Fluorescent probe for labeling of synthetic/endogenous polyP
Polyphosphate (polyP), an inorganic linear polymer of orthophosphate residues linked by high-energy phosphoanhydride bonds. [2][3]

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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- Fluorescence response to polyP standards: In HEPES buffer (12.5 mmol/L, pH 8.0) containing 3% DMSO and 30 μmol/L JC-D7, the fluorescence intensity of polyP (chain length n=45) responded linearly to polyP concentrations from 0.5 to 36 μmol/L (R² = 0.9995), with a detection limit below 0.5 μmol/L. Linearity decreased when polyP concentrations exceeded ~50 μmol/L, with fluorescence maximum at ~100-120 μmol/L. [2]
- Effect of JC-D7 concentration: Varying JC-D7 concentration from 20 to 40 μmol/L did not substantially change the sensitivity of the fluorescence response. Fluorescence signals varied by approximately 2.1 ± 0.6%, and the slopes of linear calibration curves varied by 3.8 ± 1.4%. [2]
- Effect of polyP chain length: The chain length of polyP (n = 14, 45, 60, and 130) did not substantially affect the linear fluorescence response. Fluorescence signals varied by an average of 5.0%, and the slopes varied by approximately 5.7 ± 1.0%. [2]
- Comparison with DAPI and PPX methods: In wild-type Chlamydomonas reinhardtii (polyP-accumulating), JC-D7 gave polyP estimates consistent with PPX and DAPI methods. In a mutant strain lacking polyP, JC-D7 and PPX both indicated undetectable polyP, while DAPI produced false-positive signals due to DNA/RNA interference. In Vogesella perlucida bacteria, JC-D7 results were comparable to PPX (p = 0.74), whereas DAPI overestimated polyP. JC-D7 gave reliable measurements for marine algae samples where PPX failed. [2]
- Interference from lysis buffer components: The designed plankton lysis buffer (containing proteinase K, NaCl, Triton X-100, Tween 20, EDTA in Tris-HCl) decreased JC-D7-polyP fluorescence sensitivity by ~16%, but the detection limit (still <0.5 μmol/L) and linear range (0.5-30 μmol/L) were not affected. The detection limit could be lowered to ~0.2 μmol/L with reduced sensitivity. High NaCl concentrations (≥300 mmol/L) increased the detection limit, decreased sensitivity, and narrowed the linear range. EDTA substantially suppressed fluorescence from chondroitin sulfate (a sulfated polysaccharide) when stained with JC-D7, reducing interference to <3% at 550 nm emission. [2]
- Staining of polyP in cultured astrocytes: Cultured mouse astrocytes were incubated with 5 μmol/L JC-D7 in HBSS for 30 minutes in a CO₂ incubator, then washed, fixed, and imaged. JC-D7 was used to visualize intracellular polyP distribution. [3]

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].

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].

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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.

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.

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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.

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- PolyP staining in cultured astrocytes (from literature [3]): Cultured mouse astrocytes grown on coverslips were incubated with 5 μmol/L JC-D7 dissolved in HBSS for 30 minutes in a CO₂ incubator. After incubation, cells were washed three times with HBSS and fixed with 4% paraformaldehyde for 15 minutes. Cells were then further incubated with Hoechst nuclear dye, washed, and mounted on glass slides with Fluoromount-G. Fluorescence images of JC-D7 were acquired using an excitation laser at 488 nm with a confocal microscope (60× objective lens, zoom 2.5, Z-interval 0.3 μm). This procedure was used to visualize intracellular polyphosphate distribution. [3]
- PolyP quantification in cultured algae and bacteria (from literature [2]): Cultured cells (e.g., C. reinhardtii, V. perlucida) were collected by filtration or centrifugation, and polyP was extracted using either the phenol-chloroform method or the plankton lysis buffer method. The extracted polyP samples (or polyP standards) were stained with a working JC-D7 solution (60 μmol/L JC-D7 in HEPES buffer) at a 1:1 ratio (v/v), resulting in a final staining mixture containing 30 μmol/L JC-D7 and 3% DMSO in 12.5 mmol/L HEPES buffer. Samples were incubated for 5-10 minutes, and fluorescence intensities were measured at excitation 405 nm and emission 535 nm using a microplate reader. [2]

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.

[1]. In situ investigation of mammalian inorganic polyphosphate localization using novel selective fluorescent probes JC-D7 and JC-D8. ACS Chem Biol. 2014 Sep 19;9(9):2101-10.
[2]. Quantification of Polyphosphate in Environmental Planktonic Samples Using a Novel Fluorescence Dye JC-D7. Environ Sci Technol. 2024 Aug 13;58(32):14249-14259.
[3]. Astrocytes modulate brain phosphate homeostasis via polarized distribution of phosphate uptake transporter PiT2 and exporter XPR1. Neuron. 2024 Sep 25;112(18):3126-3142.e8.

Inorganic polyphosphate (polyP) is a polymer composed of many orthophosphates linked by phosphate anhydride bonds. Recent studies have shown that polyP plays an important role not only in microbial function but also in the pathological and physiological functions of higher eukaryotes, including mammals. However, the amount of polyP in mammalian cells is much lower than that in microorganisms, making its study an experimental challenge. This article introduces the identification of a novel fluorescent probe that can specifically label polyP synthesized in vitro as well as endogenous polyP in living cells. These probes exhibit high selectivity for polyP labeling and are insensitive to a variety of ubiquitous organic polyphosphates (especially RNA). Using these probes, we achieved real-time detection of polyP released from lysosomes in living cells. In addition, we also detected elevated polyP levels in cells carrying Parkinson's disease-related mutations. [1]

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- JC-D7 is a polyP-specific fluorescent probe originally discovered for visualizing polyP in mammalian cells. In these studies, it is adapted for quantitative detection of polyP in environmental planktonic samples and cultured astrocytes. [2][3]
- Unlike the traditional DAPI method, JC-D7 staining is not significantly affected by interference from nucleic acids (DNA/RNA) or sulfated polysaccharides, especially when combined with the plankton lysis buffer containing EDTA. This allows for accurate quantification of polyP in complex environmental samples where the DAPI method often produces overestimations (>100% of total particulate P). [2]
- The JC-D7 fluorescence quantification method is described as sensitive, easy to operate, stable, and capable of working in various extractants and buffers. It is recommended for polyP quantification in all planktonic samples (algae and bacteria, freshwater and marine), as it is the most stable and reliable method compared to DAPI and PPX for environmental applications. [2]

C28H31BRCL2N4O

590.381943941116

588.105

C, 56.96; H, 5.29; Br, 13.53; Cl, 12.01; N, 9.49; O, 2.71

1036271-54-1

909715-05-5 (cation);1036271-54-1 (bromide);

154724048

Solid powder

2

3

10

36

706

0

C[N+]1=C(N(C2=CC(=C(C=C21)Cl)Cl)CCCCCC(=O)NCCN)/C=C/C3=CC=CC4=CC=CC=C43.[Br-]

DSNPFMKJUXAPGB-IERUDJENSA-N

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+;

1-[5-(2-Amino-ethylcarbamoyl)-pentyl]-5,6-dichloro-3-methyl-2-(2-naphthalen-1-yl-vinyl)-3H-benzoimidazol-1-ium Bromide

JC-D7; JC D7; 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

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

>10 mM in DMSO

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.)