Luminescent enzyme substrate; Marine luciferases (specifically Renilla-type luciferase, EC 1.13.12.5) and calcium-regulated photoproteins (e.g., aequorin). It acts as the primary luciferin substrate for the enzyme-catalyzed reaction: Coelenterazine h + O₂ → Excited Coelenteramide h monoanion + CO₂ + Light . In vitro, the peak emission is approximately 466 nm .

1. Solution Preparation [4]
1.1 Stock Solution Preparation
Solvent: Methanol or ethanol
Concentration: 10 mM (optimize according to experimental conditions).
1.2 Working Solution Preparation
Dilute with PBS or cell culture medium to 10 µM (optimize according to experimental conditions).
Note: Stock and working solutions should be prepared immediately before use and stored protected from light.

2. Cell Staining
2.1 Transfect HeLa cells with BRAC or G5A.
2.2 Add 10 µM Coelenterazine h to the cell plate.
2.3 Incubate the plate with the dye in a cell culture incubator for 1–4 hours (optimize according to experimental conditions).
2.4 Detect by fluorescence microscopy (Ex/Em = 437/466 nm).

Coelenterazine h (1–10 μM) can be used as a luminescence substrate for RLuc8 [4].
In measurements of Ca²⁺ binding kinetics using BRAC, 5 nM BRAC protein was rapidly mixed with 20 μM Coelenterazine h in buffers containing various Ca²⁺ concentrations. The Venus emission intensity (530 nm) of BRAC was then monitored at 1 kHz [4].

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The drift observed in both RLuc8 and Venus signals might be caused by the uptake and consumption of coelenterazine-h or the change in cell shape.[4]
We then measured the Ca2+-association kinetics of BRAC by stopped-flow photometry system. However, the time course data we obtained was composed of at least two exponential decay components (ι<0.1 sec) which were thought to be derived from both Ca2+ and coelenterazine-h binding to BRAC.[4]

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The genetic transformation of the higher plant Nicotiana plumbaginifolia to express the protein apoaequorin has recently been used as a method to measure cytosolic free calcium ([Ca2+]i) changes within intact living plants (Knight, M. R., A. K. Campbell, S. M. Smith, and A. J. Trewavas. 1991. Nature (Lond.). 352:524-526; Knight, M. R., S. M. Smith, and A. J. Trewavas. 1992. Proc. Natl. Acad. Sci. USA. 89:4967-4971). After treatment with the luminophore coelenterazine the calcium-activated photoprotein aequorin is formed within the cytosol of the cells of the transformed plants. Aequorin emits blue light in a dose-dependent manner upon binding free calcium (Ca2+). Thus the quantification of light emission from coelenterazine-treated transgenic plant cells provides a direct measurement of [Ca2+]i. In this paper, by using a highly sensitive photon-counting camera connected to a light microscope, we have for the first time imaged changes in [Ca2+]i in response to cold-shock, touch and wounding in different tissues of transgenic Nicotiana plants. Using this approach we have been able to observe tissue-specific [Ca2+]i responses. We also demonstrate how this method can be tailored by the use of different coelenterazine analogues which endow the resultant aequorin (termed semi-synthetic recombinant aeqorin) with different properties. By using Coelenterazine H, which renders the recombinant aequorin reporter more sensitive to Ca2+, we have been able to image relatively small changes in [Ca2+]i in response to touch and wounding: changes not detectable when standard coelenterazine is used. Reconstitution of recombinant aequorin with another coelenterazine analogue (e-coelenterazine) produces a semi-synthetic recombinant aequorin with a bimodal spectrum of luminescence emission. The ratio of luminescence at two wavelengths (421 and 477 nm) provides a simpler method for quantification of [Ca2+]i in vivo than was previously available. This approach has the benefit that no information is needed on the amount of expression, reconstitution or consumption of aequorin which is normally required for calibration with aequorin[2].

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In cell culture, Coelenterazine H exhibits 4- to 8-fold higher Renilla luciferase (Rluc) activity compared to native coelenterazine in stably expressing cells . For calcium studies, the apo-aequorin-Coelenterazine H complex (photoprotein) is used; binding of Ca²⁺ triggers rapid oxidation of the substrate, resulting in a flash of light proportional to Ca²+ concentration. It is particularly sensitive for detecting small changes in Ca²⁺ levels .

In living mice expressing Rluc, administration of Coelenterazine H via intravenous or intraperitoneal injection produces strong bioluminescent signals due to rapid oxidation . Optimal imaging results are highly dependent on the delivery route and time post-injection . The RLuc-Coelenterazine H pair is a classic combination for studying gene expression and tumor growth in small animal models .

