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

指南 (以下是我们推荐的方案。此方案仅提供指南，应根据您的具体需要进行修改)。
1. 溶液配置^[1][2]
1.1 储存液
保存：分装后 -20°C 或 -80°C 避光保存，避免反复冻融。
1.2 工作液的配制
用 PBS 或者无血清培养基稀释 (根据实验优化)。可根据实际情况稀释相应的储备液。注意，如果溶剂为 DMSO，则必须考虑 DMSO 的细胞毒性，并应制备溶剂对照；如果溶剂为纯水，则工作液在加入细胞前需要过滤并除菌。
注意：工作液现配现用，避光。

2. 细胞标记 (例如 A549 细胞) ：
2.1 将 2 mL 生长的种子细胞以 3×10⁴ (因细胞而异) 的密度接种到 35 毫米玻璃底培养皿中
2.2 加入 Ac4ManNAz (50 μM，终浓度) ，孵育 3 天 (在细胞表面产生叠氮基团)。
2.3 用 DPBS (pH 7.4) 洗涤细胞两次。
2.4 用 Cyanine5 DBCO (20 μM，终浓度) 在 37°C 孵育细胞 1 h。
2.5 DPBS (pH 7.4) 冲洗细胞，甲醛-戊二醛联合固定液 25°C 固定 15 min。
2.6 DPBS 洗涤细胞两次 (pH 7.4) 并用 DAPI 染色细胞以标记细胞核。
2.7 使用共聚焦激光扫描显微镜测量 Cyanine5 DBCO 荧光 (Ex=635 nm，Em=650-700 nm)。
注意:
1) 在用 Ac4ManNAz 处理后，Cyanine5 DBCO 可以靶向细胞表面人工引入的叠氮基团超过 3 天。
2) 程度 Cyanine5 DBCO 标记具有剂量依赖性。

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

929.05

C₅₃H₅₉F₆N₄O₂P

2360411-64-7

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]