Materials Required

• Azide labeled cells or mice
• DBCO detection reagent
• PBS buffer (pH 7.4)
• Optional: Fixative, Hoechst 33342, DAPI, coverslips/microscope slides, mounting media

Material Preparation

Growth medium, cell density, cell type variations, and other factors may influence labeling. Investigators are encouraged to determine the optimal concentration of the azide-labeling reagent as well as labeling time individually for each cell type on a small-scale first. Metabolic labeling should be carefully assessed for each cell line of interest.

1. Cell labeling with DBCO detection reagent

1. 1. Wash azide metabolically labeled cells twice with PBS
   2. Incubate with 15 µM DBCO detection reagent in growth media for 1 hour at 37°C
   3. Wash the cells 3 times with PBS
   4. Cells are ready for analysis

Note: If background is high – incubate the cells in DBCO detection reagent-free culture media for 1–2 hours prior the analysis.

2. Mice labeling with DBCO detection reagent

1. 1. Inject 200 µL of 25 µM AFDye-DBCO (5 nmol) in tail vein of azide metabolically labeled mice, proceed to analysis

It is possible to inject cancer cells labeled with DBCO detection reagent directly in blood stream to observe tumor formation.

Selected References:

1. Lee, S., et al. (2014). Labeling and Tracking Method without Distorted Signals by Phagocytosis of Macrophages. Theranostics, 4 (4), 420-31. [PubMed]
2. Murrey, H. E, et al. (2015). Systematic Evaluation of Bioorthogonal Reactions in Live Cells with Clickable HaloTag Ligands: Implications for Intracellular Imaging. J Am Chem Soc., 137 (35), 11461-75. [PubMed]
3. Kim, K., et al. (2016). Bioorthogonal Copper Free Click Chemistry for Labeling and Tracking of Chondrocytes In Vivo. Bioconjug Chem., 27 (4), 927-36. [PubMed]