Cell Labeling with ManNAz and GlcNAz

Labeling of cells via this method largely followed previously established protocols.^{28 (link)} In both suspended and adherent modifications, cells were cultured in complete DMEM media supplemented with 40 μM ManNAz or GlcNaz (0.4% DMSO v/v), for 72 h at 37 °C and 5% CO₂. For adherent labeling, culture medium was removed and cells rinsed twice with phosphate-buffered saline (PBS). Cells were then incubated in Dylight 650-Phosphine (1% DMSO v/v) and incubated for 3 h at 37 °C and 5% CO₂ and then washed twice with PBS. Cells were rinsed once more with PBS before use. For suspended modification, cells were detached using a cell scraper. For 4 × 10⁶ cells, following additional centrifugation, the pellet was rinsed with 1 mL of 2% fetal bovine serum (FBS) in Hank’s Buffered Saline Solution (HBSS), and then resuspended in 400 μL of 100 μM Dylight 650-Phosphine (1% DMSO v/v) and incubated for 3 h at 37 °C and 5% CO₂. Cells were centrifuged at 1500 rpm for 5 min, and the pellet was rinsed twice with 1 mL of 2% FBS in HBSS and once with 1 mL of PBS before use. Images of cells were acquired using a Nikon Point Scanning C2+ confocal microscope with excitation at 488 and 650 nm. For experiments employing a DMSO control, populations of cells were treated with DMSO for analogous times at the same concentrations (0.4% for 72 h and 1% for 3 h).