Cy5-DBCO

Method for Cy5-DBCO Cell Labeling and Tracing^[1]

This protocol is adapted from research data and provided for reference only. Adjustments may be necessary based on specific experimental requirements.

1. Reagent Preparation: Dissolve tetraacetylated N-azidoacetyl-D-mannosamine (Ac4ManNAz) in complete cell culture medium to prepare a 50μM working solution. Dissolve Cy5-DBCO in a suitable buffer (DPBS, pH=7.4) to prepare a working concentration (20μM for in vitro experiments, 25μM for in vivo injection).

2. Target Cell Azide Labeling: Seed target cells at an appropriate density and culture them in complete medium containing 50μM Ac4ManNAz for 3 days to metabolically introduce azide groups onto the cell surface.

3. Cy5-DBCO Labeling and Detection (In Vitro): Wash the Ac4ManNAz-treated cells twice with DPBS (pH=7.4). Add the Cy5-DBCO working solution (20μM) and incubate at 37°C for 1 hour. Wash the cells thoroughly with DPBS to remove unbound probe. Subsequently, fix the cells (with formaldehyde-glutaraldehyde fixative), stain the nuclei (with DAPI), and perform fluorescence imaging analysis using a confocal laser scanning microscope (Ex=635nm, Em=650-700nm). Quantitative analysis can also be performed using flow cytometry.

4. Cy5-DBCO Labeling and Detection (In Vivo): Directly inject target cells treated with Ac4ManNAz for 3 days into the target organ of a live animal. Three days after cell transplantation, inject the Cy5-DBCO solution (25μM, 200μL) into the mouse via the tail vein. Four hours after probe injection, use an in vivo imaging system for whole-body fluorescence imaging to observe specific signals at the target organ. Alternatively, dissect the organ for ex vivo imaging using a fluorescence imaging station or prepare frozen sections for microscopic observation.

Precautions:

(1) Unreacted, free Cy5-DBCO is primarily cleared via the renal route within 4 hours post-injection, which helps reduce background signal.

(2) For your safety and health, please wear a lab coat and disposable gloves during operation.

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