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Purity: = 84%
JC-D7 (JCD7; JC D7) is a novel and selective fluorescent probe/dye used for specific labeling of synthetic polyphosphate (polyP) in vitro as well as endogenous polyP in living cells. It demonstrated high selectivity for the labeling of polyP that was not sensitive to a number of ubiquitous organic polyphosphates, notably RNA. Use of JC-D7 allowed the real time detection of polyP release from lysosomes in live cells. Furthermore, use of JC-D7 enabled the detection of increased levels of polyP in cells with Parkinson's disease related mutations. Inorganic polyphosphate (polyP) is a polymer composed of many orthophosphates linked together by phosphoanhydride bonds. In addition to its important role in the function of microorganisms, polyP plays multiple important roles in the pathological and physiological function of higher eukaryotes, including mammalians. However, due to the siginificantly lower abundance of polyP in mammalian cells when comparing to microorganisms, its investigation poses an experimental challenge. Inorganic polyphosphate (polyP) is a linear polymer made up of many orthophosphates linked together by phosphoanhydride bonds identical to the ones found in ATP. PolyP is a biological macromolecule, which has been found in all studied organisms ranging from bacteria to humans. One of the key challenges faced by the field of polyP studies is the very limited number of methods for direct detection and investigation of polyP in mammalian cells and tissues.JC-D7 is a novel highly selective polyP sensor identified from a benzimidazolinium dye library and its application for analytical polyP assay of purified polymer as well as polyP staining in live cells and tissue samples.
| Targets |
Fluorescent probe for labeling of synthetic/endogenous polyP
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|---|---|
| ln Vitro |
Inorganic polyphosphate (polyP) is a polymer composed of many orthophosphates linked together by phosphoanhydride bonds. Recent studies demonstrate that in addition to its important role in the function of microorganisms, polyP plays multiple important roles in the pathological and physiological function of higher eukaryotes, including mammalians. However, due to the dramatically lower abundance of polyP in mammalian cells when comparing to microorganisms, its investigation poses an experimental challenge. Here, we present the identification of novel fluorescent probes that allow for specific labeling of synthetic polyP in vitro as well as endogenous polyP in living cells. These probes (JC-D7, JC-D8) demonstrate high selectivity for the labeling of polyP that was not sensitive to a number of ubiquitous organic polyphosphates, notably RNA. Use of these probes allowed us to demonstrate the real time detection of polyP release from lysosomes in live cells. Furthermore, we have been able to detect the increased levels of polyP in cells with Parkinson's disease related mutations [1].
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| ln Vivo |
Using the novel indicators, we have been able to detect increased level of polyP in brain slices obtained from an animal model of Parkinson’s disease. This finding demonstrates the potential for practical use of the probes (JC-D7 and JC-D8) in live samples. However, care should be taken and specific protocols developed for each particular set of experimental conditions [1].
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| Enzyme Assay |
Primary Screening
Fluorescence intensities were measured using a SpectraMax M2 plate reader in a 96-well plate. JC compounds were dissolved to a final concentration of 10 μM (20 mM HEPES buffer, pH 7.4, containing 1% (v/v) DMSO) and incubated with different analytes at different serial concentration in 20 mM HEPES buffer (pH 7.4). The excitation wavelength was set at 390 nm, and the emission spectra were recorded from 450 to 700 nm. The fluorescence fold increase ratios were determined by referring the maximum fluorescence intensity of JC compounds in the presence and absence of analytes. The quantum yield of JC-D7 before and after the addition polyphosphate is 0.007 and 0.38, respectively. Whereas, the quantum yield of JC-D8 in the absence and presence of polyphosphate is 0.008 and 0.37, respectively. Because of the comparatively low quantum yield of JC-D7 than JC-D8, the earlier has slightly higher fold increase [1]. Selectivity Study [1] Benzimidazolium compounds were transferred to Greiner 96 well black polypropylene plates (final concentration as 10 μM) and tested against DNA and RNA 100 μg/mL, other analytes: sodium phosphate, ATP, AMP, GTP, GMP, CTP, and CMP 1 mM, and heparin sodium and polyP is 20 μg/mL in 20 mM HEPES buffer (pH = 7.4) with 1% DMSO. Fluorescent spectra were recorded on a SpectraMax M2 fluorescent plate reader with excitation at 390 nm (cutoff: 420 nm), emission 450 to 700 nm. |
| Cell Assay |
Live Cell Imaging [1]
