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].
Solution Concentration: 10 mM

Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs).
1. Solution preparation^[1][2]
1.1 Storage solution
Storage: After aliquoting, store at -20°C or -80°C in the dark and avoid repeated freezing and thawing.
1.2 Preparation of working solution
Dilute with PBS or serum-free medium (optimized according to the experiment). The corresponding stock solution can be diluted according to the actual situation. Note that if the solvent is DMSO, the cytotoxicity of DMSO must be considered, and a solvent control should be prepared; if the solvent is pure water, the working solution needs to be filtered and sterilized before adding cells.
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 2.3 days (generate azide groups on the surface of cells).
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℃.
2.5. Rinse cells with DPBS (pH 7.4) and fix with formaldehyde-glutaraldehyde combined fixative for 15 min at 25℃.
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.

929.05

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]