Protein expression, purification and Ca2+ titration in vitro[4]
Recombinant BRAC protein with N-terminal polyhistidine tags was expressed in Escherichia coli [JM109(DE3)] at 23°C, purified using an Ni-NTA column. Emission spectra of BRAC were measured using a spectrophotometer and a microplate reader. Final concentration of 1–10 µM coelenterazine-h was used as the luminescent substrate for RLuc8. Ca2+ titration was performed by reciprocal dilution of Ca2+-free and Ca2+-saturated buffers prepared using O,O′-bis(2-aminoethyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid (EGTA), N-(2-Hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (EDTA-OH), or Nitrilotriacetic acid (NTA) in 100 mM KCl, 10 mM MOPS (pH 7.2). Free Ca2+ concentrations were calculated by using 0.15, 4.3 and 170 µM as the K d value of EGTA, EDTA-OH and NTA for Ca2+, respectively [20]. A Ca2+ titration curve was used to calculate apparent K d value by non-linear regression analysis. The averaged data from eight independent measurements were fitted to the Hill equation using Origin7 software.

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Measurement of Ca2+ binding kinetics[4]
Measurements of Ca2+ binding kinetics of BRAC were performed by using stopped-flow photometry system consisting of RX.2000 rapid mixing stopped-flow unit and FP-750 spectrophotometer. Emission intensity of Venus (530 nm) from BRAC were monitored at 1 kHz just after rapid mixing of 5 nM BRAC protein with 20 µM coelenterazine-h in various concentration of Ca2+ buffer. In this experiment, we did not mix coelenterazine-h with BRAC prior to measurement to avoid undesirable consumption of coelenterazine-h by Rluc8 in BRAC during sample preparation. Thus, time course of emission intensity in the stopped-flow experiments consists of three components of kinetics derived from Ca2+ binding to BRAC, coelenterazine-h binding to BRAC, and catalytic oxidation of coelenterazine-h by BRAC. To estimate the catalytic oxidation of coelenterazine-h by Rluc8 in BRAC, we measured time course of emission intensity change after mixing BRAC with 20 µM coelenterazine-h in Ca2+-free solution, and used the obtained data as a “base line”. Then, we measured time course of both association and dissociation of Ca2+ to and from BRAC by mixing 1 volume of BRAC in Ca2+-free buffer with 25 volume of solution containing 1.69 µM Ca2+, and 1 volume of BRAC in 1.69 µM Ca2+ solution with 25 volume of Ca2+-free buffer, respectively. The averaged data from at least 5 independent measurements were used for following analysis. The averaged time course data for association and dissociation kinetics were subtracted by the base line to remove the fraction derived from autonomous catalytic oxidation of coelenterazine-h by BRAC. Then, the time constants (ι) were calculated by means of curve fitting in single exponential equation using the data from 0.2 sec to 2.0 sec to minimize contribution of signal derived from association of coelenterazine-h with BRAC just after mixing. Measurements of Ca2+ binding kinetics of YC3.60 were performed as shown previously. In the stopped-flow experiment, final Ca2+ concentration was controlled by reciprocal dilution of Ca2+-free and Ca2+-saturated buffers prepared using O,O′-bis(2-aminoethyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid (EGTA) in 100 mM KCl, 10 mM MOPS (pH 7.2). Free Ca2+ concentration in every solution was confirmed with Ca2+-sensitive electrode which is calibrated with a CaCl2 standard solution.

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Kinetic/Structural Characterization: Purified Rluc or apophotoprotein is mixed with the substrate in a buffer system (e.g., Tris-HCl or PBS) at room temperature. The reaction is initiated by injection of oxygen or Ca²⁺ solution into the cuvette containing the enzyme-substrate complex. Luminescence is measured immediately (flash kinetic mode) using a luminometer or stopped-flow spectroscopy . Spectral analysis is conducted to determine peak emission (approx. 466 nm) .

Cell culture and transfection[4]
Hela cells were cultured in a homemade 35-mm glass-bottom dish in DMEM containing 10% fetal bovine serum. Cells were transfected with plasmids by means of Lipofectamine 2000. At 1 to 2 days after transfection, cells expressing BRAC or G5A were subjected to imaging. 10 µM coelenterazine-h were added to the culture medium just before observation of BRAC and 1–4 hours before observation of G5A.

