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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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6 protocols using coelenterazine

1

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".
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2

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.
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3

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.
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4

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 described60 (link).
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5

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.
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6

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.
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