The target of Carboxy-SNARF 1 is not a specific protein but rather the hydrogen ions (protons) in the intracellular or extracellular environment. As a pH indicator, its fluorescence properties are directly modulated by the concentration of H+ ions (pH). The mechanism involves a reversible protonation/deprotonation of the phenolic hydroxyl group in the fluorophore. At low pH (acidic), the probe is protonated, and the emission maximum is around 590 nm (orange-red). At high pH (basic), the probe is deprotonated, and the emission maximum shifts to around 640 nm (deep red). The ratio of fluorescence intensities at 640 nm and 590 nm (or the ratio of emission at two wavelengths) is proportional to pH, following a sigmoidal function. The pKa of Carboxy-SNARF 1 is approximately 7.5, making it ideal for measuring physiologically relevant pH changes (pH 6.5-8.5). The probe is not toxic at typical loading concentrations (1-20 uM). Unlike single-wavelength indicators (e.g., BCECF), the ratiometric measurement eliminates artifacts due to dye leakage, photobleaching, and differences in cell loading. Thus, Carboxy-SNARF 1 is a preferred tool for precise pHi measurements. The AM ester form is cell-permeable and can be loaded into cells without disrupting the plasma membrane. After intracellular cleavage, the free acid form is trapped and remains functional for several hours.

In vitro characterization of Carboxy-SNARF 1 involves determining its fluorescence properties in solutions of known pH. To generate a calibration curve, the free acid form of Carboxy-SNARF 1 is dissolved in buffers of different pH values (e.g., pH 6.0, 6.5, 7.0, 7.5, 8.0, 8.5) at a constant concentration (e.g., 1-10 uM). The fluorescence emission spectrum is recorded using a fluorometer with excitation at 540 nm (or 488 nm). The emission intensities at 590 nm and 640 nm are measured, and the ratio (F₆40/F₅₉0) is plotted against pH. The resulting curve is fitted with a sigmoidal (Boltzmann) equation to determine the pKa and the dynamic range. The probe shows high sensitivity to pH with minimal response to other ions (e.g., Ca2+, Mg2+). It is also resistant to photobleaching and has a high quantum yield. In cells, the pKa may shift slightly due to the intracellular environment (e.g., viscosity, protein binding), so an in situ calibration (using ionophores such as nigericin in buffers of known pH) is recommended to accurately convert the ratio to pHi. The dye is stable at room temperature for short periods but should be stored frozen at -20degC to prevent degradation. The AM ester is particularly susceptible to hydrolysis, so it should be stored desiccated and protected from light. In cell-free assays, the probe is often used as a reference or standard.

In vivo applications of Carboxy-SNARF 1 are limited to ex vivo measurements (e.g., in isolated cells, tissue slices, or small organisms like C. elegans). The probe is not typically used for whole-body imaging in animals because it is not targetable and does not possess tissue-penetrating properties. However, it can be used in ex vivo tissue samples to measure pH in specific cell types. For example, hippocampal brain slices can be loaded with Carboxy-SNARF-1 AM to measure pH changes during neuronal activity. In zebrafish embryos, the probe can be microinjected to measure pH in specific regions. The probe has also been used to measure pH in perfused isolated organs (e.g., kidneys, hearts). In all these applications, the ratiometric approach allows for accurate pH measurements in thick samples, as the ratio is independent of path length. For live-cell imaging in whole animals, other pH indicators with longer wavelengths (e.g., near-infrared) are preferred. Therefore, the in vivo “activity” is not defined as a therapeutic effect but rather as the ability to accurately report pH in real time. The probe is non-toxic at the concentrations used (1-10 uM for loading, 1-2 hours). The AM ester is sometimes associated with cytotoxicity due to the accumulation of the acid byproduct (acetic acid), but this can be minimized by using low concentrations and thorough washing. The in vivo half-life of the free acid form is determined by cellular export (via organic anion transporters), which varies by cell type. In most cells, the dye remains trapped for 2-6 hours.

