The ability to detect cell surface proteins using fluorescent dye-labeled antibodies is crucial for the reliable identification of many cell types. However, the different types of cell surface proteins used to identify cells are currently limited in number because they need to be expressed at high levels to exceed background cellular autofluorescence, especially in the shorter wavelength region. Herein, we report on a new method (CLAMP: quinone methide-based <u>c</u>atalyzed signa<u>l</u> <u>amp</u>lification) in which the fluorescence signal is amplified by an enzymatic reaction that strongly facilitates the detection of cell surface proteins on living cells. We used β-galactosidase as an amplification enzyme and designed a substrate for it, called MUGF, which contains a fluoromethyl group. Upon removal of the galactosyl group in MUGF by β-galactosidase labeling of the target cell surface proteins, the resulting quinone methide group-containing product was found to be both cell membrane permeable and reactive with intracellular nucleophiles, thereby providing fluorescent adducts. Using this method, we successfully detected several cell surface proteins including programmed death ligand 1 protein, which is difficult to detect using conventional fluorescent dye-labeled antibodies.

The ability to detect cell surface proteins using fluorescent dye-labeled antibodies is crucial for the reliable identification of many cell types. However, the different types of cell surface proteins used to identify cells are currently limited in number because they need to be expressed at high levels to exceed background cellular autofluorescence, especially in the shorter wavelength region. Herein, we report on a new method (CLAMP: quinone methide-based <u>c</u>atalyzed signa<u>l</u> <u>amp</u>lification) in which the fluorescence signal is amplified by an enzymatic reaction that strongly facilitates the detection of cell surface proteins on living cells. We used β-galactosidase as an amplification enzyme and designed a substrate for it, called MUGF, which contains a fluoromethyl group. Upon removal of the galactosyl group in MUGF by β-galactosidase labeling of the target cell surface proteins, the resulting quinone methide group-containing product was found to be both cell membrane permeable and reactive with intracellular nucleophiles, thereby providing fluorescent adducts. Using this method, we successfully detected several cell surface proteins including programmed death ligand 1 protein, which is difficult to detect using conventional fluorescent dye-labeled antibodies.