CY7-SE triethylamine does not bind to a biological target; it is a chemical labeling reagent. The succinimidyl ester (SE) group reacts specifically with primary amines (-NH2) on lysine residues or protein N-termini under mild conditions to form stable covalent amide bonds. The CY7 fluorophore provides NIR fluorescence for detection and imaging.

Protocol 1: Preparing proteins 1) Please prepare the protein (antibody) concentration to 2 mg/mL in order to achieve the greatest labeling effect. 2) The protein solution has a pH of 8.5±0.5. One milligram of sodium bicarbonate (1M) should be added to the pH if it is less than 8.0. 3) The labeling efficiency will be significantly decreased if the protein content is less than 2 mg/mL. The range of suggested final protein concentration is 2–10 mg/mL for best labeling efficiency. 4) To ensure optimal labeling efficacy, the protein needs to be in a buffer free of ammonium ions and primary amines, like Tris or glycine. 2. Dye preparation (using, for instance, CY3-NHS ester) To create a 10 mM stock solution, fill the CY3-NHS ester vial with anhydrous DMSO. Use a pipette or vortex to thoroughly mix. 3. The dosage calculation for dye. The amount of protein to be labeled determines how much CY3-NHS ester is needed for the reaction; the ideal molar ratio for CY3-NHS is roughly 10. For instance: Assuming that 500 μL 2 mg/mL IgG (MW=150,000) is the necessary marker protein, dissolve 1 mg of CY3-NHS ester in 100 μL DMSO to get the 5.05 μL of CY3-NHS ester that is needed. The following is the precise calculating procedure: 1) mg/mL(IgG) ×mL(IgG)/MW(IgG) = 2 mg/mL × 0.5 mL/150,000 mg/mmol = 6.7×10-6 mmol 2) moles(CY3-NHS ester) = moles(IgG) × 10=6.7 × 10-6 mmol×10=6.7 × 10-5 mmol 3) uL(CY3-NHS ester) = mmol(CY3-NHS ester) ×MW(CY3-NHS ester)/mg/μL(CY3-NHS ester) = 6.7 ×10-5 mmol ×753.88 mg/mmol/0.01 mg/μL=5.05 μL (CY3-NHS ester) 4. Start the coupling reaction. 1) Add the required quantity of recently made CY3-NHS ester (10 mg/mL). A 0.5 mL protein sample should be added gradually to the solution, gently shaken to combine, and then quickly centrifuged to gather the material at the reaction tube's bottom. To prevent denaturation and inactivation of protein samples, do not mix them equally. 2) After placing the reaction tube in a dark location, gently incubate it for 60 minutes at room temperature. After 10 to 15 minutes, gently reverse the response. 5. Make the conjugate pure An example of a SepHadex G-25 column purification process for a dye-protein conjugate is provided below. br/> First, prepare the SepHadex G-25 column as directed by the manufacturer. 2) Fill the SepHadex G-25 column to the brim with the reaction mixture (from "Run conjugation reaction"). 3) Add PBS (pH 7.2–7.4) as soon as the sample is running below the top resin surface. To finish the purification of the chosen sample, add additional PBS (pH 7.2–7.4). The appropriate dye-protein conjugate-containing fractions were combined.

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In vitro, CY7-SE triethylamine is used as a fluorescent labeling agent. It is added to solutions containing the target protein or peptide, where the SE group reacts with exposed amines. The resulting labeled biomolecules can be used in assays such as flow cytometry, fluorescence microscopy, or immunoassays, where the CY7 label allows detection in the NIR channel with minimal background autofluorescence from biological samples.

CY7-SE triethylamine is not administered directly for in vivo activity. It is used to label targeting ligands (e.g., antibodies, peptides) that are then administered intravenously to animal models. The CY7 fluorophore provides NIR fluorescence, enabling deep-tissue imaging in vivo due to reduced light scattering and absorption at NIR wavelengths. This allows non-invasive tracking of biodistribution and tumor accumulation.

