Fluorescent dye

Stock Solution Preparation
1. Protein Preparation
To achieve optimal labeling efficiency, prepare the protein (antibody) at a concentration of 2 mg/mL.
(1) The pH of the protein solution should be 8.5±0.5. If the pH is below 8.0, adjust it using 1 M sodium bicarbonate.
(2) If the protein concentration is below 2 mg/mL, the labeling efficiency will significantly decrease. For optimal labeling efficiency, the final protein concentration should range between 2-10 mg/mL.
(3) The protein must be in a buffer free of primary amines (such as Tris or glycine) and ammonium ions, as these can interfere with labeling efficiency.
2. Dye Preparation
Dilute the anhydrous DMSO with CY dye to prepare a 10 mM stock solution. Mix thoroughly using a glass tube or vortex.
Note: It is recommended to aliquot the CY stock solution and store it at -20°C or -80°C, protected from light.
Before use, activate the dye with a condensation solution (500 μg/mL) before proceeding with the labeling experiment.
3. Calculation of Dye Working Solution Volume
The amount of CY dye required for the labeling reaction depends on the amount of protein to be labeled. The optimal molar ratio of CY dye to protein is approximately 10.
Example: If the protein to be labeled is 500 μL of 2 mg/mL IgG (MW=150,000), and 1 mg of CY dye is dissolved in 100 μL of DMSO, the detailed calculation for the required volume of CY dye (using CY3-NHS ester as an example) is as follows:
(1) mmol (IgG) = mg/mL (IgG) × mL (IgG) / MW (IgG) = 2 mg/mL × 0.5 mL / 150,000 mg/mmol = 6.7×10^-6 mmol
(2) mmol (CY3-NHS ester) = mmol (IgG) × 10 = 6.7×10^-6 mmol × 10 = 6.7×10^-5 mmol
(3) μL (CY3-NHS ester) = mmol (CY3-NHS ester) × MW (CY3-NHS ester) / mg/μL (CY3-NHS ester) = 6.7×10^-5 mmol × 917.05 mg/mmol / 0.01 mg/μL

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Instruction for Use
1. Labeling Reaction
(1) Take the calculated amount of freshly prepared 10 mM CY dye and activate it (approximately 10 μL of stock solution mixed with 50 μL of 500 μg/mL condensation solution). Slowly add the activated dye to 0.5 mL of the protein sample solution. Gently mix by shaking, then briefly centrifuge to collect the sample at the bottom of the reaction tube. Avoid vigorous mixing to prevent denaturation or inactivation of the protein. (2) Place the reaction tube in a light-protected area and incubate at room temperature with gentle shaking for 60 minutes. Every 10–15 minutes, gently invert the tube several times to ensure thorough mixing of the reactants and to enhance labeling efficiency.
2. Protein Purification and Desalting
The following protocol uses a Sephadex G-25 column for purifying the dye-protein conjugate as an example.
(1) Prepare the Sephadex G-25 column according to the manufacturer’s instructions.
(2) Load the reaction mixture onto the top of the Sephadex G-25 column.
(3) When the sample runs just below the surface of the resin, immediately add PBS (pH 7.2-7.4).
(4) Continue adding PBS (pH 7.2-7.4) to the column to complete the purification. Collect the fractions containing the desired dye-protein conjugate.

Cy5.5-labeled Factor VIIa was created for tumor imaging. Cy5.5 tagged with these inhibitory proteins localized to tumor xenografts for at least 14 days, whereas unbound Cy5.5 did not localize to any xenografts. This method of visualizing anti-tissue factors in VECs can be utilized to detect initial tumors and metastases, monitoring, and in vivo therapy responses [1]. pH/temperature-sensitive magnetic nanoconductor (Cy5.5-Lf-MPNA nanoconductor) associated with Cy5.5-labeled lactoferrin was developed as a promising imaging agent for preoperative MRI and intraoperative fingerprint imaging of stellate tumors [2].

