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| Targets |
Cy 3 (Non-Sulfonated) (triethylamine) does not bind to a biological target; it is a chemical labeling reagent. As a non-sulfonated Cy3 dye, it has a hydrophobic character compared to sulfonated versions. The dye can be used for non-covalent (electrostatic or hydrophobic) interactions or covalently incorporated via functional groups (if present; the triethylamine salt form is typically for storage and the dye may be used as a free acid or NHS ester). The Cy3 fluorophore provides green fluorescence for detection.
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| ln Vitro |
Guidelines: The following is the suggested course of action. This protocol should be customized to meet your unique requirements, as it just offers guidelines. Protein Labeling [2]: 1. For 50 minutes at 37 °C, add 100 μL of protein to Cy 3 Non-Sulfonated Triethylamine in 96 Black Micro-Wells. Proceed with the regular protocol for incubating the cells. 2. Use BioTek to scan the plate and gather data at 470 nm for excitation and 515 nm and 565 nm for emission wavelengths.
In vitro, Cy 3 (Non-Sulfonated) triethylamine can be used for high-density single-particle tracking of living cells without the use of UV illumination and cytotoxic additives. A typical protein labeling protocol: add 100 microL of protein (e.g., antibody at 0.1-1 mg/mL) to Cy 3 Non-Sulfonated triethylamine in 96-well black microplates. Incubate at 37degC for 50 minutes. Then proceed with the standard protocol for incubating the cells. Scan the plate and gather data at 470 nm excitation and 515 nm and 565 nm emission wavelengths. |
| ln Vivo |
Cy 3 (Non-Sulfonated) triethylamine is not administered directly for in vivo activity. The non-sulfonated version is more hydrophobic than its sulfonated counterpart, which affects solubility and biodistribution. It may be used for labeling lipophilic biomolecules or for applications where minimal charge is desired. For in vivo imaging, the sulfonated (water-soluble) version is typically preferred. This product is more suitable for in vitro and ex vivo labeling applications.
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| Enzyme Assay |
For in vitro protein labeling (via NHS ester form, if the triethylamine salt is converted or the dye is obtained as an NHS ester): Dissolve 1 mg of protein (e.g., bovine serum albumin, BSA, or antibody) in 1 mL of 0.1 M sodium bicarbonate buffer (pH 8.3). Prepare a solution of the Cy3 NHS ester (synthesized from the triethylamine salt) in anhydrous DMSO at 1-10 mM concentration. Add 5-20 equivalents of the Cy3 NHS ester to the protein solution (e.g., 10 microL of 10 mM Cy3 in DMSO for 1 mg/mL protein solution; DMSO concentration <5%). Incubate at room temperature for 1 hour with gentle mixing. Protect the reaction from light. Remove unreacted dye by dialysis or size-exclusion chromatography (e.g., Sephadex G-25). The degree of labeling (DOL) is calculated by measuring absorbance at 550 nm (Cy3) and 280 nm (protein).
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| Cell Assay |
For cell-based assays, Cy3-labeled proteins (e.g., antibodies, lectins) are used for fluorescence microscopy. Cells (e.g., 5×10⁴-1×10⁵ cells/well in 8-well chamber slides) are seeded and cultured overnight. The Cy3-labeled probe is added to the culture medium at a final concentration of 1-20 microg/mL and incubated for 1-4 hours at 37degC. For live-cell imaging, cells are washed with PBS (3×) and imaged immediately using a fluorescence microscope with filter sets for Cy3 (Ex ~540-560 nm, Em ~570-590 nm). For fixed-cell imaging, cells are washed, fixed with 4% paraformaldehyde (15 minutes at room temperature), washed again, counterstained with DAPI or Hoechst 33342 for nuclei, mounted, and imaged. For studies requiring single-particle tracking, cells can be incubated with the Cy3-labeled probe at low concentrations (e.g., 0.1-1 microg/mL) to achieve single-molecule resolution, and imaging is performed using total internal reflection fluorescence (TIRF) microscopy or confocal microscopy with high temporal resolution. The non-sulfonated Cy3 dye may exhibit more hydrophobic interactions, which could affect membrane partitioning and cellular uptake compared to sulfonated versions. This property may be advantageous for certain applications such as lipophilic drug tracking or membrane labeling.