Protocol for in vitro calibration: To prepare a pH calibration curve, dissolve Carboxy-SNARF 1 free acid in a series of pH calibration buffers (e.g., 100 mM MES, HEPES, or Tris, with 100 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 1 uM nigericin). The buffers should cover the range of pH 5.5 to 8.5 in 0.5 pH unit increments. Add the dye to a final concentration of 1-10 uM. Incubate for 10 minutes at room temperature to equilibrate. Using a fluorometer or a fluorescence plate reader (excitation: 540 nm or 488 nm), record the emission spectra from 570 nm to 700 nm. Alternatively, measure the emission intensity at 590 nm and 640 nm. Calculate the ratio (F640/F590). Plot the ratio against pH and fit a sigmoidal curve using the equation: Ratio = R_min + (R_max - R_min)/(1 + 10^{pKa - pH}), where R_min and R_max are the ratios at extreme pH values. The pKa is determined from the curve. For in situ calibration, the same procedure is performed on cells loaded with Carboxy-SNARF-1 AM, using buffers containing 10 uM nigericin and 10 uM valinomycin (to equilibrate pH across the membrane) and clamping external pH to known values. The F640/F590 ratio in cells is then measured, and the pH is calculated by interpolation from the calibration curve. Ensure that the dye concentration is within the linear range and that the measurements are performed under identical instrumental settings. The protocol for in vitro binding is not applicable, as the probe does not bind to a specific receptor.

In vitro cellular experiments using Carboxy-SNARF 1 are performed to measure intracellular pH (pHi). The AM ester form is used for live-cell loading. For adherent cells (e.g., HeLa, HEK293, primary neurons), cells are seeded on glass coverslips or in 96-well optical plates and allowed to adhere overnight. The culture medium is removed, and the cells are incubated with 1-10 uM Carboxy-SNARF-1 AM (from a 1-10 mM stock in DMSO) in HBSS (Hanks' Balanced Salt Solution) with 0.02% Pluronic F-127 to aid dispersion, for 30-60 minutes at 37degC in the dark. After loading, the cells are washed twice with HBSS to remove extracellular dye and allow de-esterification (15-30 minutes at 37degC). For suspension cells (e.g., lymphocytes, leukemia cells), 1-2 × 10⁶ cells are loaded in a similar manner and washed by centrifugation. The loaded cells are then mounted on a microscope stage or placed in a fluorometer cuvette. Fluorescence imaging: Use a confocal or fluorescence microscope with excitation at 540 nm (or 488 nm). Emitted fluorescence is collected at two channels: a “green” channel (e.g., 590/20 nm) and a “red” channel (e.g., 640/20 nm). Ratiometric images are acquired at time intervals (e.g., every 30 seconds). For flow cytometry, use a flow cytometer with excitation at 488 nm and detectors for FL-2 (590 nm) and FL-3 (640 nm). Record the ratio on a cell-by-cell basis. To change pH, add agents such as NH4Cl to induce alkalosis, or propionate/acetate to induce acidosis. For drug studies, add the drug of interest and monitor pHi changes over time. Calibration at the end of each experiment is performed by adding 10 uM nigericin and 10 uM valinomycin in buffers of known pH (as described above). The ratio-pH calibration curve is used to convert the fluorescence ratio to absolute pH values. Data analysis: for each cell or well, calculate the F640/F590 ratio, then use the calibration curve to compute pH. The average pH and changes over time are plotted. This protocol is widely used in cell physiology, apoptosis research, and drug discovery.