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Glioma is the most common primary brain tumor and causes a disproportionate level of morbidity and mortality across a wide range of individuals. From previous clinical practices, definition of glioma margin is the key point for surgical resection. In order to outline the exact margin of glioma and provide a guide effect for the physicians both at pre-surgical planning stage and surgical resection stage, pH/temperature sensitive magnetic nanogels conjugated with Cy5.5-labled lactoferrin (Cy5.5-Lf-MPNA nanogels) were developed as a promising contrast agent. Due to its pH/te mperature sensitivity, Cy5.5-Lf-MPNA nanogels could change in its hydrophilic/hydrophobic properties and size at different pH and temperatures. Under physiological conditions (pH 7.4, 37 °C), Cy5.5-Lf-MPNA nanogels were hydrophilic and swollen, which could prolong the blood circulation time. In the acidic environment of tumor tissues (pH 6.8, 37 °C), Cy5.5-Lf-MPNA nanogels became hydrophobic and shrunken, which could be more easily accumulated in tumor tissue and internalized by tumor cells. In addition, lactoferrin, an effective targeting ligand for glioma, provides active tumor targeting ability. In vivo studies on rats bearing in situ glioma indicated that the MR/fluorescence imaging with high sensitivity and specificity could be acquired using Cy5.5-Lf-MPNA nanogels due to active targeting function of the Lf and enhancement of cellular uptake by tailoring the hydrophilic/hydrophobic properties of the nanogels. With good biocompatibility shown by cytotoxicity assay and histopathological analysis, Cy5.5-Lf-MPNA nanogels are hopeful to be developed as a specific and high-sensitive contrast agent for preoperative MRI and intraoperative fluorescence imaging of glioma [4].

Subcutaneous inoculation of cancer cells [2]
U87EGFRviii glioma cells, MiaPaCa and ASPC-1 pancreatic cancer cells at 106 cells/0.1 mL, and KB-V1 SCC cells at 3 × 106 cells/0.1 mL, were inoculated subcutaneously suspended in PBS. An aliquot of Cy5.5-FFRck-fVIIa or unconjugated Cy5.5 containing approximately 0.03 mg of Cy5.5/0.1 mL/mouse was injected intravenously into the lateral tail vein of athymic nude mice when all tumors reached 0.5-1.0 cm in diameter.

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Conjugation of Cy5.5 with factor VIIa, anti-TF antibody, FFRck-fVIIa and paclitaxel-FFRck-fVIIa [2]
Factor VIIa (5 mg/mL), FFRck-fVIIa (ASIS, Batch NLDP013: 7 mg/mL), and anti-TF antibody (1 mg/mL) were dissolved in distilled water and dialyzed in 2 liters of 0.1 M Na-carbonate buffer (pH8.8) for 48 hours. Cy5.5 (10 mg) was dissolved in 3 mL of 100% DMSO. An aliquot of Cy5.5 was added to the following proteins in approximately the indicated Cy5.5 : protein ratios: fVIIa (1.5 : 1), FFRck-fVIIa (2 : 1), paclitaxel-FFRck-fVIIa (2 : 1) and anti-TF antibody (2 : 1), based on calculations following the manufacturer’s instruction. The mixtures were stirred gently for 1-1.5 hours at room temperature. The resulting Cy5.5-protein conjugates were separated from unconjugated Cy5.5 by a Sephadex G25-150 column previously equilibrated with 0.1 M Na-carbonate buffer (pH 8.8). In a typical experiment, 1.8 mg of fVIIa in 0.6 ml in 0.1M sodium-bicarbonate buffer, pH8.8 was incubated with 1 mg of Cy5.5 mono-NHS ester in DMSO in 0.3 ml at room temperature for 1 h. Cy5.5-fVIIa and free Cy5.5 dye were separated using the Sephadex G25-150 column (8 ml). 0.3 ml (0.324 mL =6 drops)/fraction was collected (1 drop = 54 μL) for fractions 2-6, containing Cy5.5-fVIIa. Then fractions 7-14 with no color were eluted at 1ml/fraction. Free Cy5.5 dye was eluted from fractions 15-21 and thereafter. Absorbance reading at A280 and A678 identified fractions containing Cy5.5-fVIIa (protein) and free CY5.5 dye (no protein). Fractions with higher protein were determined using a Micro BCA protein assay kit and pooled. The protein concentration of the pooled fraction (1 mL total volume) typically was 0.7 mg/mL. The Cy5.5 to fVIIa ratio was calculated as 1.24:1, using extinction coefficients for fVIIa and Cy5.5 dye, 1.39 × 10 5 M-1cm-1 and 2.5 × 105 M-1cm-1, respectively. The ratio of Cy5.5 to anti-TF antibody was calculated as 1.86:1, using the extinction coefficient 1.7 × 105 M-1cm-1 for the antibody, as determined by following the manufacturer’s manual.