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Live Cell Imaging Protocol: Cells are seeded in culture plates. A working solution of Coelenterazine H is prepared by diluting a 10 mM stock (in ethanol or methanol) to a final concentration of 1–20 μM in PBS or HBSS . The culture medium is replaced with this substrate solution, and the cells are incubated at 37°C for 10-60 minutes . For Ca²⁺ monitoring, cells expressing apoaequorin are treated with the substrate to regenerate the active photoprotein; luminescence is then recorded upon agonist stimulation to measure Ca²⁺ flux .

In Vivo Imaging (Mice): Preparation involves dissolving Coelenterazine H in a minimal amount of 100% ethanol (e.g., 20 μL of a 5 mg/mL stock) containing 2% v/v 3N HCl, followed by dilution in PBS to the desired volume . For intravenous injection via the tail vein, the recommended dosage is 1.5–100 μg per mouse (25–150 μL volume) . Animals are anesthetized and imaged immediately (within 1-5 minutes) post-injection using a charge-coupled device (CCD) camera as the signal peak is transient . Intraperitoneal injection results in slower kinetics and a more sustained signal compared to IV administration .

Coelenterazine H is characterized by extremely rapid pharmacokinetics in vivo. It undergoes "active dioxetanone" breakdown within seconds of contact with oxygen and luciferase, resulting in a sharp peak of light production followed by rapid clearance (flash kinetics) . The substrate demonstrates significant auto-oxidation in serum due to binding to albumin, which limits its circulation time . The speed of distribution is heavily dependent on the injection route (IV is fastest) .

Standard bioluminescent substrates like Coelenterazine H are generally considered low-toxicity reagents for research applications when used at recommended doses (e.g., microgram levels in mice). Specific LD50 data for Coelenterazine H alone is not typically cited in standard biochemical protocols. Toxicity is more dependent on the handling of ancillary solvents (Ethanol/HCl). While generally safe for acute imaging, its high instability and rapid oxidation often degrade the compound before long-term toxicity becomes a concern . It is not intended for human therapeutic use .

[1]. New bioluminescent coelenterazine derivatives with various C-6 substitutions. Org Biomol Chem. 2017 Aug 23;15(33):7008-7018.

[2]. Imaging calcium dynamics in living plants using semi-synthetic recombinant aequorins. J Cell Biol. 1993 Apr;121(1):83-90.

[3]. Five colour variants of bright luminescent protein for real-time multicolour bioimaging. Nat Commun. 2016 Dec 14:7:13718.

[4]. Auto-luminescent genetically-encoded ratiometric indicator for real-time Ca2+ imaging at the single cell level. PLoS One. 2010 Apr 1;5(4):e9935.

Coelenterazine H belongs to the phenolic and imidazopyrazine classes and has the function of fluorescein. It is derived from the hydride of imidazo[1,2-a]pyrazine.

Chemical Name: 2-Deoxycoelenterazine .
Molecular Formula/Weight: C₂₆H₂₁N₃O₂ / 407.46 g/mol .
Spectral Properties: Excitation maximum ~437 nm; Emission maximum ~466 nm .
Stability: Highly sensitive to air oxidation and light. Stock solutions should be stored at -20°C in an inert gas (Argon) to prevent degradation . It is not recommended to pre-dissolve in DMSO; use ethanol or methanol .
Applications: BRET (Bioluminescence Resonance Energy Transfer), Ca²⁺ detection, Dual-Luciferase Reporter assays, and ROS detection .

C26H21N3O2

407.463845968246

407.163

C, 76.64; H, 5.19; N, 10.31; O, 7.85

50909-86-9

135398664

Yellow to brown solid powder

1.3±0.1 g/cm3

593.5±60.0 °C at 760 mmHg

141ºC

312.7±32.9 °C

0.0±1.7 mmHg at 25°C

1.675

4.6

2

4

5

31

555

0

OC1=C(CC2C=CC=CC=2)N=C2C(CC3C=CC=CC=3)=NC(C3C=CC(=CC=3)O)=CN21

MGTUVUVRFJVHAL-UHFFFAOYSA-N

InChI=1S/C26H21N3O2/c30-21-13-11-20(12-14-21)24-17-29-25(22(27-24)15-18-7-3-1-4-8-18)28-23(26(29)31)16-19-9-5-2-6-10-19/h1-14,17,30-31H,15-16H2

2,8-dibenzyl-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3-ol

Coelenterazine h; 50909-86-9; Renilla luciferin; 2-Deoxycoelenterazine; Coelenterazine-h; 2,8-dibenzyl-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one; Luciferin (Renilla); H-Coelenterazine;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.  (2). This product is not stable in solution, please use freshly prepared working solution for optimal results.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)