Dibenzocyclooctyne-Cy5.5

The following plans are based on literature, please modify them to meet your specific needs.
In vitro cell labeling^[1]
1. MDA-MB-231 and HCT-116 cells were each seeded at a density of 1×10⁵ cells onto a 35mm confocal dish.
2. Cells were then incubated with various concentrations of Ac4ManNAz (0–20μM) for 24h to generate azide groups on the cell surface via metabolic engineering.
3. Cells were washed twice with DPBS and treated with 10μM Dibenzocyclooctyne-Cy5.5 for 2h to visualize azide expression on the cell surface.
In vivo labeling^[2]
1. ALN-PEG-N3 (80mg; dissolved in 8mL of PBS) and Dibenzocyclooctyne-Cy5.5 (31.9mg; dissolved in 2mL of DMSO) were reacted for 24h.
2. The reactants were dialyzed against water containing 30% EtOH using a dialysis membrane (MWCO; 3500 Da) for 1 day and further dialyzed against distilled water for additional 2 days.
3. Centrifugation (4000rpm, 20min) of the dialyzed solution to remove the precipitated materials (unreacted Dibenzocyclooctyne-Cy5.5).
4. Lyophilize the separated supernatant to obtain ALN-PEG-Cy5.5.
Note: ALN (Specific calcium ion-targeting ligand) -PEG-Cy5.5 has a high affinity for bone minerals containing calcium phosphate and is used to visualize intracellular calcium ion levels and normal bone tissue.

References:
[1] Lim, Gyeong Taek et al. “Bioorthogonally surface-edited extracellular vesicles based on metabolic glycoengineering for CD44-mediated targeting of inflammatory diseases.” Journal of extracellular vesicles vol. 10,5 (2021): e12077.
[2] Lim YG, Kim HJ, Kim JW, Park K. Calcium-binding near-infrared fluorescent nanoprobe for bone tissue imaging. Journal of Industrial and Engineering Chemistry. 2020;89:442-447.