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| Targets |
The compound has no specific biological target. It is a fluorescent labeling reagent used to covalently tag primary amines on biomolecules. The NHS ester is the reactive moiety that reacts with the ε-amino group of lysine residues or the N-terminal amine of proteins or peptides. The Cy3.5 fluorophore is then used for detection. The dye does not have a therapeutic mechanism.
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| ln Vitro |
The compound is not a drug and has no direct biological activity. Its optical properties are characterized by UV-Vis and fluorescence spectroscopy. In PBS (pH 7.4), the absorption maximum (λmax) is 591 nm, and the fluorescence emission maximum (λem) is 604 nm. The dye has high photostability. Conjugation to proteins does not significantly alter their biological activity. The labeling efficiency is typically 1-10 fluorophores per protein. The dye is not cytotoxic at the concentrations used for labeling (0.1-10 uM).
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| ln Vivo |
Cy3.5 NHS ester is used for in vivo imaging after conjugation to targeting ligands (e.g., antibodies, peptides). The conjugate is administered intravenously to mice, and fluorescence imaging is performed using a red filter set (λex 580-600 nm, λem 600-620 nm). The compound itself is not administered; the conjugate is the imaging agent. No therapeutic activity is reported.
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| Enzyme Assay |
Not applicable. Cy3.5 NHS ester is a fluorescent dye, not a standard enzyme inhibitor. Its purity (>95%) is assessed by HPLC. The structure is confirmed by ¹H NMR and mass spectrometry (ESI-MS, expected [M+H]+ m/z 736). Physicochemical properties: MW 735.75, red solid, soluble in DMSO and DMF, insoluble in water. For conjugation to primary amines, the dye (1-10 mg/mL in DMSO) is added to the protein or peptide solution (1-10 mg/mL in 0.1 M bicarbonate buffer, pH 8.5) at a molar ratio of 5-20:1 (dye:protein). After incubation for 1-4 h at room temperature, the conjugate is purified by size-exclusion chromatography.
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| Cell Assay |
For cellular imaging, cells are incubated with a Cy3.5-conjugated antibody or peptide (1-10 ug/mL) for 1-4 h at 37degC. After washing, the cells are imaged by fluorescence microscopy using a red filter set (λex 540-580 nm, λem 590-620 nm). The mean fluorescence intensity (MFI) is quantified by flow cytometry. For cytotoxicity, cells are treated with Cy3.5 NHS ester (1-100 uM) for 24-48 h, and viability is measured by MTT assay. The CC₅0 is expected to be >100 uM.
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| Animal Protocol |
For in vivo imaging, female BALB/c nude mice (6-8 weeks old, n=5/group) bearing subcutaneous tumor xenografts are injected intravenously (tail vein) with a Cy3.5-conjugated targeting probe (10-50 ug/mouse). At 1, 4, 8, 24, 48 h post-injection, mice are anesthetized, and whole-body fluorescence imaging is performed using an IVIS Spectrum imaging system (λex 580 nm, λem 620 nm). The fluorescence intensity in the tumor region is quantified. Mice are sacrificed, and the tumors and major organs are harvested for ex vivo imaging.
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| ADME/Pharmacokinetics |
Not applicable. Cy3.5 NHS ester is an imaging reagent, not a drug. It is not intended for systemic administration as a free dye. The dye is rapidly cleared from the bloodstream. For research use, it is stored as a powder at -20degC (protected from light). It is soluble in DMSO.
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| Toxicity/Toxicokinetics |
For Cyanine 3.5 bromide, hazard statements: H315 (Causes skin irritation), H319 (Causes serious eye irritation), H335 (May cause respiratory irritation). Signal word: Warning. Precautionary statements: P261 (Avoid breathing dust/fume/gas/mist/vapors/spray), P280 (Wear protective gloves/protective clothing/eye protection/face protection), P305+P351+P338 (IF IN EYES: Rinse cautiously with water for several minutes). The dye is light-sensitive; protect from light. Storage: at -20degC, sealed, protect from light.
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| References |
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| Additional Infomation |
Cyanine 3.5 bromide (Cy3.5 NHS ester bromide) is a research-grade red fluorescent dye used for labeling peptides, proteins, and oligonucleotides. It is not an FDA-approved drug. It is used in fluorescence microscopy, flow cytometry, and microarray analysis. For research use only, not for diagnostic or therapeutic applications.
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| Molecular Formula |
C42H44BRN3O4
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|---|---|
| Molecular Weight |
734.72
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| Related CAS # |
Cyanine 3.5 chloride;2231670-86-1
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| Appearance |
Yellow to brown solid powder
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| Synonyms |
Cy3.5 NHS ester bromide; Cy 3.5 bromide
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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 | 1.3611 mL | 6.8053 mL | 13.6106 mL | |
| 5 mM | 0.2722 mL | 1.3611 mL | 2.7221 mL | |
| 10 mM | 0.1361 mL | 0.6805 mL | 1.3611 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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