Coelenterazine

Manufactured by Santa Cruz Biotechnology

Coelenterazine is a chemiluminescent substrate used in bioluminescence assays. It is a naturally occurring compound found in various marine organisms, such as jellyfish and shrimp. Coelenterazine is commonly used as a reporter molecule in cell-based assays to measure various biological activities, including protein-protein interactions, gene expression, and cell signaling pathways.

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Lab products found in correlation

Envision plate reader, by PerkinElmer (3 mentions) Adenosine triphosphate (atp), by Cytiva (2 mentions) Infinite f200 pro illuminometer, by Tecan (1 mentions)

6 protocols using coelenterazine

Bioluminescent Cytosolic Calcium Monitoring

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A bioluminescent method based on the reaction of cytosolic Ca²⁺ with
the genetic probe aequorin in wild type cells was employed as previously
described 12 (link). Briefly, 100 μl of conidia
at a concentration of 2x10⁶ cells/ml, 5 μM coelenterazine (Santa Cruz
Biotechnology) and minimal medium were added to white opaque 96-well plates and
incubated for 6 hours at 26°C, in the dark, without agitation. Luminescence (in
RLU, relative light units) was captured over time on a Bio-Tek Synergy HT
microplate reader. Maximum levels of luminescence, determined in extra wells by
measuring luminescence during 3 mins after the injection of 100 μl of 3M
CaCl₂ in 20% ethanol, were used to normalize each experiment
(cytosolic Ca²⁺ levels (arbitrary units) = experimental RLU values /
total emitted luminescence). Quantification was performed by summing the
normalized values and data are expressed as mean ± SEM. The values from control
DMSO-treated samples were subtracted from staurosporine-treated samples to
obtain the "staurosporine-induced amplitude of response".

Gonçalves A.P., Monteiro J., Lucchi C., Kowbel D.J., Cordeiro J.M., Correia-de-Sá P., Rigden D.J., Glass N.L, & Videira A. (2014). Extracellular calcium triggers unique transcriptional programs and modulates staurosporine-induced cell death in Neurospora crassa. Microbial Cell, 1(9), 289-302.

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Mitochondrial Ca2+ Dynamics Monitored

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Ca²⁺ measurements were performed by co-transfecting HeLa cells in a six-well plate with low-affinity mitochondrial aequorin (mtAeqmut) with the two SPLICS moieties in a 1.5:1.5:1 ratio favouring SPLICS. Forty-eight hours post transfection, cells were re-plated into a 96-wells plate (PerkinElmer). mtAeqmut was reconstituted by incubating cells for 1.5 h with 5 µM coelenterazine (Santa Cruz Biotech.) in modified Krebs Ringer Buffer (KRB: 125 mM NaCl, 5 mM KCl, 400 mM KH₂PO₄, 1 mM MgSO₄, 20 mM Hepes, pH 7.4) supplemented with 5 mM glucose at 37 °C. Luminescence measurements were carried out using a PerkinElmer EnVision plate reader equipped with two injector units. After reconstitution, cells were placed in 70 µl of KRB solution and luminescence from each well was measured for 1 min. During the experiment, 100 µM histamine at the final concentration were first injected to activate Ca²⁺ transients, and then a hypotonic, Ca²⁺-rich, digitonin-containing solution was added to discharge the remaining aequorin pool. Output data were analyzed and calibrated with a custom-made macro-enabled Excel workbook.

Cieri D., Vicario M., Giacomello M., Vallese F., Filadi R., Wagner T., Pozzan T., Pizzo P., Scorrano L., Brini M, & Calì T. (2017). SPLICS: a split green fluorescent protein-based contact site sensor for narrow and wide heterotypic organelle juxtaposition. Cell Death and Differentiation, 25(6), 1131-1145.

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Measuring cAMP Levels in HEK-293 Cells

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To detect cAMP levels, we transfected a Nano-lantern cAMP plasmid (1 µg) and human GPR37 plasmid (1 µg) into HEK-293 cells. BRET activity was measured upon addition of 100 µl/well for a final concentration of 10 µM Coelenterazine (Santacruz), 10 µM drug, and 100 µM forskolin (Enzo, # BML-CN100). Bioluminescent activity was measured using an infinite F200 Pro Illuminometer (Tecan Trading AG, Switzerland). The data displayed indicate the values normalized to basal signal levels.

Bang S., Donnelly C.R., Luo X., Toro-Moreno M., Tao X., Wang Z., Chandra S., Bortsov A.V., Derbyshire E.R, & Ji R.R. (2021). Activation of GPR37 in macrophages confers protection against infection-induced sepsis and pain-like behaviour in mice. Nature Communications, 12, 1704.

