C2 point scanning confocal microscope

Biovision’s EZClick^™ protein synthesis kit # K715-100 was conducted according to manufacturer’s protocol. Normal mouse fibroblast (ATCC: CRL-2907) cells were plated at 2× 10⁴ per well in 8 well 1% gelatin coated ibidi μ-slides (80826) and incubated at 37 °C for 12 h in phenol red–free IMDM medium supplemented with 10% FBS. The cells were treated with CA (50 μM) for 2 h before click reaction, along with DMSO controls. Cycloheximide (20 nM) was used as a positive control and added 30 min prior to click reaction and staining protocol. Images shown were taken with a Nikon C2 point-scanning confocal microscope at 20x magnification and whole image montage was quantified with Gen5 3.04 Software (Winooski, VT, USA).

Cells were labeled in either an adherent or suspended manner. For adherent labeling, culture medium was removed and cells rinsed twice with phosphate-buffered saline (PBS). Cells were then incubated in 2 mM Sulfo-NHS-LC-Biotin for 30 min at ambient temperature and then washed twice with 100 mM glycine, once with PBS, and then incubated in 2.5 μg/mL Avidin-FITC or Streptavidin-Dylight for 30 min at 37 °C/5% CO₂. Cells were rinsed once more with PBS before use. For suspended labeling, cells were harvested via trypsinization, centrifugation, and resuspension, followed by counting. For 6 × 10⁶ cells, the cell pellet was rinsed twice with 1 mL of PBS, centrifuging and removing supernatant for each wash. Cells were resuspended in 2 mL of 2 mM Sulfo-NHS-LC-Biotin and incubated for 30 min at ambient temperature. Cells were then centrifuged at 1500 rpm for 5 min, and the pellet was washed twice with 2 mL of 100 mM glycine and once with 1 mL of PBS. Cells were resuspended in 2 mL of 2.5 μg/mL FITC-Avidin or Streptavidin-Dylight 680 for 30 min at 37 °C and 5% CO₂. Cells were centrifuged at 1500 rpm for 5 min, and the pellet was rinsed 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.

Cells were labeled in either an adherent or suspended manner. For adherent labeling, culture medium was removed and cells rinsed twice with phosphate-buffered saline (PBS). Cells were then incubated in 100 μM Sulfo-NHS-Cyanine5 (Lumiprobe) for 1 h at 37 °C and 5% CO₂, and then washed twice with 100 mM glycine, once with PBS. Cells were rinsed once more with PBS before use. For suspended labeling, the cell monolayer was treated with 0.25% trypsin for detachment, centrifuged, resuspended, and counted. For 4 × 10⁶ cells, the pellet was rinsed twice with 1 mL of PBS, centrifuging and removing supernatant for each wash. Then, cells were suspended in 400 μL of 100 μM Sulfo-NHS-Cyanine5 (Lumiprobe) and incubated for 1 h at 37 °C and 5% CO₂. Cells were centrifuged at 1500 rpm for 5 min, and the pellet was rinsed twice with 2 mL of 100 mM glycine 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.

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).