Figure 4.

Simultaneous labeling of two cell surface chemical reporters. Distinct metabolic targets can be simultaneously imaged using tetrazine and azido reporters. (A) MDA-MB-231 cells were cultured in the presence of both Ac₄ManNTz (25 μmol/L) and Ac₄ManNAz (25 μmol/L) for 72 h, followed by concurrent treatment with DBCO-FITC (25 μmol/L, 30 min at 37 °C) to covalently tag cell surface azides and TCO-Biotin (0.25 mmol/L, 30 min at 37 °C), streptavidin-AF647 to tag tetrazines, respectively. Cells were then mounted to cover slides and counter stained with DAPI and imaged via confocal microscopy. (B) Representative images are shown. Red: Cy5, green: FITC, blue: DAPI. Scale bar: 10 μm.