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Monitoring Mitochondrial Calcium Dynamics in HeLa Cells

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After 48 h of transfection mitochondrial low affinity aequorin (mtAEQ) was reconstituted by incubating cells for 1.5 h with 5 μM wt coelenterazine (Santacruz) in DMEM supplemented with 1% fetal bovine serum at 37 °C in a 5% CO₂ atmosphere. After reconstitution, cells were transferred to the chamber of a purpose-built luminometer, and Ca²⁺ measurements were started in Krebs-Ringer modified buffer (KRB,125 mM NaCl, 5 mM KCl, 1 mM Na₃PO₄, 1 mM MgSO₄, 5.5 mM glucose, 20 mM HEPES, pH 7.4, 37 °C) medium supplemented with 1 mM CaCl₂ by stimulating HeLa cells with 100 μM histamine. The experiments were terminated by cell permeabilization with 100 μM digitonin in a hypotonic Ca²⁺-rich solution (10 mM CaCl₂ in H₂O) to discharge the remaining unused aequorin pool. The light signal was collected and calibrated off-line into Ca²⁺ concentration values using a computer algorithm based on the Ca²⁺ response curve of mitochondrial aequorin, as previously described^{60 (link)}.

Vicario M., Cieri D., Vallese F., Catoni C., Barazzuol L., Berto P., Grinzato A., Barbieri L., Brini M, & Calì T. (2019). A split-GFP tool reveals differences in the sub-mitochondrial distribution of wt and mutant alpha-synuclein. Cell Death & Disease, 10(11), 857.

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Measuring Mitochondrial Calcium Uptake

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The recombinant Ca²⁺-sensitive photoprotein aequorin specifically targeted to the mitochondrial matrix (52 (link)) was employed to measure Ca²⁺ uptake by the mitochondria which reflects the Ca²⁺ microdomains generated at the mouth of the InsP3 channels upon their opening (53 (link)). For cytosolic and mitochondrial calcium measurements using the aequorin method, OBs were plated into a 96-well plate at 50–60% of confluence and transduced for 48h in their culture medium with adenovirus expressing cytosolic or mitochondrial aequorin (cytAEQ or mtAEQ). 48h post adenoviral transduction cells were incubated (1 hr, 37°C) with the prosthetic group coelenterazine (5 μM, 1 h at 37°C, Santa Cruz Biotechnology) in CaCl₂-containing Krebs-Ringer buffer (KRB - CaCl₂, 125 mM NaCl, 1 mM Na₃PO₄, 1 mM MgSO₄, 1 mM CaCl₂, 5.5 mM glucose, 5 mM KCl, 20 mM HEPES, pH 7.4). After aequorin reconstitution, cells were placed in 70 μl of KRB-CaCl₂ and cytosolic or mitochondrial Ca²⁺ transients were evoked by applying 100 μM ATP (Amersham Biosciences) in KRB. Cytosolic and mitochondrial calcium measurements were carried out using a PerkinElmer EnVision plate reader equipped with two injector units. Output data were analyzed and calibrated with a custom-made macro-enabled Excel workbook.

Joiner W.J., Wang L.Y., Tang M.D, & Kaczmarek L.K. (1997). hSK4, a member of a novel subfamily of calcium-activated potassium channels. Proceedings of the National Academy of Sciences of the United States of America, 94(20), 11013-11018.

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Measuring Mitochondrial Calcium Dynamics

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The recombinant Ca 2+ -sensitive photoprotein aequorin specifically targeted to the mitochondrial matrix (52) was employed to measure Ca 2+ uptake by the mitochondria which reflects the Ca 2+ microdomains generated at the mouth of the InsP3 channels upon their opening (53) . For cytosolic and mitochondrial calcium measurements using the aequorin method, OBs were plated into a 96-well plate at 50-60% of confluence and transduced for 48h in their culture medium with adenovirus expressing cytosolic or mitochondrial aequorin (cytAEQ or mtAEQ). 48h post adenoviral transduction cells were incubated (1 hr, 37°C) with the prosthetic group coelenterazine (5 μM, 1 h at 37°C, Santa Cruz Biotechnology) in CaCl 2 -containing Krebs-Ringer buffer (KRB -CaCl 2 , 125 mM NaCl, 1 mM Na 3 PO 4 , 1 mM MgSO 4 , 1 mM CaCl 2 , 5.5 mM glucose, 5 mM KCl, 20 mM HEPES, pH 7.4). After aequorin reconstitution, cells were placed in 70 μl of KRB-CaCl 2 and cytosolic or mitochondrial Ca 2+ transients were evoked by applying 100 μM ATP (Amersham Biosciences) in KRB.
Cytosolic and mitochondrial calcium measurements were carried out using a PerkinElmer EnVision plate reader equipped with two injector units. Output data were analyzed and calibrated with a custom-made macro-enabled Excel workbook.

Besio R., Contento B.M., Garibaldi N., Filibian M., Sonntag S., Shmerling D., Tonelli F., Biggiogera M., Brini M., Salmaso A., Jovanovic M., Marini J.C., Rossi A, & Forlino A. (2023). CaMKII inhibition due to TRIC-B loss-of-function dysregulates SMAD signaling in osteogenesis imperfecta. Matrix biology : journal of the International Society for Matrix Biology, 120.

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