SHY-SY5 cells, human skin fibroblasts, whole Drosophila brains, mixed primary brain cultures, or acute brain slices were loaded for 30 min at RT with 5 μM JC-D7 or JC-D8 in a HEPES-buffered salt solution (HBSS) composed (mM): 156 NaCl, 3 KCl, 2 MgSO4, 1.25 KH2PO4, 2 CaCl2, 10 glucose, and 10 HEPES, pH adjusted to 7.35 with NaOH. Confocal images were obtained using a Zeiss 710 CLSM microscope equipped with a META detection system and a 40× oil immersion objective. JC-D7/D8 fluorescence was determined with excitation at 405 nm and emission above 450 nm. Illumination intensity was kept to a minimum (at 0.1–0.2% of laser output) to avoid phototoxicity and the pinhole set to give an optical slice of ∼2 μm. For images in experiments comparing levels of fluorescence in different cells, the imaging setting were kept at the same level. For better visual representation, the different false-color was chosen. The DAPI-polyP fluorescence was detected with excitation 405 nm and emission between 480 and 520 nm. The images were analyzed using Zeiss software. Toxicity Experiments [1] For toxicity assays, cells were exposed to 20 μM propidium iodide (PI) and 4.5 μM Hoechst 33342 (Molecular Probes, Eugene, OR) for 30 min prior to imaging. The PI is excluded from viable cells and exhibits a red fluorescence following a loss of membrane integrity, while the Hoechst 33342 labels all nuclei blue. This allows expression of the number of dead (red stained) cells as a fraction of the total number of nuclei counted. Each experiment was repeated four or more times using separate cultures. |
| Animal Protocol |
Preparation of Acute Brain Slices [1]
All mouse experiments were carried out in compliance with institutional ethical and welfare standards and under Home Office regulation. Slices were prepared using standard procedures, as previously described. Briefly, transverse acute brain slices (100–200 μm) were prepared from 20 to 24-week-old WT, PINK1 KO, and LRRK2 KO C57BL/6 mice. The animals were euthanized by cervical dislocation, brains were collected, and tissue was immediately sliced at 4 °C using a vibratome (Leica VT1200S). The tissue slices were cut and maintained in physiological saline at RT (24 °C) for ∼1 h before imaging. |
| References | |
| Additional Infomation |
Inorganic polyphosphate (polyP) is a polymer composed of many orthophosphates linked together by phosphoanhydride bonds. Recent studies demonstrate that in addition to its important role in the function of microorganisms, polyP plays multiple important roles in the pathological and physiological function of higher eukaryotes, including mammalians. However, due to the dramatically lower abundance of polyP in mammalian cells when comparing to microorganisms, its investigation poses an experimental challenge. Here, we present the identification of novel fluorescent probes that allow for specific labeling of synthetic polyP in vitro as well as endogenous polyP in living cells. These probes demonstrate high selectivity for the labeling of polyP that was not sensitive to a number of ubiquitous organic polyphosphates, notably RNA. Use of these probes allowed us to demonstrate the real time detection of polyP release from lysosomes in live cells. Furthermore, we have been able to detect the increased levels of polyP in cells with Parkinson's disease related mutations.[1]
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| Molecular Formula |
C28H31BRCL2N4O
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|---|---|
| Molecular Weight |
590.381943941116
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| Exact Mass |
588.105
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| Elemental Analysis |
C, 56.96; H, 5.29; Br, 13.53; Cl, 12.01; N, 9.49; O, 2.71
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| CAS # |
1036271-54-1
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| Related CAS # |
909715-05-5 (cation);1036271-54-1 (bromide);
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| PubChem CID |
154724048
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| Appearance |
Solid powder
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
10
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| Heavy Atom Count |
36
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| Complexity |
706
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C[N+]1=C(N(C2=CC(=C(C=C21)Cl)Cl)CCCCCC(=O)NCCN)/C=C/C3=CC=CC4=CC=CC=C43.[Br-]
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| InChi Key |
DSNPFMKJUXAPGB-IERUDJENSA-N
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| InChi Code |
InChI=1S/C28H30Cl2N4O.BrH/c1-33-25-18-23(29)24(30)19-26(25)34(17-6-2-3-12-27(35)32-16-15-31)28(33)14-13-21-10-7-9-20-8-4-5-11-22(20)21;/h4-5,7-11,13-14,18-19H,2-3,6,12,15-17,31H2,1H3;1H/b14-13+;
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| Chemical Name |
1-[5-(2-Amino-ethylcarbamoyl)-pentyl]-5,6-dichloro-3-methyl-2-(2-naphthalen-1-yl-vinyl)-3H-benzoimidazol-1-ium Bromide
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| Synonyms |
JC-D7; JC D7; EX-A5208; AKOS040752173; 1-(6-((2-Aminoethyl)amino)-6-oxohexyl)-5,6-dichloro-3-methyl-2-(2-(naphthalen-1-yl)vinyl)-1H-benzo[d]imidazol-3-ium bromide; 1-[5-(2-Amino-ethylcarbamoyl)-pentyl]-5,6-dichloro-3-methyl-2-(2-naphthalen-1-yl-vinyl)-3H-benzoimidazol-1-ium Bromide; N-(2-aminoethyl)-6-[5,6-dichloro-3-methyl-2-[(E)-2-naphthalen-1-ylethenyl]benzimidazol-3-ium-1-yl]hexanamide;bromide; JCD7
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
>10 mM in DMSO
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|---|---|
| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6938 mL | 8.4691 mL | 16.9382 mL | |
| 5 mM | 0.3388 mL | 1.6938 mL | 3.3876 mL | |
| 10 mM | 0.1694 mL | 0.8469 mL | 1.6938 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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