Fluorescence imaging of metabolically labeled cells

4T1 cells (2 × 10⁴ cells per well) in 1 ml of DMEM were seeded into a six-well plate with glass coverslips, and Ac₄ManNAz was added with a final concentration of 25 μM. After incubation for 48 h, cells were washed with PBS three times, followed by incubation with 50 μM DBCO-Cy5 in Opti-MEM for 30 min. After washing, cells were stained with the Membrane Green and DAPI following the manuals, fixed with 4% paraformaldehyde, and imaged under a fluorescence microscope.