ratio in Trypanosoma
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Changes in the ATP/ADP ratio and total cellular ATP were determined in BSF T. brucei cells using the d-luciferin-luciferase bioluminescent enzymatic reaction (assay kit Sigma MAK135) according to manufacturer’s instructions. Briefly, from each sample 1 × 106 cells were harvested and washed once with PBS supplemented with 6 mM glucose (PBS-G). Pellets were resuspended in PBS-G and the mixture transferred into a microtiter plate (96-well white flat-bottom). The bioluminescence signal was recorded in an Orion II microplate luminometer (Titertek Berthold) and analyzed using Prism (8.0) (GraphPad Software).
3
Luciferase Assay for PPARγ Binding
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To test the direct binding of PPARγ to its putative target regions downstream the Pex16-transcription start site (TSS) a luciferase reporter assay was performed. Target sequences R1 (1052–1676 bp downstream Pex16-TSS) and R2 (2160–2554 bp downstream Pex16-TSS) were cloned into luc2- luciferase reporter vector pGL4.26 (Promega). Cos7 cells were transfected with MetafectenePro (Biontex Laboratories GmbH) in 24-well plates according to the manufacturer’s protocol at a ratio of MetafectenePro to DNA 2.5:1 (μl:μg). 200 ng of pGL4.26-R1/R2 or empty vector were co-transfected with 100 ng/well pCMX-PPARγ2 and 100 ng/well pCMX-RXRα expression vectors. In controls, 200 ng of empty pCMX vector were co-transfected. Co-transfection of renilla luciferase reporter vector pGL4.75 in a ratio of 1:50 to the luc2-reporter vectors served as control for varying transfection efficiencies. Luciferase reporter assays were performed 48 h after transfection using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol. Luminescence measurement was done using “Berthold Orion II” luminometer. Luciferase activity was normalized to renilla luciferase activity.
4
Measuring Gaussia Luciferase Activity
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To measure the GLuc activity in infected cell cultures, we used a microplate luminometer (Orion II; Berthold) with an injector system. Fifty microliters of culture supernatant was plated into a 96-well opaque white plate (Nunc) and, by using the injector, was mixed with 50 μl 1-μg/ml native coelenterazine (a 1-mg/ml stock in methanol [Sigma] diluted in PBS). Enzymatic activity was measured in the well mode with the following sequence: injection (50 μl), with no shaking, delay for 0 s, measurement at 10 s. The values were analyzed with GraphPad Prism (version 5.0) software after using the remove baseline function.
5
Measuring Cell Viability under ER Stress
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Cell viability was measured with an ATP Determination Kit (Invitrogen™, cat. no. A22066). Cells were seeded on a 96 well plate. After 24 h, cells were transfected with PEI. To induce ER stress, cells were treated with 500 nM Thapsigargin. After 48 h, cells were lysed and ATP was determined according to the manufacturer’s instructions. Firefly luciferase activity was measured using an Orion II microplate reader (Berthold Technologies) and normalized cell viability was calculated by dividing the measured luciferase activity with the luciferase activity of cells treated with DMSO.
6
Evaluating TP73 Transactivation Activity
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U-2 OS cells were plated in 48-well plates and grown overnight under standard conditions. Cells were then transiently co-transfected with Renilla luciferase reporter plasmid (pLightSwitch; Switch Gear Genomics) and TP73-expressing plasmids using FuGENE HD Transfection reagent (Promega) following manufacturer′s instructions. For normalization, an empty vector pLightSwitch (Switch Gear Genomics) was used. Twenty-four hours after transfection, cells were lysed in Passive Lysis Buffer (Promega). Luciferase activities were measured in cell lysates using Renilla Juice Luciferase Assay Kits and coelenterazine as substrate (PJK). Luminescent signal was measured on a microplate reader luminometer Orion II (Titertek-Berthold).
7
Functional Aequorin Biosensor Reconstitution
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A stock of f-coelenterazine (Biotium, Fremont, CA, USA) was prepared at 5 mM in methanol, 5 μL aliquots were prepared and frozen at −80 °C. To reconstitute functional aequorin, embryos injected with the mRNA of each biosensor were dechorionated at 4 hpf, transferred to a 24-well plate coated with agarose, and incubated (10 embryos per well) with 50 μM f-coelenterazine in E3 medium (28.5 °C in darkness) for 3–23 h, as indicated. To quantitatively determine the functional aequorin at different time points, total light emission from embryos of 7, 10 and 27 hpf was measured. Embryos were transferred to a 96-well opaque plate (1 embryo/well) in a microplate luminometer equipped with automatic injectors (Orion II, Titertek Berthold, Pforzheim, Germany). A Fast Kinetics protocol was designed with a total reading time of 1 min per well (1.2 s of light signal integration). An injection volume of 50 µL 0.5% Triton X-100 was added per well (0.25% final concentration) to trigger the release of luminescence.
8
Intracellular ATP Quantification Protocol
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The intra-cellular ATP level was measured by ATP Assay Kit from Beyotime Biotechnology. MDA-MB-231 cells were collected and then lyzed by ATP releasing buffer on ice. According to the protocols, the standard curve of the absorbance to ATP concentrations was measured. We may then determine the ATP concentration using luminometer Orion II (Berthold DS, Bleichstr, Pforzheim, Germany).
9
Quantifying ADP/ATP Ratio and ATP Levels
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Both the ADP/ATP ratio and the total cellular ATP were estimated using the bioluminescence-based ADP/ATP assay kit (Sigma MAK135) following the manufacturer's protocol. In brief, 1 × 106 cells per sample were harvested and washed once with PBS-G (1× PBS plus 6-mM glucose). Cells were resuspended in 10 μl of 1× PBS-G and transferred into a white flat-bottom 96-well microtiter plate. Luminescence was recorded in an Orion II microplate luminometer (Titertek-Berthold). The ATP levels and calculated ADP/ATP ratios of RNAi-induced/tetracycline-depleted cells were normalized to those of control cells and expressed in percentage. The values were plotted and analyzed statistically using GraphPad Prism 8.0 software.
10
Luciferase Assay for TLR2 Ligand Activity
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Luciferase assaying for analysis of TLR2 ligand activity has been described earlier [3 (link)]. In short, TLR2 expression and luciferase reporter plasmids were transiently transfected into largely TLR deficient human embryonic kidney fibroblastoid HEK293 cells to assess TLR2 specific P/DAMP driven NF-κB activation [17 (link)]. Twenty μl of blood plasma in 80 ml RMPI 1640 was added to individual wells of 96 well cell culture plates, which were incubated at standard incubation conditions for 16 hours upon which cells were lysed. Reporting and control luciferase activities in cellular lysates were measured with a 96-well plate luminescence reader (Orion II, Titertek-Berthold, Bad Wildbad, Germany).
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