Coelenterazine sulfate was prepared by reacting Coelenterazine (Gold Biotechnology, #CZ10) with sulfur trioxide according to protocol adapted from Teranishi, K. 2002 [10 ]. Commercially available compounds were obtained from Sigma-Aldrich (unless specified otherwise) and used without further purification. Briefly, Coelenterazine (2.0 mg, 4.7 μmol) was dissolved in anhydrous pyridine (0.5 mL) and sulfur trioxide pyridine complex (4.0 mg, 25.1 μmol) was added. The reaction mixture sonicated in an ultrasonic bath at 20°C for 3 min (protected from light). The reaction completion was monitored by LC/MS on Agilent 6120 Single Quad System 1200 Series equipped with a Waters Atlantis T3 C18 column (2.1 x 150 mm, 5 μm particle size). After completion, the reaction mixture was poured into a 2 M ammonia solution in methanol (0.5 mL) at 0°C. The solvent was removed under vacuum at 20°C. The crude product was redissolved in 1:9 acetonitrile/water (2 mL) and purified by reversed-phase HPLC on an Agilent 1100 Preparative-scale Purification System equipped with a VYDAC 218TP series C18 polymeric reversed-phase column (22 x 250 mm, 10 μm particle size) at a flow rate of 20 mL/min and using an acetonitrile/water gradient of 5–95% over 40 min (absorbance was monitored at 263 and 433 nm). ESI-MS [M]−m/z 502.1 (calc. for C26H20N3O6S–m/z 502.1078). The yield after HPLC purification was 55% (Fig 4 ).
Coelenterazine
Manufactured by GoldBio
Sourced in United States
Coelenterazine is a bioluminescent compound commonly used as a substrate for luciferase reporter assays. It serves as a light-emitting cofactor in various luminescent systems, enabling the detection and quantification of cellular processes and gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Spark multimode microplate reader, by Tecan (2 mentions)
Costar 96 well white solid plate, by Corning (2 mentions)
Atlantis t3 c18 column, by Waters Corporation (1 mentions)
Photon imager, by Biospace (1 mentions)
Forskolin, by Merck Group (1 mentions)
Isoproterenol hydrochloride, by Merck Group (1 mentions)
5 n ethylcarboxamidoadenosine neca, by Merck Group (1 mentions)
D luciferin sodium salt, by GoldBio (1 mentions)
8 bromo camp, by Santa Cruz Biotechnology (1 mentions)
H 89 dihydrochloride hydrate, by Merck Group (1 mentions)
Centro xs3 lb 960, by Berthold Technologies (1 mentions)
Ivis lumina 2 in vivo imaging system, by PerkinElmer (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Glycerol, by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
White bottomed 96 well plate, by Corning (1 mentions)
Fluostar optima luminometer, by BMG Labtech (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Sodium ascorbate, by Merck Group (1 mentions)
Sodium iodide, by Merck Group (1 mentions)
17 protocols using coelenterazine
1
Synthesis of Coelenterazine Sulfate
2
Subcutaneous Implantation of 3D Printed Constructs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Control and gene activated 3D printed constructs were implanted subcutaneously into the back of nude mice (Balb/c; Harlan, United Kingdom) as previously described with two samples from the same group inserted in each of two pockets [26] (link). The constructs were harvested after three weeks for the in vivo luciferase expression study, and after four weeks for the therapeutic genes studies. Mice were euthanized by CO2 inhalation and the animal protocol was reviewed and approved by the Ethics Committee of Trinity College Dublin and the Health Products Regulatory Authority (HPRA). For the in vivo luciferase imaging, a protocol previously developed by Tannous et al (2009) was followed [27] (link). Briefly, previous to administration, coelenterazine (Gold Biotechnology, USA), prepared in acid methanol at a concentration of 5 mg/mL, was dissolved in PBS to a final concentration of 0.5 mg/mL under minimal light conditions, immediate after the mice were anesthetized using isoflurane, 50 μL of the coelenterazine mix were injected in each of the pockets. Immediately after coelenterazine injection, photon counts were acquired using a real-time bioluminescence imaging system (PhotonImager; Biospace lab, France) over five minutes.
