Adult E. scolopes were collected in shallow water off the shores of Oahu Hawaii and bred and maintained as previously described (Montgomery & McFall‐Ngai, 1993 ) in an aquatic facility containing recirculating artificial seawater at the University of Connecticut (Schleicher & Nyholm, 2011 ). Juvenile animals were colonized with V. fischeri ES114 by addition of 3,000 colony forming units per milliliter (CFUs/ml) to filter‐sterilized artificial seawater (FSASW) for up to 24 hr. Colonization by V. fischeri was determined by monitoring luminescence using a FB12 single tube luminometer (Titertek Berthold, Huntsville Alabama).
Fb12 single tube luminometer
Manufactured by Berthold Technologies
Sourced in United States
The FB12 single tube luminometer is a laboratory instrument designed to measure luminescence. It provides quantitative data on light emission from samples, enabling analysis of biochemical and biological processes.
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6 protocols using fb12 single tube luminometer
1
Breeding and Colonization of Squid
2
Extraction and Quantification of ATP and ADP
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The extraction of ATP and ADP was modified from Dordas et al. (2003) (link) as described earlier (Cochrane et al., 2017 (link)). Fresh leaf biomass (100 mg) was rapidly frozen in liquid nitrogen and homogenized in 1 mL of 2.4 M HCl using a mortar and pestle. The homogenate was then transferred to a microcentrifuge tube, neutralized using 5 M KOH and centrifuged (20,000 g, 10 min, 4°C). ATP and ADP in the supernatant were then measured using a luciferase-based bioluminescent assay (EnzyLightTM ADP/ATP ratio Assay Kit ELDT-100 by BioAssay Systems, CA, United States) and FB 12 Single Tube Luminometer by Titertek-Berthold (Berthold Detection Systems, GmbH, Germany).
3
NADPH-Driven ROS Production in Rat Aorta
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NADPH-dependent ROS production was measured in aortic rings from rats (n=6–8/group) as previously described [15] (link). Aortic rings were transferred to luminescence vials containing 1 ml of Hanks’ buffer, pH 7.2. After equilibration and background counts, a non-redox-cycling concentration of lucigenin (5 µM) and b-NADPH (12 µM) was automatically added and the luminescence counts were measured continuously for 15 min. in a Berthold (FB12 single-tube luminometer) at 37 °C. Background signals from the aortic rings were subtracted from the b-NADPH-driven signals and the results were normalized for the dry weight and reported as lucigenin chemiluminescence/mg of dry tissue.
4
Vascular ROS Formation Assay
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To further validate the possible effects of MMP-2 on vascular ROS formation, we carried out the lucigenin fluorescence assay as previously detailed [35] (link). Briefly, fresh intact aortic rings incubated with MMP-2 or vehicle in absence or presence of the MMP inhibitors doxycycline 100 µM or GM6001 1 µM. Aortic rings were transferred to luminescence vials containing 1 ml of Krebs buffer, pH 7.4. After equilibration and background counts, lucigenin (5 μM) and NADPH (300 μM) were added to the vials and the luminescence count was measured continuously for 15 min in a Berthold FB12 single tube luminometer at 37 °C. Background signals from aortic rings were subtracted from the NADPH-driven signals and the results were normalized for the dry weight and reported as RLU/mg of dry aorta.
5
Transient Transfection and Luciferase Assay
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For transient transfections, HEK 293T cells were transfected with CRE-RLuc along with Flag-EWSR1-ATF1 using Lipofectamine2000 (Life Technologies) following the manufacturer’s instructions. Twenty-four hours post transfection, the cells were lysed using Renilla luciferase lysis buffer, and the Renilla luciferase activity was measured using Renilla luciferase assay system (Promega) in FB12 single tube luminometer (Berthold). For reporter assay in DTC-1 cells with stable CRE-RLuc integration, the cells were treated with indicated drugs at different concentrations for 24 h. The remaining Renilla luciferase activity was measured as above. All the luciferase activity was normalized to protein concentration in each well and expressed as RLuc/μg protein.
6
Luciferase Assay for SNF2PH Depletion
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Luciferase assays were carried out using the Luciferase Assay System (Promega®) following the manufacturer's instructions from bloodstream form culture (3 × 106 cells) of control and SNF2PH depleted cells. Lectures were performed in a FB 12 Single Tube Luminometer (Titertek‐Berthold) with pre‐established parameters (2 s of delay time/10 s temp).
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