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In vitro cytotoxicity [4]
C6 cells and NIH/3T3 mouse embryo fibroblast cell line (NIH/3T3) were used for cell viability studies according to a previous report [14]. The medium containing Cy5.5-Lf-MPNA nanogels or MPNA nanogels was added in a dilution series (cell medium containing 0, 25, 50, 75 and 100 µg/mL Fe). The control was the culture medium without the nanogels. After 24, 48 and 72 h of incubation, 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/mL) was added to wells. After incubation for 4 h, formazan crystals were solubilized by 100 μL of isobutanol in the incubator overnight. The absorbance of each well was read on a microplate reader (1420 multilabel counter) at 560 nm. The relative cell viability (%) related to the control wells containing cell culture medium without nanoparticles was calculated by [A]test/[A]control × 100%, where [A]test is an absorbance value of the tested cell, and [A]control is an absorbance value of the control group. The average result was calculated from 6 samples.

In vivo biocompatibility [4]
The normal rats were randomly divided into four groups (n = 9). The rats in one group were left without any treatment as the control. The rats in other three groups were injected with saline, Cy5.5-Lf-MPNA nanogels and MPNA nanogels (12 mg Fe/kg body weight) via the tail vein, respectively. At the time of the 21st day post-injection, rats were euthanized. 4 mL of blood was collected from femoral artery and sent to the clinical laboratory of Huazhong University of Science and Technology Hospital for the important biological function analysis immediately. Meanwhile, rats were perfused with sodium chloride (250 mL), and various tissues (heart, liver, spleen, lung, kidney and brain) were collected for histological examination. All the tissues were stained with H&E according to the standard clinical laboratory protocol and reviewed by a pathologist with expertise in veterinary pathology.

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Imaging Cy5.5 near infrared in vivo [2]
Imaging of Cy5.5-labeled fVIIa, FFRck-fVIIa, paclitaxel-FFRck-fVIIa and anti-TF antibody was monitored over time by detecting Cy5.5 in the whole animal according to the instructions of the IVIS Lumina Imaging System 100 Series (Xenogen). Standard filter set pairs for Cy5.5 were selected in the Filter Lock box and ensured that the excitation (615-665 nm) and emission (695-770 nm) filters were properly paired for Cy5.5. Imaging was carried out daily for up to 26 days after the injection (Figures 2-5). Mice were anesthetized by an intraperitoneal injection of the mixture of ketamine (50 mg/mL), xylazine (20 mg/mL) and sterile distilled water mixed at a ratio of 8, 1 and 9 volumes according to the IACUC approved protocol at Emory University. In Figure 5, tumors and normal organs were individually dissected and imaged. Cy5.5 was imaged at 2 days after the i.v. injection using the IVIS Imaging System 100 Series located in the Department of Animal Facility according to the manufacturer’s instructions.

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

[2]. Visualizing cancer and response to therapy in vivo using Cy5.5-labeled factor VIIa and anti-tissue factor antibody. J Drug Target. 2015 Apr;23(3):257-65.

[3]. Fundamentals in the chemistry of cyanine dyes: A review. Dyes and Pigments, 145, 505–513. doi:10.1016/j.dyepig.2017.06.029.

[4]. pH/temperature sensitive magnetic nanogels conjugated with Cy5.5-labled lactoferrin for MR and fluorescence imaging of glioma in rats. Biomaterials. 2013 Oct;34(30):7418-28.