3
Pharmacological Modulation of cAMP Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
(-)-Isoproterenol hydrochloride was purchased from Sigma-Aldrich (Cat #I6504), dissolved in water/100 mM ascorbic acid to 10 mM stock, and used at 1 μM final concentration. 5′-(N-Ethylcarboxamido)adenosine (NECA) was purchased from Sigma-Aldrich (Cat #119140), solubilized in DMSO to 10 mM stock, and used at 10 μM final concentration. Forskolin was purchased from Sigma-Aldrich (Cat #F6886), dissolved in DMSO to 10 mM stock, and used at 10 μM final concentration. Shield-1 ligand for stabilization of DD-tagged proteins was purchased from Takara Bio (Cat # 632189) and added to the cell medium to 1 μM final concentration. The cell-permeable cAMP analog, 8-Bromo-cAMP, was purchased from Santa Cruz Biotechnology (Cat #sc-201564), dissolved in DMSO and used at 1 mM final concentration within 1 month of re-suspension. The CREB inhibitor compound, 666–15, was purchased from Tocris Bioscience (Cat #5661), resuspended in DMSO and used at 100 nM final. At doses higher than 100 nM, 666–15 was toxic in our cell line. H89 dihydrochloride hydrate was purchased from Sigma-Aldrich (Cat #B1427), resuspended in DMSO to 10 mM stock, and used at 10 μM final concentration. D-luciferin sodium salt (Cat #LUCNA) and coelenterazine (Cat #CZ) were purchased from GoldBio and resuspended to 100 mM in 10 mM Hepes, and 10 mM in ethanol, respectively, and stored protected from light.
4
ER Microsome Fusion Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ER microsome fusion assays using isolated mammalian microsomes were performed as described for the ER microsome fusion assay using isolated yeast microsomes (Lee et al., 2015 (link); Lee et al., 2019 (link)) with modifications. The standard ER microsome fusion reactions contained 1 μg of Gluc1 microsomes, 1 μg of Gluc2 microsomes, reaction buffer (10 mM PIPES-KOH, pH 6.8, 5 mM MgCl2, and 200 mM sorbitol), an energy-regenerating system (0.5 mM MgCl2, 0.5 mg/ml creatine kinase, and 14.5 mM creatine phosphate), 10 μM coenzyme A, and 200 nM GST-ZIP. Fusion reactions were initiated by adding 1 mM GTP (Roche) and then incubated at 37°C. After 90 min, fusion reaction mixtures were mixed with coelenterazine (40 μM; GoldBio), and luminescence was measured using a luminometer (Centro XS3 LB 960; Berthold Technologies). ER microsome fusion assays using microsomes isolated from yeast cells expressing human atlastins were performed as previously described (Lee et al., 2015 (link); Lee et al., 2019 (link)).
5
Bioluminescence Imaging of Candida albicans in Wound
6
Dual Luciferase Reporter Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The supernatant was collected from triplicate wells of cells at the appropriate time points and 20 µl was transferred to 20 µl assay buffer (25 mM Tris Phosphate [pH7.8] containing 1% BSA and 30% glycerol, all Sigma‐Aldrich) in a white‐bottomed 96‐well plate (Corning, NY, USA) in technical triplicates. VLuc samples were assayed detecting photonic emissions at 460 nm after addition of 5 nM vargulin (Gold Biotechnology, Olivette, USA) and NLuc photonic emissions at 454 nm after addition of 2 mM coelenterazine (Gold Biotechnology) which had been incubated on ice for 45 minutes with 0.1 M KI and 10 mM EDTA (Sigma‐Aldrich) using a FLUOstar Optima luminometer (BMG Labtech, Ortenberg, Germany). NLuc values were divided by VLuc values before the average fold change over control ± standard deviation was plotted graphically.
p values were calculated using analysis of variance (One‐way ANOVA) followed by Bonferroni's multiple comparison post hoc test.
p values were calculated using analysis of variance (One‐way ANOVA) followed by Bonferroni's multiple comparison post hoc test.
