Overview of the SBFI AM Na⁺-Sensitive Fluorescent Indicator and Its Applications
SBFI, fully named sodium-binding benzofuran isophthalate, is a Na⁺-selective fluorescent indicator. It can be used to assess Na⁺ gradients in purified mitochondria, monitor intracellular Na⁺ levels, measure Na⁺ efflux from cells, and, in combination with other fluorescent indicators, analyze the relationships between Na⁺ and Ca²⁺/Mg²⁺ concentrations, intracellular pH, and membrane potential changes.
Although the affinity (Kd) of SBFI for Na⁺ is lower than that of typical Ca²⁺ indicators (such as the affinity of Fura-2 for Ca²⁺), SBFI is still sufficiently sensitive to detect Na⁺ at physiological concentrations in the presence of other monovalent cations. Upon Na⁺ binding, the excitation spectrum of SBFI shifts, enabling ratiometric measurements using 340/380 nm excitation. SBFI can therefore be used together with Fura-2 on the same instrumentation and filter sets.
In solutions containing physiological concentrations of K⁺/Na⁺ (approximately 135 mM), the dissociation constant (Kd) of SBFI for Na⁺ is about 11.3 mM; in the absence of K⁺, the Kd for Na⁺ is about 3.8 mM. SBFI exhibits roughly 18-fold higher selectivity for Na⁺ over K⁺.
In practical applications, the AM ester form of SBFI (SBFI AM) is generally used rather than the free acid form. The free form of SBFI is charged and cannot freely cross the plasma membrane. For intracellular Na⁺ measurements, it must be introduced by microinjection, electroporation, or other delivery methods, which are technically demanding and less reproducible. In contrast, SBFI AM carries acetoxymethyl (AM) ester groups that temporarily mask the charges, rendering the molecule neutral and lipophilic so that it can passively diffuse across the cell membrane. Once inside the cell, intracellular esterases gradually hydrolyze the AM groups (the “esterase hydrolysis incubation” step described below), regenerating the charged, free form of SBFI. The free SBFI is retained within the cell and regains its Na⁺-responsive fluorescent properties. Consequently, loading cells with the membrane-permeant SBFI AM and then relying on esterase-mediated release of free SBFI is essential for achieving stable and reproducible intracellular Na⁺ imaging.
Physicochemical Properties and Storage Conditions
This product is the acetoxymethyl ester (AM ester) form of SBFI (CAS No. 129423-53-6). It is cell-permeant and can be loaded into cells by simple incubation. Typical loading concentrations are 5–10 µM with incubation times of 40 min to 4 h, which may be adjusted according to specific experimental requirements and cell type.
1. Physicochemical Properties
1. Chemical name: 4,4’-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,2-benzofurandiyl)]bis-1,3-benzenedicarboxylic acid 1,1’,3,3’-tetrakis[(acetyloxy)methyl] ester
2. Synonym: Sodium-binding Benzofuran Isophthalate Acetoxymethyl ester
3. CAS No.: 129423-53-6
4. Molecular formula: C₅₆H₅₈N₂O₂₃
5. Molecular weight: 1127.07
6. Ex/Em: 340, 380 / 500 nm
7. Appearance: pale yellow to dark yellow or dark orange solid or viscous/oily material
8. Solubility: soluble in DMSO (10 mM)
2. Storage and Transport
(a) Storage: store at -20 °C under dry, light-protected conditions. Stable for two years.
(b) Transport: ship with ice packs.
Preparation of SBFI AM Working Solutions and Intracellular Na⁺ Imaging Workflow
The following procedures are provided for reference only. Experimental conditions should be optimized according to actual requirements and relevant literature.
1) Preparation of SBFI AM stock solution
Prepare a 1–10 mM SBFI AM stock solution. For example, before the experiment, allow a vial containing 50 µg SBFI AM to equilibrate to room temperature for at least 20 min, then briefly centrifuge to collect the powder. Add 8.8723 µL anhydrous DMSO to fully dissolve the dye and obtain a 5 mM stock solution. If the stock is not used up in a single experiment, aliquot and store at low temperature (frozen).
2) Preparation of 25% (w/v) Pluronic F-127
Weigh 100 mg Pluronic F-127 powder and add 400 µL DMSO to prepare a 25% (w/v) Pluronic F-127 DMSO stock solution. Dissolution can be facilitated by incubating at 40–50 °C for 20–30 min. The solution can be stored at room temperature and does not require refrigeration. If crystals precipitate during storage, reheat to dissolve; this does not affect use. (Other concentrations of Pluronic F-127 may also be prepared, provided that the final concentration of Pluronic F-127 in the working solution is < 0.1% (w/v).)
3) Premixing SBFI AM with Pluronic F-127
Immediately before the experiment, thoroughly mix the SBFI AM stock solution with an equal volume of 25% (w/v) Pluronic F-127.
Note: SBFI AM has poor aqueous solubility and must be premixed with Pluronic F-127 to promote its dispersion and entry into the aqueous loading buffer. The two components should be mixed fresh before use and are not recommended for long-term storage after mixing.
4) Preparation of working solution and cell loading
Dilute the premixed solution into an appropriate volume of cell loading buffer (e.g., physiological buffer such as PBS, or serum-free cell culture medium) to obtain the desired working concentration, then add to the cells for probe loading. The incubation temperature can be either room temperature or 37 °C. Typical loading conditions are 5–10 µM for 40 min–4 h. The optimal loading concentration and incubation time should be determined based on relevant literature and the specific cell type.