In vivo animal experiments using Carboxy-SNARF 1 are not common, as the probe is not suited for whole-body imaging due to its visible wavelength emission and the need for local loading. However, it can be used in ex vivo tissue slices or in small transparent organisms. For example, in Caenorhabditis elegans, the AM ester can be loaded by incubating worms in M9 buffer containing 10 uM Carboxy-SNARF-1 AM and 0.02% Pluronic F-127 for 1-2 hours at 20degC. The worms are then washed and placed on an agarose pad on a slide. Ratiometric imaging can be performed using a confocal microscope with 540 nm excitation and detection at 590 nm and 640 nm. The pH in specific cells (e.g., pharyngeal muscle) can be measured before and after stressors (e.g., heat shock, drug treatment). For tissue slices (e.g., brain slices, kidney slices), the slices are incubated with 5-10 uM Carboxy-SNARF-1 AM for 30-60 minutes in oxygenated artificial cerebrospinal fluid (ACSF) at 37degC, washed, and then placed in a perfusion chamber on a microscope. Imaging is performed as described. For isolated perfused organs (e.g., heart, kidney), the organ is perfused with dye-containing buffer, and pH changes are measured using a fiber-optic probe or by collecting surface fluorescence. These ex vivo protocols allow for the measurement of tissue pH under controlled conditions. For in vivo use in mice, the dye would need to be injected into a specific tissue or organ, and then a fiber-optic probe can be used to measure fluorescence. This is not a standard application. The AM ester may cause adverse effects if injected systemically due to the release of acetaldehyde and formaldehyde from the AM group. Therefore, it is not recommended for systemic administration. The compound is generally non-toxic in the described ex vivo applications at the concentrations used.

Pharmacokinetic data for Carboxy-SNARF 1 are not applicable, as it is not intended for systemic administration. The free acid form is membrane-impermeant and is rapidly excreted if introduced into the circulation. The AM ester form is cell-permeable and is hydrolyzed by esterases in the plasma and within cells. If injected into an animal, the AM ester would be rapidly cleared and metabolized, with the free acid being excreted in the urine. Therefore, no PK studies have been performed for this compound. The compound is stable as a solid when stored at -20degC, protected from light. For solution, the AM ester should be stored at -80degC in small aliquots to prevent hydrolysis, as it is sensitive to moisture. The free acid form can be stored at -20degC in solution for several months. The compound is not metabolized by CYP450 enzymes; it is simply de-esterified and then excreted. The half-life in cells is determined by the rate of export (via MRP or other organic anion transporters). For most cells, the dye remains trapped for 2-6 hours. The quantum yield and photostability are excellent, allowing for long-term imaging studies. The probe does not bind to serum proteins to a significant extent, so its intracellular concentration is directly proportional to the loading concentration. For in vitro use, the dye is typically used at 1-10 uM. The cellular concentration can be estimated, but for ratiometric measurements, absolute concentration is not needed. Thus, PK is not a concern for typical biological applications.

No acute or chronic toxicity data are available for Carboxy-SNARF 1 because it is a fluorescent probe and not a therapeutic agent. In cellular assays, it is generally considered non-toxic at concentrations used for pH measurements (1-10 uM for the AM ester). However, the AM ester releases acetic acid upon hydrolysis, which can acidify the cytoplasm and cause toxicity if the loading concentration is too high or the loading time too long. Therefore, it is recommended to keep the AM ester concentration below 20 uM and the loading time under 1 hour. Many labs use 5 uM for 30 minutes and then wash. The free acid form is not toxic to cells up to 100 uM, as it is membrane-impermeant and remains in the extracellular medium. In animal studies, the AM ester is not injected systemically due to its potential to cause metabolic acidosis and the release of formaldehyde. For ex vivo studies, the concentration of the AM ester used (1-10 uM) is well tolerated by tissue slices and isolated organs. No genotoxicity, carcinogenicity, or reproductive toxicity studies have been reported. The compound is considered a laboratory chemical, and standard safety precautions should be followed: wear gloves, safety glasses, and a lab coat. The AM ester is sensitive to moisture and light; handle with care. The compound is not classified as a hazardous substance under GHS, but it may cause eye irritation if powder contacts eyes. In case of skin contact, wash with soap and water. For disposal, follow institutional guidelines for chemical waste. The probe does not contain any heavy metals or toxic elements. The LD₅0 has not been determined.

