Fig 1: Principle of USB method and comparison of USB and Ab labelling methods. (A) Principle of the USB and Ab methods (top). In the USB method, cell surface proteins (represented by small rectangles on the cell) are universally biotinylated with S-NHS-biotin and treated with hashtag DNA-tagged streptavidin. In the Ab method, cells are treated with biotin-conjugated antibody against specific cell surface protein (small rectangles on the cell) followed by the hashtag DNA-tagged streptavidin. If the specific protein is not expressed on some cells, those cells are not labelled by the Ab method (bottom right), whereas the USB method can label all the cells (bottom left). (B) Efficiency of cell labelling examined by flow cytometry. The USB labelling is highly efficient, with 99.6% of undifferentiated (top left) and 96.4% of differentiated cells (top right) being PE positive. In contrast, using the Ab method, 87.8% of undifferentiated cells (bottom left) and 9.9% of differentiated cells (bottom right) were PE positive. (C) Cell labelling by the USB method (top panel) and the Ab method (bottom panel) confirmed by fluorescence microscopy. In this case, the Ab method uses an antibody against mouse CDH1. CDH1 is known to be expressed in the undifferentiated ES cells, while the expression is limited to some cells in differentiated states. (Left) Undifferentiated ES cells; (right) differentiated cells (12 days after induction of differentiation). ph, phase-contrast images; PE, fluorescence images of TotalSeq-PE-streptavidin; arrowhead, PE negative cells in left panel; arrow, PE positive cells in right panel.