The optimal stock solution preparation 1. Protein preparation: Add protein (antibody) at a concentration of 2 mg/mL to achieve the labeling effect. 1) The protein solution's pH should be 8.5±0.5. In case the pH falls below 8.0, utilize 1 M carbon dioxide. 2) The labeling efficiency will be significantly decreased if the protein content is less than 2 mg/mL. The ideal labeling efficiency can be achieved by ensuring that the protein content ranges from 2 to 10 mg/mL. 3) To ensure optimal labeling efficacy, the protein needs to be in a clear buffer that contains primary amines (such Tris or glycine) and ammonium ions. 2. Dye preparation: To generate a 10 mM stock solution, add anhydrous DMSO to CY dye. Using a glass tube or a vortex, thoroughly mix. After being aliquoted, it is advised to store CY storage solution at -20°C or -80°C in the dark. 3. Amount of dye working solution The amount of labeled protein determines how much CY dye is needed for the labeling reaction. The ideal amount of CY dye and protein to utilize is as follows: Assume 500 μL of labeled IgG (2 mg/mL; MW = 150,000). Dissolve a tube of 1 mg CY dye in 100 μL of DMSO. This means that 3.95 μL of CY volume is needed. Using Cy5.5-SE as an example, the calculation procedure is as follows in detail: 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 (Cy5.5-SE) = mmol (IgG) × 10 = 6.7×10-6 mmol×10 = 6.7×10-5 mmol 3) μL (Cy5.5-SE) = mmol (Cy5.5-SE) ×MW (Cy5.5-SE) / mg/μL (Cy5.5-SE) = 6.7×10-5 mmol× 590.15 mg/mmol / 0.01 mg/μL = 3.95 μL (Cy5.5-SE) Usage method 1. Labeling reaction 1: Take the predicted volume of fresh vector. With a gentle shake to mix, slowly add 10 mg/mL CY dye to 0.5 mL protein sample solution. Centrifuge for a brief period of time to gather sampling. 2) Under the starting conditions, place the reaction tube in a dark location and gently shake it on the left side for 60 minutes. Every week, set aside the reaction vial at the bottom for ten to fifteen minutes. Turn the two reactants over a few times gently to ensure complete mixing and increase labeling effectiveness. 2. The desalting strategy for peach rosette is as follows, with an example utilizing the SepHadex G-25 column ring dye conjugate. 1) As directed by the manufacturer, prepare SepHadex G-. 2) Fill the SepHadex G-25 column to the brim with the reaction mixture. 3) Add PBS (pH 7.2–7.4) as soon as the sample dips below the upper resin surface. 4) To finish column sealing, add additional PBS (pH 7.2–7.4) to the necessary sample. Put the parts together that contain the chosen dye-protein combination.

Factor VIIa, Cy5.5-labeled, was created for tumor imaging. For at least 14 days, Cy5.5 that had been tagged with these inhibitory proteins localized to tumor xenografts; unbound Cy5.5, however, did not localize to any xenografts. Anti-tissue factors in VECs can be imaged using this technique, which can be employed for surveillance, in vivo therapy response, and primary cancer and metastatic detection [1]. Cy5.5-Lf-MPNA nanoconductor, a pH/temperature-sensitive magnetic nanoconductor linked with Cy5.5-labeled lactoferrin, was developed as a promising imaging agent for intraoperative fingerprint imaging of stellate tumors and preoperative magnetic resonance imaging [2].

[1]. 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.

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

[3]. 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.

[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.

C45H47N3O16S4

1014.12518811226

1013.18

442912-55-2

Cy5.5;210892-23-2;Cy5.5-SE (DIPEA);Cy5.5-SE TEA

131879370

Light blue to blue solid powder

10.279

3

17

15

68

2550

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)ON7C(=O)CCC7=O)(C)C

DOMDXTIMIZCSNC-UHFFFAOYSA-N

InChI=1S/C45H47N3O16S4/c1-6-46-33-18-16-29-31(23-27(65(52,53)54)25-35(29)67(58,59)60)42(33)44(2,3)37(46)13-9-7-10-14-38-45(4,5)43-32-24-28(66(55,56)57)26-36(68(61,62)63)30(32)17-19-34(43)47(38)22-12-8-11-15-41(51)64-48-39(49)20-21-40(48)50/h7,9-10,13-14,16-19,23-26H,6,8,11-12,15,20-22H2,1-5H3,(H3-,52,53,54,55,56,57,58,59,60,61,62,63)

(2E)-2-[(2E,4E)-5-[3-[6-(2,5-dioxopyrrolidin-1-yl)oxy-6-oxohexyl]-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

2934.99.9001

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.

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 (~98.61 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.)