Fig. 1: NHS-ester-based amine-reactive chemistry enables rapid, uniform, and high-density protein labeling for prolonged live imaging of the cell membrane.

a The general principle of membrane protein labeling via N-hydroxysuccinimide (NHS) ester chemistry. The NHS-dye ester is covalently coupled to the exposed amine groups of proteins on the outer surface of cells. b The schematic illustration of the procedural steps for NHS-ester-dye labeling of live cells in suspension within 5 min. c The total-internal-reflection fluorescence (TIRF) image of a DC2.4 cell labeled with AF647 NHS-ester. d DiO labeling results in heterogeneous membrane staining. The merged TIRF images of the DC2.4 cells co-labeled with DiO and NHS-AF647 (left) and the single color TIRF images (right). The NHS-ester labeling is shown in magenta, DiO labeling is shown in green and colocalized cells appear in white. e A representative TIRF image of dendritic cells labeled for proteins with NHS-AF647. f TIRF images of the same region in (e) in the presence of trypan blue. g Intensity line profiles across lines before (black solid line) and after exposure (blue solid line) to trypan blue marked as i-ii in (e, f). arb. units refer to arbitrary units. h Venn diagram showing the overlap between plasma membrane proteins enriched with NHS-ester-biotin vs. total plasma membrane proteins, indicating that 91.3% of the membrane proteins are labeled by NHS-ester conjugation (n = 3 technical replicates). i Representative images from a time-lapse video showing DC2.4 cells reversing the direction of migration by expanding their retraction fibers. Time points are presented in the format hh:mm:ss. The orange dashed line indicates the direction of their migration. The complete image sequences are shown in Supplementary Movie 1. j Representative images from a time-lapse video captured a DC2.4 cell (marked cell 1) contacting another DC2.4 cell (marked cell 2). The yellow dashed line indicates the direction of migration. The color bar represents image intensity (arbitary units). The complete image sequences are shown in Supplementary Movie 2-3. Scale bars: 10 μm.