[5]. A Unique Recombinant Fluoroprobe Targeting Activated Platelets Allows In Vivo Detection of Arterial Thrombosis and Pulmonary Embolism Using a Novel Three-Dimensional Fluorescence Emission Computed Tomography (FLECT) Technology. Theranostics. 2017 Feb 26;7(5):1047-1061.

Many small-molecule organic compounds possess properties that make them suitable for in vivo fluorescence imaging. Among the most promising candidates are anthocyanins, squaric acid cyanins, boron dipyrrole methylene, porphyrin derivatives, hydroporphyrins, and phthalocyanines. Recent design and synthesis efforts have focused on improving the optical properties of these compounds (e.g., shifting absorption and emission peaks to longer wavelengths and increasing brightness), as well as enhancing their stability and water solubility. The most significant advances include the development of encapsulated anthocyanin dyes with improved stability and water solubility, squaric acid cyanin rotaxanes with improved stability, long-wavelength absorbing boron dipyrrole methylene, long-wavelength absorbing porphyrins and hydroporphyrin derivatives, and water-soluble phthalocyanines. Recent advances in luminescence and bioluminescence have enabled the application of self-emissive fluorophores in in vivo imaging. The development of novel hydroporphyrin energy transfer binary compounds holds promise for further advancing in vivo multicolor imaging techniques. [1] We developed a specific technique for in vivo tumor imaging using Cy5.5-labeled coagulation factor VIIa (fVIIa), coagulation-deficient FFRck-fVIIa, paclitaxel-FFRck-fVIIa, and anti-tissue factor (TF) antibodies. FVIIa is a natural ligand for TF. We utilized the fact that in tumor tissues, vascular endothelial cells (VECs) (rather than normal tissues) aberrantly express TF due to the induction of vascular endothelial growth factor (VEGF). Under physiological conditions, TF is expressed by stromal cells and the outer layer of blood vessels (smooth muscle and adventitia), but not by VECs. We hypothesized that labeled fVIIa or anti-TF antibodies could be used for in vivo tumor vascular imaging. To verify this, researchers developed Cy5.5-labeled fVIIa, FFRck-fVIIa, paclitaxel-FFRck-fVIIa, and anti-TF antibodies and injected them into nude mice with xenografts, including glioma U87EGFRviii, pancreatic cancer ASPC-1 and Mia PaCa-2, and squamous cell carcinoma KB-V1. Cy5.5 labeled with these target proteins specifically localized to the xenograft tumors for at least 14 days, while unlabeled Cy5.5 did not localize to any xenograft tumors or organs. This method of imaging TF in tumor vascular endothelial cells (VECs) may help detect primary tumors and metastases, as well as monitor in vivo treatment responses. [2] In this review article, some important basic principles of cyanine dye chemistry are explained. These include the structure and resonance forms of cyanine dyes, naturally occurring cyanine dyes, different classes of cyanine dyes, and the formation mechanisms of cyanine dyes. This article covers metynyl cyanine dyes, apocyanine dyes, styryl cyanine dyes (semi-cyanine dyes), azastyryl cyanine dyes, azasemi-cyanine dyes, cyanine dyes (acyclic cyanine dyes and cyclic cyanine dyes), squaric acid cyanine dyes (aromatic squaric acid cyanine dyes and heterocyclic squaric acid cyanine dyes), spectro-sensitization evaluation of cyanine dyes, solvent color change evaluation of cyanine dyes, halogen color change evaluation of cyanine dyes, cyanine dyes for CD-R and DVD-R, cyanine dyes as fluorescent labels for nucleic acid research, the mechanism of action of dimethynyl cyanine dyes, and the mechanism of action of apocyanine dyes. In addition, the introduction section of this article highlights some important uses and applications of cyanine dyes. There has been little attention paid to systematic reviews of the basic principles, knowledge and/or understanding of cyanine dye chemistry, and such studies are lacking in the chemical literature. [3] Advances in drug development are highly dependent on preclinical in vivo animal studies. Small animal imaging is crucial for identifying new disease biomarkers and evaluating drug efficacy. This article reports for the first time the use of a three-dimensional fluorescence bioimaging technique called fluorescence emission computed tomography (FLECT) to detect a novel recombinant fluorescent probe. This probe is safe, easy to prepare on a large scale, and stable before scanning. This novel fluorescent probe (Targ-Cy7) consists of a single-chain antibody fragment (scFvTarg) that specifically binds to activated platelets and is conjugated to the near-infrared (NIR) dye Cy7 for detection. Following carotid artery injury in mice, the injected fluorescent probe circulates and binds within platelet-rich thrombi. Fluorescence emission computed tomography (FLECT) imaging detects the in vivo binding of the fluorescent probe to the thrombus, compared to a non-targeted control fluorescent probe. Analyzed FLECT images quantify the NIR signal and localize it to the site of vascular injury. Further validation of the detected fluorescence using a two-dimensional IVIS® Lumina scanner revealed significant NIR fluorescence at both the in vivo thrombus formation site and in vitro damaged carotid arteries. Furthermore, fluorescence levels in different organs were quantified to assess their biodistribution, indicating the highest uptake of the fluorescent probe in damaged arteries. Subsequently, this in vivo animal imaging technique was successfully applied to monitor changes in the response to treatment to induced thrombosis over time. This suggests that longitudinal FLECT scanning has the potential to evaluate the efficacy of candidate drugs in preclinical studies. In addition to intravascular thrombosis, we have also demonstrated that this non-invasive FLECT imaging technique can detect pulmonary embolism in vivo. In summary, this report describes a novel fluorescence-based preclinical imaging technique. The method employs an easily prepared, non-radioactive recombinant fluorescent probe. This provides a unique tool for studying the mechanisms of thromboembolic diseases and will greatly facilitate the in vivo testing of antithrombotic drugs. Furthermore, its non-radioactive, low-cost, high-sensitivity nature, along with the rapid development of optical scanning technology, makes this fluorescence imaging technique highly attractive for future clinical applications. [5]

