JC-D7

Protocol Guide (The following is a recommended experimental protocol. This protocol is for reference only and should be adjusted according to your specific needs).

1. Stock Solution Preparation
1.1 Solvent: Dissolve JC-D7 in DMSO (pure DMSO or 100% v/v).
1.2 Recommended concentrations: Prepare JC-D7 stock solutions in DMSO at 1 mM, 5.9 mg/mL, or 50 mM as needed.

2. Working Solution Preparation
2.1 Diluents:
2.1.1 HBSS: 10 mM HEPES/NaOH (pH 7.35-7.4), 156 mM NaCl, 3 mM KCl, 2 mM MgSO₄, 1.25 mM KH₂PO₄, 2 mM CaCl₂, and 10 mM glucose.
2.1.2 Assay buffer for cell-free/in vitro polyP detection: 20 mM HEPES buffer, pH 7.4, containing 1% (v/v) DMSO.
2.1.3 HEPES buffer for staining extracted polyP: 25 mM HEPES, pH 8.0.
2.2 Working concentrations:
2.2.1 Live cell and tissue staining: 5 μM JC-D7 in HBSS.
2.2.2 In vitro polyP fluorescence detection: 10 μM JC-D7 in 20 mM HEPES buffer (pH 7.4) containing 1% (v/v) DMSO.
2.2.3 Cell-free polyP detection: Dilute JC-D7 in assay buffer to a maximum of 50 μM, then mix with polyP solution in equal volumes. For interference assays, use 50 μM JC-D7 in assay buffer, then add equal volume to the pre-incubated polyP mixture, resulting in a final JC-D7 concentration of 25 μM in the reaction system. For yeast extract detection, use 20 μM JC-D7 in assay buffer, then mix with diluted extract in equal volumes.
2.2.4 Plant tissues and Chlamydomonas reinhardtii: 100 μM JC-D7 in HBSS.
2.2.5 PolyP extracted from plankton samples or polyP standards: Prepare working staining reagent at 60 μM JC-D7 in 25 mM HEPES (pH 8.0); mix with samples or standards at 1:1 (v/v) to obtain a final staining mixture containing 30 μM JC-D7 and 3% DMSO in 12.5 mM HEPES buffer.
2.3 Note: Adjust working solution concentrations as needed; prepare fresh before use.

3. Staining Procedure
3.1 Live cells and tissues:
Sample types include SH-SY5Y cells, human skin fibroblasts, intact Drosophila brain, mixed primary brain cultures, and acute brain slices.
Primary neurons and astrocytes can be imaged before and after addition of JC-D7.
For acute brain slices, prepare 100-200 μm transverse sections from 20-24 week-old mice, collect brain tissue, section using a vibratome at 4 °C, and maintain slices in physiological saline at room temperature (24 °C) for approximately 1 h before imaging.
Incubate samples with 5 μM JC-D7 in HBSS at room temperature for 30 min.
For probe uptake, add 5 μL of 1 mM JC-D7 DMSO stock solution to 0.5 mL imaging medium; cells begin to rapidly take up the indicator and reach a steady-state plateau fluorescence within 8-10 min, but loading for approximately 30 min is recommended for optimal staining.

3.2 Cell-free polyP, in vitro polyP, and yeast extract samples:
Cell-free polyP solutions (0-1000 µg/mL) can be analyzed in 96-well polystyrene microplates.
A polyP chain length mixture at 10 µg/mL can be used for interference testing.
S. cerevisiae extracts should be diluted 1:500 in assay buffer before staining.
Intact yeast cells are not loaded with JC-D7 for direct intracellular polyP determination; extracts are used for JC-D7-based polyP detection.
For in vitro polyP fluorescence detection, use purified polyP in 20 mM HEPES buffer (pH 7.4).
For cell-free detection, mix equal volumes of JC-D7 working solution (up to 50 μM in assay buffer) and polyP solution (0-1000 µg/mL) in 96-well polystyrene microplates to prepare a 200 µL reaction mixture, then incubate at 30°C for 30 min.
For in vitro polyP fluorescence detection, incubate 10 μM JC-D7 with different series concentrations of analyte in 20 mM HEPES buffer (pH 7.4) containing 1% (v/v) DMSO.
For yeast extract detection, mix 100 µL of extract dilution with 100 µL of JC-D7 (20 µM), then incubate at 30°C for 30 min.
For interference detection, pre-incubate 50 µL of polyP chain length mixture (10 µg/mL) with 50 µL of four-fold concentrated test substance pre-diluted in double-distilled water at 30°C for 30 min; then add 100 µL of 50 µM JC-D7 and incubate at 30°C for 30 min.
JC-D7 incubation uses 30°C; 37°C causes a slight decrease in fluorescence.
Use HEPES buffer to maintain pH around 7.4; avoid pH 3.0 and 9.0 for cell extract samples, as JC-D7 signal decreases.
Measure JC-D7-stained samples promptly or within a 20-min time frame to mitigate potential signal decrease.

