Cyanine5 DBCO hexafluorophosphate (solution) 

Cyanine5 DBCO (DBCO-Cy5) hexafluorophosphate (solution) 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].
Solvent and concentration: DMSO: 10 mM

Guide (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs).
1. Preparation of solution preparation^[1][2]
1.1 Storage solution
Dissolve the Cyanine5 DBCO powder in DMSO (dimethyl sulfoxide) to create a stock solution, with a recommended concentration of 20 mM.
1.2 Preparation of working solution
Dilute the stock solution to 20 μM using PBS (Phosphate-Buffered Saline) or serum-free medium immediately before use.
Note: To enhance solubility, heat the tube to 37°C and use an ultrasonic bath if needed.


2. Cell Labeling (e.g. A549 cells)
2.1 Add 2 mL of growing seed cells at a density of 3×10⁴ (varies depending on the cells) to a 35 mm glass-bottomed petri dish.
2.2 Add Ac4ManNAz (50 μM, final concentration), and incubate for 3 days (forming azide groups on the cell surface).
2.3 Wash the cells twice with DPBS (pH 7.4).
2.4 Incubate the cells with Cyanine5 DBCO (20 μM, final concentration) at 37°C for 1 hour.
2.5 Wash the cells with DPBS (pH 7.4), and fix them with a combined fixative of formaldehyde and glutaraldehyde at 25°C for 15 minutes.
2.6 Wash the cells twice with DPBS (pH 7.4) and stain them with DAPI to label the cell nuclei.
2.7 Use a confocal laser scanning microscope to measure the fluorescence of Cyanine5 DBCO (Ex = 635 nm, Em = 650-700 nm).
Note:
1) After treatment with Ac4ManNAz, Cyanine5 DBCO can target the azide groups artificially introduced onto the cell surface for more than 3 days.
2) The degree of Cyanine5 DBCO labeling is dose-dependent.

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

929.05

C₅₃H₅₉F₆N₄O₂P

2360411-64-7

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.

Please store the product under the recommended conditions in the Certificate of Analysis.

• [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]