7
Quantifying L1 Retrotransposition In Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To quantitatively assess retrotransposition activity in cells, 4T1-tetL1(GLucAI) were plated at 106 per 10-cm plate, and the expression of the integrated tetL1-GLucAI cassette was induced by the addition of doxycycline (Doxy) to a final concentration of 400 ng/mL in the culture medium. After 48 h, cells were collected for RNA isolation (SI Appendix, Materials and Methods ), and GLuc activity in the conditioned medium (indicative of retrotransposition) was measured using a microplate luminometer immediately following the addition of an equal volume of 2x GLuc reagent (50 μM coelenterazine (GoldBio) in D-PBS containing 300 mM sodium ascorbate (Sigma) and 0.2% Triton X-100 (Sigma)).
8
Bioluminescence Monitoring of Tumor Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Six-week-old BALB/c nude mice were purchased from the Model Animal Research Center of Nanjing University. All animal procedures were performed in accordance to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by NIH. BEL-7404 cells were infected with Lv-Gluc (Gauss Luciferase) to produce cell strain named BEL-7404 Gluc, which stably secreted Gluc outside the cells. BEL-7404-Gluc cells were infected with appropriate lentiviruses to produce BEL-7404 Gluc_Luc, BEL-7404 Gluc_miR-26a, BEL-7404 Gluc_shGFP and BEL-7404 Gluc_shITGA5. Treated cells were injected into BALB/c nude mice through the tail vein (1 × 106 cells/mouse, five mice/group). Half an hour after injection, blood samples were withdrawn by making a small incision in the tail of awake mice. Typically, 5 μl blood was added to 1 μl 20 mM EDTA and Gluc activity was measured using a TD 20/20 luminometer (Turner Design Inc., Sunnyvale, CA, USA) which was set to inject 100 μl 100 μM coelenterazine (GOLDBIO, St. Louis, MO, USA) in DMEM and to acquire photon counts for 10 sec. Blood samples were withdrawn and photon was monitored every 2 hours for a total period of 10 hours.
9
Gaussia Luciferase Neutralization Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gaussia luciferase (GLUC) assay buffer was prepared using 10 μM coelenterazine (Gold Bio), 50 mM Sodium Iodide (Sigma-Aldrich) in phosphate-buffered saline (PBS). 100 μL of culture supernatant was harvested from the co-cultured cells as described above and transferred into a white 96-well microplate to which 100 μL of the GLUC assay buffer was present. RLU was quantified on a Biotek plate reader. Data was analyzed with non-linear regression using GraphPad Prism to generate neutralization curves and IC-50 values.
10
SARS-CoV-2 Pseudovirus Neutralization Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The SARS-CoV-2 pseudovirus neutralization titer was determined using a lentiviral SARS-CoV-2 pseudotyped virus as described in a previous study (59 (link)). Specifically, 100 μl of virus was incubated with mouse or hamster serum solutions for 1 h at 37°C, and the mixture was added to HEK293T cells expressing ACE2 in 96-well plates. At 72 h postinfection, supernatants were collected from each well for measurement of luciferase activity. Briefly, 20 μl of supernatant was transferred to a new 96-well plate and mixed with 20 μl of Gluc substrate (0.1 M Tris [catalog no. T6066; Millipore Sigma] pH 7.4, 0.3 M sodium ascorbate [catalog no. S1349; Spectrum], and 10 μM coelenterazine [catalog no. CZ2.5; GoldBio]). Luminescence was immediately read by a plate reader. A nonlinear regression of x-y analyses was performed and fitted with an inhibition curve. The 50% inhibitory concentration (IC50) titer was calculated.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!