3.3 PolyP extracted from plankton samples or polyP standards:
Use polyP extracted from plankton samples or polyP standards; JC-D7 working solution is used to stain polyP in samples or standards at a 1:1 ratio (v/v).
This method is applicable to cultured and environmental samples in freshwater and marine environments.
Mix samples or standards with JC-D7 working solution at a 1:1 ratio (v/v) to obtain a final staining mixture containing 30 μM JC-D7 and 3% DMSO in 12.5 mM HEPES buffer.
Incubate for 5-10 min.

3.4 Plant tissues and Chlamydomonas reinhardtii:
Incubate plant tissues or Chlamydomonas reinhardtii WT 2C strain (mt+) with JC-D7 in HBSS buffer.
Perform live cell imaging in root, hypocotyl, and leaf epidermal cells.
For fixed plant tissues, vacuum-fix roots in PBS buffer containing 4% (v/v) paraformaldehyde, 0.1% (v/v) Triton X-100, and 1 mM DTT, then sequentially wash twice in PBS and twice in HBSS buffer.
Arabidopsis lines are grown on 1/2MS medium for 8-9 days, then stained with JC-D7.
For transient expression, infiltrate Agrobacterium tumefaciens cell cultures into Nicotiana benthamiana leaves; image epidermal cells by confocal microscopy after incubation at room temperature for 3 days.
Chlamydomonas reinhardtii cells are grown in TRIS-acetate-phosphate medium; -sulfur medium is prepared by replacing sulfate with the corresponding chloride salts.
Apply JC-D7 at 100 µM in HBSS at 25°C for 1.5 h.
For fixed plant tissues, sequentially wash twice in PBS and twice in HBSS buffer.

4. Controls
4.1 Positive control: Overexpression of mitochondria-targeted GFP-labeled polyphosphate kinase (mgPPK) increases JC-D7 fluorescence in SH-SY5Y cells.
4.2 Negative control: Overexpression of mitochondria-targeted GFP-labeled exopolyphosphatase (mgPPX) decreases JC-D7 fluorescence in mitochondria of cultured cells. Lines expressing mPPK1-mCit show no colocalization with JC-D7-stained particles.
4.3 Autofluorescence control: Control experiments show low levels of autofluorescence.
4.4 Buffer control: Buffer controls are used for comparison with JC-D7/polyP fluorescence. Buffer controls are used for comparison with Budit spectra in yeast extract experiments.