5) Esterase hydrolysis incubation
At the end of the loading period, replace the loading solution with probe-free cell loading buffer and incubate for an additional 20–60 min to ensure that intracellular esterases fully hydrolyze the AM groups of SBFI AM, generating the free form of SBFI.
6) Washing
Wash the cells at least once with physiological buffer or serum-free culture medium to reduce extracellular background fluorescence.
7) Fluorescence measurement
Use appropriate fluorescence instrumentation to collect signals at excitation wavelengths of 340 nm and 380 nm and an emission wavelength of 500 nm (the emission detection window may be set within 450–550 nm as needed). Intracellular Na⁺ concentration can be estimated from changes in the 340/380 nm excitation ratio.
Additional Notes
1. If the cell loading buffer uses a bicarbonate-based buffering system, the loading process should be carried out in an atmosphere containing 5% CO₂ to prevent alkalinization due to CO₂ loss.
2. If the cell loading buffer contains serum, the working concentration of the AM probe should be increased appropriately to compensate for the loss of probe caused by binding to serum proteins.
3. For AM-type probes with molecular weights close to or exceeding 1000, it is generally necessary to incubate the cells in probe-free loading buffer for an additional 20–60 min to allow intracellular esterases to fully cleave the AM groups.
4. Optimal loading times and probe concentrations may vary between cell types. Please consult relevant literature and perform empirical optimization before formal experiments.
5. The effective intracellular Kd of the probe may differ from its Kd in solution. The intracellular Kd of SBFI can be calibrated using channel-forming antibiotics (such as gramicidin) to perform in situ calibration, as needed, depending on the specific experimental requirements.
Recommended Aladdin Products
Aladdin Catalog No. | Product Name | CAS / Specification | Role in the Experiment |
– | SBFI AM (Sodium-binding Benzofuran Isophthalate, AM ester) | 129423-53-6 | Primary probe; a cell-permeant Na⁺ fluorescent indicator that enters cells in the AM ester form and, after esterase hydrolysis to free SBFI, is used for intracellular Na⁺ ratiometric imaging (340/380 nm). |
SBFI (Sodium-binding Benzofuran Isophthalate, free acid) | 124549-08-2 | Non–cell-permeant Na⁺ indicator dye; generally used in cell-free solutions or introduced into cells via microinjection, electroporation, or other delivery methods. | |
Pluronic F-127 (Poloxamer F-127) | 9003-11-6 BioReagent for cell culture Granular waxy | Nonionic surfactant; premixed at equal volume with SBFI AM to enhance dispersion and solubility of the AM probe in aqueous solutions and to improve cell loading efficiency. The final concentration should be < 0.1% (w/v). | |
Dimethyl sulfoxide (DMSO, anhydrous) | 67-68-5, anhydrous grade | Organic solvent; used to dissolve SBFI AM to prepare 1–10 mM stock solutions and to dissolve Pluronic F-127 for the 25% (w/v) stock solution. Requires anhydrous conditions, light protection, and low-temperature storage. | |
PBS (Phosphate-buffered saline) | liquid, modified, without calcium chloride and magnesium chloride, sterile-filtered, 1×, pH 7.1–7.5 | Used as one of the “cell loading buffers” to prepare SBFI AM working solutions and for cell incubation/washing, providing near-physiological ionic strength and pH. | |
Serum-free cell culture medium (RPMI serum-free formulation) | RPMI 1640 medium; sterile-filtered, BioReagent, endotoxin-tested, for cell culture | Used as a serum-free “cell loading buffer” for incubating cells and loading SBFI AM. When serum-containing media are used, the working concentration of the AM probe should be increased appropriately. | |
S656979 | Sodium bicarbonate (NaHCO₃) | Animal-free, low endotoxin, for cell culture, ≥99.5% | Component of bicarbonate-based media/buffer systems. Note: when using bicarbonate-buffered solutions, loading should be performed in a 5% CO₂ atmosphere to avoid alkalinization due to CO₂ loss. |
Serum (e.g., newborn calf serum, NCS) | BioReagent, sterile | Used as a medium supplement. | |
Fura-2 AM (Ca²⁺ ratiometric fluorescent probe, Fura-2 acetoxymethyl ester) | 108964-32-5, ≥95% (HPLC) | Ratiometric Ca²⁺ indicator; can share the 340/380 nm dual-excitation imaging system with SBFI, enabling simultaneous monitoring of Na⁺ and Ca²⁺. | |
Gramicidin (channel-forming ionophore) | 1405-97-6≥95% | Channel-forming antibiotic/ionophore; used to equilibrate intra- and extracellular Na⁺ for in situ calibration of SBFI ratio signals, allowing determination of the effective intracellular Kd and standard curve. |
References
a) Negulescu PA et al. Intracellular ion activities and membrane transport in parietal cells measured with fluorescent dyes. Methods Enzymol. 1990;192:38–81.
b) Harootunian et al. Ratio imaging using a newly developed fluorescent sodium indicator in rat fibroblasts. FASEB J. 1988;2:A728.
c) Harootunian et al. Fluorescent ratio imaging of cytosolic free Na⁺ in fibroblasts and lymphocytes. J. Biol. Chem. 1989.
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