Fluorescence Signal Amplification by Using β-Galactosidase for Flow Cytometry; Advantages of an Endogenous Activity-Free Enzyme.

We previously proposed using a hydrolysis enzyme for fluorescent signal amplification in flow cytometric detection of antigen proteins, which was named the catalyzed reporter penetration (CARP) method. In this method, antigen proteins are labeled with enzyme-modified antibodies, and then fluorophore-modified substrates stain cells by penetrating the cell membrane upon hydrolysis of the substrate. We proved the concept by using alkaline phosphatase (AP) as the hydrolysis enzyme.

Alkaline Phosphatase-Catalyzed Amplification of a Fluorescence Signal for Flow Cytometry.

Despite the expanding use of flow cytometry, its detection limit is not satisfactory for many antigen proteins with low copy numbers. Herein, we describe an alkaline phosphatase (AP)-based technique to amplify the fluorescence signal for cell staining applications. We designed a fluorescent substrate that acquires membrane permeability upon dephosphorylation by AP.