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| Animal Protocol |
Cy 3 (Non-Sulfonated) triethylamine is not typically used in animal studies as a standalone agent. The labeled conjugate (e.g., Cy3-labeled antibody) can be administered intravenously (via tail vein) to mice (6-8 weeks, 20-25 g) for imaging of superficial targets (e.g., subcutaneous tumors). A typical dose is 50-100 microg of labeled protein in 100-200 microL PBS. Because Cy3 emits in the visible range (Ex/Em ~550/570 nm), tissue penetration is limited; it is suitable for imaging subcutaneous or surface tissues, but not for deep tissue imaging. In vivo imaging is performed using an imaging system equipped with Cy3 filter sets (excitation 540-560 nm, emission 570-590 nm). Mice are anesthetized with isoflurane during imaging. Imaging is performed at various time points (e.g., 1, 6, 12, 24 hours post-injection). Ex vivo imaging of harvested organs can be done at the terminal time point to assess biodistribution.
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| ADME/Pharmacokinetics |
Cy 3 (Non-Sulfonated) (triethylamine) has a molecular weight of 1255.06 Da (for the triethylamine salt form). The compound is supplied as a solid powder and should be stored at -20degC, protected from light and moisture. Powder is stable for up to 3 years at -20degC and 2 years at 4degC. Stock solutions in DMSO (e.g., 10 mM) should be stored at -80degC for up to 6 months or at -20degC for up to 1 month. Solubility: The non-sulfonated form is less water-soluble than sulfonated versions; it is typically dissolved in DMSO or DMF for conjugation reactions. The triethylamine counterion is used to neutralize the dye (likely present as the carboxylic acid or sulfonic acid free acid form) to improve handling and stability. For in vivo applications, the labeled conjugate (not the free dye) is used, and solubility in saline or PBS depends on the conjugate itself. For conjugation, the dye is typically used in DMSO solution.
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| Toxicity/Toxicokinetics |
Cy 3 (Non-Sulfonated) triethylamine is considered non-toxic at typical research concentrations (microM range). Standard laboratory safety practices should be followed: use of gloves, lab coat, and eye protection. This product is for research use only and not for human diagnostic or therapeutic applications. No significant toxicity has been reported. The non-sulfonated form may have slightly different biological properties (e.g., increased cell membrane partitioning) compared to sulfonated versions, which could influence toxicity at high concentrations; however, at labeling concentrations, no adverse effects are expected.
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| References |
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| Additional Infomation |
Cy 3 (Non-Sulfonated) (triethylamine) (Cyanine3 triethylamine) is a non-sulfonated cyanine dye with a triethylamine counterion. Key features: (1) Non-sulfonated structure, making the dye more hydrophobic and suitable for applications where minimal charge interactions are preferred; (2) Excitation/emission maxima: Ex ~470 nm, Em at 515 nm and 565 nm (bimodal emission spectrum is typical for Cy3 in some formulations); (3) Can be used for high-density single-particle tracking in living cells without the need for UV excitation; (4) The triethylamine counterion is used for salt formation and stabilization. This product is typically used as a fluorescent label for proteins and nucleic acids. Purity is typically ≥98% by HPLC. Related CAS for the free base (non-sulfonated Cy3): 146397-20-8.
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| Molecular Formula |
C49H65N5O14S2
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| Molecular Weight |
1255.06
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| Related CAS # |
Cy 3 (Non-Sulfonated);146397-20-8
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| Appearance |
Solid powder
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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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| 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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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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 | 0.7968 mL | 3.9839 mL | 7.9677 mL | |
| 5 mM | 0.1594 mL | 0.7968 mL | 1.5935 mL | |
| 10 mM | 0.0797 mL | 0.3984 mL | 0.7968 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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