Enzymatic activity in single cells.


Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark. Electronic address: [Email]


With the increasing recognition of the importance in addressing cell-to-cell variations for the understanding of complex biological systems, single cell analyses are becoming increasingly important. Presented in this chapter is a highly sensitive approach capable of measuring human topoisomerase 1 (TOP1) activity in single CD133 positive DLD-1 cells. The method termed On-Slide "Rolling circle Enhanced Enzyme Activity Detection (REEAD)" relies on the specific capture and lysis of CD133 positive cells on glass slides dual functionalized with anti-CD133 antibodies and a specific DNA primer. The On-Slide REEAD was demonstrated to be directly quantitative. Furthermore, the method allowed for the highly sensitive detection of TOP1 activity in single CD133 positive DLD-1 cells. The described protocol is expected to open for new possibilities in the single cell research, particularly for the investigations of chemoresistance of the cancer stem cells.


Cancer,Cancer stem cells,Enzyme activity,Human topoisomerase 1,Microscopic slides,Rolling circle amplification,Single cell detection,

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