Cyanine5 DBCO hexafluorophosphate  (Synonyms: DBCO-Cy5 hexafluorophosphate; Cyanine5 dibenzocyclooctyne hexafluorophosphate)

Cyanine5 DBCO (DBCO-Cy5) hexafluorophosphate is a low-toxicity azide reactive probe (NIR fluorescent dye), for imaging azide-labeled biomolecules via a copper-free "click-through" reaction. Cyanine5 DBCO hexafluorophosphate has no apparent cytotoxicity or animal toxicity and shows no damage to the physiological functions of cells other than the target cells (azide-labeled cells). Cyanine5 DBCO hexafluorophosphate can be used to label and track cells in vitro and in vivo (Ex=635 nm, Em=650-700 nm)^[1][2].

Guide (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs)^[1][2].
1. Solution preparation:
1.1 Preparation of storage solution
Solvent: DMSO.
Concentration: 20 mM is recommended.
1.2 Preparation of working solution
Dilute to 20 μM with PBS or serum-free medium (optimized according to the experiment).
(Note: The working solution should be prepared and used immediately, and should be protected from light.)
2. Cell labeling (A549 cells for example):
2.1. Seed cells onto 35-mm glass-bottom dishes at a density of 3×10⁴ (varies by cell) in 2 mL of growth media.
2.2. Add Ac4ManNAz (50 μM, final concentration), incubate for 3 days (generate azide groups on the surface of cells).
2.3. Wash cells twice with DPBS (pH 7.4).
2.4. Incubate cells with Cyanine5 DBCO (20 μM, final concentration) for 1 h at 37°C.
2.5. Rinse cells with DPBS (pH 7.4) and fix with formaldehyde-glutaraldehyde combined fixative for 15 min at 25°C.
2.6. Wash cells twice with DPBS (pH 7.4) and stain cells with DAPI to label nuclei.
2.7. Measure Cyanine5 DBCO fluorescence (Ex=635 nm, Em=650-700 nm) using a confocal laser scanning microscope.
Note:
1) Artificially introduced azide groups on the cell surface can be targeted by Cyanine5 DBCO for more than 3 days after treatment with Ac4ManNAz.
2) Degree of Cyanine5 DBCO labeling is in a dose-dependent manner.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

929.03

C₅₃H₅₉F₆N₄O₂P

2360411-64-7

Solid

Dark purple to black

668

647

O=C(CCCCC[N+]1=C(/C=C/C=C/C=C2N(C)C3=C(C\2(C)C)C=CC=C3)C(C)(C)C4=C1C=CC=C4)NCCCCCC(N5C6=C(C#CC7=C(C5)C=CC=C7)C=CC=C6)=O.[F-][P+5]([F-])([F-])([F-])([F-])[F-]

Room temperature in continental US; may vary elsewhere.

-20°C, protect from light, stored under nitrogen

*In solvent : -80°C, 6 months; -20°C, 1 month (protect from light, stored under nitrogen)

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (protect from light, stored under nitrogen). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

• [1]. Kang SW, et al. Cell labeling and tracking method without distorted signals by phagocytosis of macrophages. Theranostics. 2014 Feb 12;4(4):420-31.  [Content Brief]

  [2]. Zhu L, et al. Coupling Aptamer-based Protein Tagging with Metabolic Glycan Labeling for In Situ Visualization and Biological Function Study of Exosomal Protein-Specific Glycosylation. Angew Chem Int Ed Engl. 2021 Aug 9;60(33):18111-18115.  [Content Brief]

  [3]. Song S, et al. In Situ One-Step Fluorescence Labeling Strategy of Exosomes via Bioorthogonal Click Chemistry for Real-Time Exosome Tracking In Vitro and In Vivo. Bioconjug Chem. 2020 May 20;31(5):1562-1574.  [Content Brief]