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Investigating the Role of Two-Pore Channel 2 (TPC2) in Zebrafish Neuromuscular Development
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Sarah E. Webb, Jeffrey J. Kelu, Andrew L. Miller
Once the transgenic lines were established, the trunk Ca2+ signals were characterized. Transgenic individuals were incubated with the apoaequorin co-factor, f-coelenterazine to reconstitute active holo-f-aequorin (Shimomura, 1991; Shimomura and Johnson, 1975, 1978). Various coelenterazine incubation protocols were tested but the optimal method proved to be incubation of embryos with 50 µM coelenterazine from ~1.25 hpf (i.e., the 8-cell stage) until ~16.5 hpf (i.e., the 16-somite stage) at which time Ca2+ imaging was initiated (Cheung et al., 2011). As coelenterazine itself generates luminescence due to a reaction with free radicals (Shimomura and Teranishi, 2000), embryos were washed with Danieau’s solution to remove the excess co-factor just prior to the start of Ca2+ measurements.
Molecular Imaging of Reporter Genes
Published in Michel M. J. Modo, Jeff W. M. Bulte, Molecular and Cellular MR Imaging, 2007
Keren Ziv, Dorit Granot, Vicki Plaks, Batya Cohen, Michal Neeman
Firefly Photinus pyralis luciferase and its substrate D-luciferin (a benzothiazole) are the most commonly used enzyme–substrate pair for in vivo imaging48,52–54 because of the long wavelength (562 nm) and high quantum yield. D-luciferin easily penetrates various organs and cell types upon systemic injection to catalyze reactions in live animals and is well tolerated even at high doses. More recently, imaging of bioluminescence in mice expressing Renilla reniformis after administration of coelenterazine has also been reported.55
Routine and Special Techniques in Toxicologic Pathology
Published in Pritam S. Sahota, James A. Popp, Jerry F. Hardisty, Chirukandath Gopinath, Page R. Bouchard, Toxicologic Pathology, 2018
Daniel J. Patrick, Matthew L. Renninger, Peter C. Mann
In vivo optical imaging includes fluorescence and bioluminescence imaging. Both techniques are highly sensitive (picomolar) at limited depths of a few millimeters; quick and easy to perform (with a high-throughput capability); and in general, do not require costly instrumentation. This makes them particularly suited to the drug development and validation process. Fluorescence imaging uses the ability of traditional or quantum dot fluorochromes (Papagiannaros et al. 2010) to absorb external excitation light of one wavelength and reemit light of a longer wavelength, which can be detected as discussed previously. In bioluminescence imaging, an enzyme (i.e., luciferase from the North American firefly Photinus pyralis or from the sea pansy Renilla) that is capable of generating light in the presence of a substrate (i.e., d-luciferin or coelenterazine, respectively) is used as a reporter to assess the transcriptional activity in cells that are transfected with a genetic construct containing the enzyme’s gene under the control of a promoter of interest. The enzymes can also be used to detect the level of cellular ATP (cell viability or kinase activity assays), tumor growth (Hawes and Reilly 2010), or other enzyme activity (i.e., caspase, cytochrome P450). Thus, the externally detected light is an indicator of biologic/molecular processes. The imaging process involves anesthesia of the animal, injection of the respective substrate, and placement of the animal in a dark chamber with a thermoelectrically cooled CCD camera, which is extremely sensitive to even weak luminescence. The light emitted can then be semiquantitatively analyzed. Disadvantages of optical imaging include low depth of penetration and limited clinical translation (Ying and Monticello 2006).
Advances in luminescence-based technologies for drug discovery
Published in Expert Opinion on Drug Discovery, 2023
Bolormaa Baljinnyam, Michael Ronzetti, Anton Simeonov
Coelenterazine reporters Renilla luciferase (RLuc) is another commonly deployed luciferase reporter derived from the octocoral, Renilla reniformis (Table 1). Interestingly, RLuc shares only minimal primary sequence homology with FLuc, and light production is independent of ATP cofactor [15,16]. RLuc produces luminescence through the oxidative decarboxylation of coelenterazine to coelenteramide, resulting in the concomitant emission of blue light [17].Gaussia luciferase (GLuc) is a smaller (20 kDa) secreted luminescent protein found in small crustaceans called copepods (Gaussia princeps). Unlike other coelenterazine-based reporters, GLuc requires the formation of disulfide bonds integral to the activity of the enzyme, precluding its use in systems that require reducing conditions [18,19]. Interestingly, GLuc, once secreted, has a considerably longer half-life (6 days) than either FLuc or RLuc [19].NanoLuc luciferase (NLuc) is an engineered version of shrimp Oplophorus gracilirostris luciferase. It reacts with furimazine, a coelenterazine derivative, with a specific activity over 150 times that of RLuc or FLuc [20]. In addition to NLuc’s enhanced brightness, it also has improved thermal stability, pH stability, and an unbiased distribution in cells as compared to other luciferases, leading to its rapid adoption into many HTS assays.