C41H44N2O14S4

917.053067207336

916.168

210892-23-2

Cy5.5 acetate;Cy5.5 TEA;Cy5.5-SE;442912-55-2

131704516

Purple to purplish red solid powder

10.819

4

15

13

61

2250

0

CCN\1C2=C(C3=C(C=C2)C(=CC(=C3)S(=O)(=O)O)S(=O)(=O)[O-])C(/C1=C/C=C/C=C/C4=[N+](C5=C(C4(C)C)C6=C(C=C5)C(=CC(=C6)S(=O)(=O)O)S(=O)(=O)O)CCCCCC(=O)O)(C)C

LIZDKDDCWIEQIN-UHFFFAOYSA-N

InChI=1S/C41H44N2O14S4/c1-6-42-31-18-16-27-29(21-25(58(46,47)48)23-33(27)60(52,53)54)38(31)40(2,3)35(42)13-9-7-10-14-36-41(4,5)39-30-22-26(59(49,50)51)24-34(61(55,56)57)28(30)17-19-32(39)43(36)20-12-8-11-15-37(44)45/h7,9-10,13-14,16-19,21-24H,6,8,11-12,15,20H2,1-5H3,(H4-,44,45,46,47,48,49,50,51,52,53,54,55,56,57)

(2Z)-2-[(2E,4E)-5-[3-(5-carboxypentyl)-1,1-dimethyl-6,8-disulfobenzo[e]indol-3-ium-2-yl]penta-2,4-dienylidene]-3-ethyl-1,1-dimethyl-8-sulfobenzo[e]indole-6-sulfonate

210892-23-2; Cy5.5; 1H-Benz[e]indolium, 2-[5-[3-(5-carboxypentyl)-1,3-dihydro-1,1-dimethyl-6,8-disulfo-2H-benz[e]indol-2-ylidene]-1,3-pentadien-1-yl]-3-ethyl-1,1-dimethyl-6,8-disulfo-, inner salt;

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 : ~100 mg/mL (~109.05 mM)
H2O : ~5 mg/mL (~5.45 mM)

Solubility in Formulation 1: ≥ 4.17 mg/mL (4.55 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 41.7 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: ≥ 4.17 mg/mL (4.55 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 41.7 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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Solubility in Formulation 3: 5 mg/mL (5.45 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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