For in vitro enzyme assays, CY7-SE triethylamine is not typically used as a substrate or inhibitor. It is used in protein labeling reactions. Labeling protocol: 100 microg of protein (e.g., antibody) at 1 mg/mL in PBS (pH 7.2-7.4) is mixed with 2-10 microL of 5-20 mM CY7-SE triethylamine in DMSO (2-20% final DMSO). The reaction is incubated for 60 minutes at room temperature with gentle mixing. Unreacted dye is removed by dialysis or size-exclusion chromatography. The degree of labeling (DOL) is determined spectrophotometrically by measuring absorbance at 750 nm and 280 nm.

For cell-based assays, CY7-labeled antibodies or peptides are used in cellular uptake and binding studies. Cells are seeded in 8-well chamber slides or 96-well plates and cultured overnight. The CY7-labeled targeting probe is added to the culture medium at a final concentration of 1-10 microg/mL and incubated for 1-4 hours at 37degC. Cells are then washed with PBS to remove unbound probe, fixed with 4% paraformaldehyde (optional), and counterstained with Hoechst 33342 for nuclei. Fluorescence imaging is performed using a microscope or plate reader with excitation/emission at 750/775 nm. To assess non-specific binding, parallel wells are co-incubated with a 50-fold excess of unlabeled antibody.

For in vivo imaging studies, CY7-labeled antibodies (e.g., anti-EGFR antibody conjugates) are administered via tail vein injection to mice (6-8 weeks old, 20-25 g) bearing subcutaneous tumor xenografts. A typical dose is 50-100 microg of labeled antibody per mouse in 100-200 microL PBS (pH 7.4). In vivo fluorescence imaging is performed at various time points (e.g., 1, 6, 12, 24, 48, 72 hours post-injection) using an NIR imaging system (excitation 745-755 nm, emission 800 nm bandpass filter). At the terminal time point, mice are euthanized, and major organs (tumor, liver, spleen, kidneys, lungs, heart) are excised for ex vivo fluorescence imaging to quantify biodistribution. Fluorescence signal is expressed as radiant efficiency [(p/s/cm2/sr)/(microW/cm2)] or as percentage of injected dose per gram of tissue.

CY7-SE triethylamine has a molecular weight of 881.11 Da (free base). The compound is supplied as a solid and should be stored at -20degC, protected from light and moisture. Stock solutions (10-20 mM) are prepared in anhydrous DMSO; once prepared, the solution should be stored at -80degC for up to 6 months or at -20degC for up to 1 month. For protein labeling, the dye is typically used in DMSO solution. The triethylamine salt form is used to stabilize the reactive NHS ester during storage.

As a reactive labeling reagent, CY7-SE triethylamine is handled with care to avoid skin contact and inhalation of dust. Standard laboratory safety practices (e.g., use of gloves, lab coat, and eye protection) should be followed. This product is intended for research use only and not for human diagnostic or therapeutic applications. No significant toxicity has been reported at typical research concentrations.

[1]. Ptaszek M. Rational design of fluorophores for in vivo applications. Prog Mol Biol Transl Sci. 2013;113:59-108.

[2]. Shindy, H. A. (2017). Fundamentals in the chemistry of cyanine dyes: A review. Dyes and Pigments, 145, 505–513. doi:10.1016/j.dyepig.2017.06.029.

CY7-SE triethylamine is classified as a fluorescent dye used in NIR imaging. The CY7 fluorophore is a heptamethine indocyanine-type cyanine dye with excellent water solubility due to sulfonate groups. The succinimidyl ester (SE) form allows covalent conjugation to primary amines via amide bond formation. The triethylamine counterion is used to stabilize the reactive NHS ester during storage. Common applications include flow cytometry, immunofluorescence (IF), fluorescence in situ hybridization (FISH), Western blotting, and in vivo imaging. The compound is also available as a free acid or as other reactive forms (e.g., NHS ester, maleimide, azide) for different labeling strategies.

C45H60N4O10S2

881.11

CY7-SE;477908-53-5

Dark purple to dark blue solid powder

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.  (2). This product is not stable in solution, please use freshly prepared working solution for optimal results.

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

DMSO :~83.33 mg/mL (~94.57 mM)
H2O :~10 mg/mL (~11.35 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (2.36 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (2.36 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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 (Please use freshly prepared in vivo formulations for optimal results.)