5. Detection and Analysis
5.1 Instrument types:
Fluorescence microplate readers are used for in vitro assays, and confocal laser scanning microscopes are used for live cell and tissue imaging. For extracted polyP detection, fluorescence intensity is measured using a microplate reader in 96-well black polypropylene plates. For confocal imaging, maintain illumination intensity at 0.1 to 0.2% of laser output to avoid phototoxicity, and set the pinhole to an optical section of approximately 2 μm. For images comparing fluorescence levels across different cells, maintain imaging settings at the same level. For cell-free assays, fluorescence measurements are performed at ambient temperature.
5.2 Wavelength settings:
In vitro microplate reader: excitation 390 nm; emission recorded from 450 to 700 nm.
Cell-free detection: excitation 390 nm, bandwidth 9 nm; emission 530 nm, bandwidth 20 nm, detector gain 50.
Yeast extract detection: emission 530 nm, bandwidth 9 nm, automatic gain adjustment.
Extracted polyP microplate reader: excitation 405 nm, emission 535 nm.
Confocal imaging: excitation 405 nm; emission above 450 nm.
Primary neuron and astrocyte imaging: excitation 405 nm; emission detection 505 to 590 nm.
JC-D7 fluorescence binding curve: emission measured at 535 nm.
Plant/Chlamydomonas confocal: excitation 405 nm, emission 480-510 nm, using a GaAsP detector.
5.3 Result analysis:
JC-D7 exhibits a turn-on fluorescence response with polyP, with a 54.28-fold increase in fluorescence. The quantum yield of JC-D7 is 0.007 before addition of polyphosphate and 0.38 after addition. The visual detection limit of JC-D7 for polyP is 2 μg/mL.
JC-D7 shows a stronger fluorescence response to longer-chain polyP compared to shorter polymers. JC-D7/polyP fluorescence increases with increasing polyP concentration and chain length, up to 40-fold higher than buffer control. PolyP with average chain lengths of 4 to 1472 P subunits is detected; at 0.5 and 1 µg/mL polyP, the signal is low (1.3-4.4 times higher than control). When polyP concentration is above 10 µg/mL and average chain length exceeds 79 P subunits, JC-D7 fluorescence saturates. JC-D7 allows semi-quantitative assessment of polyP; absolute quantification is not possible for samples with unknown chain composition.
JC-D7-stained polyP fluorescence intensity shows a linear response with polyP concentration in the range of 0.5-36 μM. The detection limit is below 0.5 μM. Linearity decreases when polyP concentration exceeds approximately 50 μM, and fluorescence intensity reaches maximum at polyP concentrations of approximately 100-120 μM. Changes in JC-D7 concentration (e.g., 20-40 μM) do not alter the sensitivity of the response, and polyP chain length has no significant effect on the linear response. Plankton lysis buffer may reduce sensitivity by approximately 16%, but the detection limit and linear range are unaffected (detection limit approximately 0.5 μM, linear range approximately 0.5-30 μM). The detection limit can be reduced to approximately 0.2 μM, although sensitivity decreases at this lower value. JC-D7 quantification is specific for polyP, with no nucleic acid interference.
JC-D7 does not respond to inorganic phosphate up to 50 mM. JC-D7 shows no significant fluorescence increase with DNA, RNA, sodium orthophosphate, ATP, AMP, GTP, GMP, CTP, or CMP. JC-D7 exhibits a turn-on response with heparin.
In yeast extracts, JC-D7 fluorescence at 530 nm is applicable, the Budit spectrum matches the yeast extract spectrum, and the signal increases with chain length and concentration, but not proportionally; the lowest Budit spectrum (polyP4-6) is 2.8 times higher than the buffer control. Yeast strains with different polyP accumulation capacities can be classified based on JC-D7 fluorescence signal.
FeSO₄, CuSO₄, CoCl₂, ZnSO₄, PbNO₃, and HgCl₂ attenuate polyP-induced JC-D7 fluorescence; EDTA reduces quenching by certain metal solutions. SDS at ≥0.1% decreases the fluorescence signal; other tested salts and detergents have no significant effect.
JC-D7 staining can reveal the localization of polyP in different compartments; suggested locations include mitochondria, membrane vesicles, or polyP particles. JC-D7 staining labels polyP particles in cells expressing PPK1-mCit; punctate JC-D7-positive structures colocalize with PPK1-mCit and PPXc-mChe in hypocotyl and root cells. In Chlamydomonas reinhardtii, JC-D7-stained polyP particles appear green, and chlorophyll autofluorescence appears magenta. JC-D7-stained structures are not reproducibly detected in Col-0 wild-type plants.
JC-D7 can record dynamic changes in intracellular polyP; FCCP (HY-100410) treatment induces a slow, gradual decrease in JC-D7 fluorescence, GPN (GPDA) (HY-16710) treatment causes a sharp increase in JC-D7 fluorescence, and Ionomycin (HY-13434) treatment causes a similar increase. JC-D7 detects higher polyP levels in PINK1- or LRRK2-deficient cells. Stained live cells can be analyzed using confocal microscopy; widefield fluorescence microscopy produces a diffuse staining pattern. JC-D7 fluorescence intensity is significantly higher in 20 mM HEPES buffer (pH 7.4) than in salt-containing buffers such as KCl, NaCl, or CaCl2.