Figure 2.

Labeling of metabolically engineered cell-surface glycoconjugates. (A) Working flowchart for engineering and detection. MDA-MB-231 cells were grown with 25 μmol/L Ac₄ManNTz (1) for three days, washed with PBS and labeled with TCO-Biotin (250 μmol/L) for 1 h at 37 °C, followed by incubation with Streptavidin-Cy5 (20 μg/mL) for 20 min at rt. (B) The chemical structure of Ac₄ManNTz (1) and Ac₄ManNPh (3). (C, D) Confocal images showing the MDA-MB-231 cells pretreated by the Ac₄ManNTz (1) could be specifically labeled on the cell surface (arrow) while the control group pretreated by the Ac₄ManNPh (3) displayed barely visible fluorescent signal. Scale bar: 20 μm. (E) Flow cytometry of cells pretreated with Ac₄ManNTz (1) and Ac₄ManNPh (3). (F) Mean Cy5 fluorescence intensity of MDA-MB-231 cells extracted from (C, D). Data were presented as mean ± SEM (n = 3) and analyzed by Student's t-test (two-tailed), ∗∗∗P ≤ 0.001.