[1]. Fluorescence behavior of the pH-sensitive probe carboxy SNARF-1 in suspension of liposomes. Photochem Photobiol. 2001 Jul;74(1):8-13.

Other information: Carboxy-SNARF 1 is a research-grade fluorescent probe for laboratory use only. It is not approved for clinical diagnostics or therapeutics. It is widely used in cell biology, physiology, and pharmacology to study intracellular pH regulation in various cell types, including neurons, cardiomyocytes, immune cells, and cancer cells. The probe is available as the free acid (carboxylate form, which is cell-impermeant) and as the AM ester (cell-permeant). The product is typically supplied as a powder (the free acid) or as a lyophilized solid (the AM ester). The CAS number is 126208-12-6 for the mixture of 5- and 6-carboxy isomers. The individual isomers are also available but are less common. The molecular weight is 453.45 g/mol. The excitation maximum is 540 nm (with a secondary peak at 488 nm). The emission maximum is pH-dependent: at pH 6.0, λem ≈ 590 nm; at pH 8.0, λem ≈ 640 nm. The pKa is approximately 7.5. The probe is soluble in DMSO and in water (for the free acid). The AM ester is soluble only in DMSO. Storage: the powder should be stored at -20degC, desiccated, and protected from light. Solutions in DMSO (10 mM) can be stored at -80degC for up to 6 months. Avoid repeated freeze-thaw cycles. For shipping, it is often stored on blue ice. The compound is not FDA-approved for any indication. It is available from chemical suppliers, but no specific supplier names are provided. Researchers should always prepare a fresh working solution for each experiment. The AM ester is prone to hydrolysis, so it is best to prepare a 10 mM stock in dry DMSO and store it in small aliquots. The working concentration should be determined empirically for each cell type, as the loading efficiency varies. For flow cytometry, 2-5 uM is often sufficient, while for microscopy, 5-10 uM may be needed. For calibration, use the ionophore nigericin (a K+/H+ exchanger) and valinomycin (a K+ ionophore) to equilibrate pH across the cell membrane. The dye can be used to monitor pH changes in response to agonists, inhibitors, or changes in extracellular pH. The ratiometric method corrects for variability in dye loading, photobleaching, and movement of cells out of focus. The dye can also be used to measure pH in acidic organelles (e.g., lysosomes) if the dye is targeted to the organelle (but the free acid form is not targeted). For lysosomal pH, other dyes (e.g., LysoSensor) are available. Carboxy-SNARF 1 is an excellent choice for cytoplasmic pH measurements. The product is intended for research use only and not for diagnostic procedures. For detailed protocols, consult the original literature or the manufacturer's instructions. As the compound is not a drug, no clinical trials have been conducted. Always refer to the safety data sheet (SDS) before use. The information provided is for educational purposes and does not constitute a recommendation for use. No specific supplier information is included. The data in this summary were compiled from publicly available information and standard protocols.

C26H20NO4

411.447468757629

410.139

126208-12-6

155885758

Dark purple to black solid powder

1

6

3

31

964

0

O1C2C=C(C=CC=2C(C2C=CC=CC=2C(=O)O)=C2C=CC3=CC(C=CC3=C12)=O)N(C)C.[2H-]

XULVEFCFZBLKOP-UHFFFAOYSA-N

InChI=1S/C26H19NO4.H/c1-27(2)16-8-11-21-23(14-16)31-25-18-12-9-17(28)13-15(18)7-10-22(25)24(21)19-5-3-4-6-20(19)26(29)30;/h3-14H,1-2H3,(H,29,30);/q;-1

2-[10-(dimethylamino)-3-oxobenzo[c]xanthen-7-yl]benzoic acid;hydride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~10 mg/mL (~22.05 mM)

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.)