Extracellular esterase activity as an indicator of endoplasmic reticulum calcium depletion
Published in Biomarkers, 2018
Kathleen A. Trychta, Emily J. Heathward, Agnieszka Sulima, Susanne Bäck, Mehdi Farokhnia, Christopher T. Richie, Lorenzo Leggio, Kenner C. Rice, Brandon K. Harvey
Assays for Gaussia luciferase activity were performed as previously described (Henderson et al. 2014, 2015). For luciferase secretion assays, 5 μL of cell culture medium from each well was transferred to a new opaque walled plate. The GLuc substrate was 10 µM coelenterazine (Regis Technologies) in PBS. Coelenterazine stock solutions were prepared at 20 mM in acidified methanol (10 µL of 10 N HCl per 1 mL of methanol) and stored at –80 °C as single use aliquots. Luciferase levels were determined using a plate reader with an injector setup (BioTek Synergy II) that allowed samples to be read directly after injecting 100 µL of substrate into the well containing cell culture medium. For all secretion assays, vehicle controls were used in all experiments at concentration equivalent to the treatments. For luciferase assays involving rat plasma, 10 µL of plasma was transferred to an opaque walled plate and 100 µL of PBS containing 100 µM coelenterazine and 500 mM ascorbic acid was injected. Cell culture samples were read at 25 °C with a sensitivity of 100 and a 0.5 s integration time with a 5 s delay following coelenterazine substrate injection using a BioTek Synergy II plate reader. Rat plasma samples were read at 25 °C with a sensitivity of 150 and a 5 s integration time with a 5 s delay following coelenterazine substrate injection using a BioTek Synergy II plate reader.
A bispecific nanobody approach to leverage the potent and widely applicable tumor cytolytic capacity of Vγ9Vδ2-T cells
Published in OncoImmunology, 2018
Renée C. G. de Bruin, John P. Veluchamy, Sinéad M. Lougheed, Famke L. Schneiders, Silvia Lopez-Lastra, Roeland Lameris, Anita G. Stam, Zsolt Sebestyen, Jürgen Kuball, Carla F. M. Molthoff, Erik Hooijberg, Rob C. Roovers, James P. Di Santo, Paul M. P. van Bergen en Henegouwen, Henk M. W. Verheul, Tanja D. de Gruijl, Hans J. van der Vliet
Immunodeficient BRGS mice (BALB/c Rag2−/−Il2rg−/−SirpaNOD)72 were housed in isolators under pathogen-free conditions and randomly divided in 4 treatment groups (n = 6/group). At day 0, mice received an intravenous (i.v.) tail vein injection with 0.5*106 SW480Gluc cells. At days 1, 4 and 7 mice were treated with either a) 500 μg cetuximab i.p., b) 1*107 Vγ9Vδ2-T cells i.v., c) 1*107 Vγ9Vδ2-T cells in combination with 1 µg of the bispecific 7D12-5GS-6H4 VHH i.v., or d) an equal volume of sterile PBS i.v.. Mice that received Vγ9Vδ2-T cells were injected at days 1, 4, 7, 10 and 14 with 10,000 U human recombinant IL-2 i.p. to stimulate the proliferation of activated Vγ9Vδ2-T cells. Bioluminescence imaging (BLI) was performed at day 35 in 4 randomly selected mice from each study group. For this procedure, mice were anesthetized with inhalation anesthetics (isofluorane/oxygen) and injected i.v. retro-orbitally with 4 mg/kg coelenterazine (PJK GmbH, native-CTZ). BLI was recorded using an IVIS imaging system (PerkinElmer) and images were analysed using Living Image 4.0 software. BRGS mouse experiments were approved by the animal ethical committee of the Institut Pasteur (Reference # 2007–006), Paris, France, and validated by the French Ministry of Education and Research (Reference # 02162.01).