Enzymatic activity in single cells

TY - CHAP

T1 - Enzymatic activity in single cells

AU - Keller, Josephine Geertsen

AU - Stougaard, Magnus

AU - Knudsen, Birgitta R.

PY - 2019/1/1

Y1 - 2019/1/1

N2 - 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.

AB - 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.

KW - Cancer

KW - Cancer stem cells

KW - Enzyme activity

KW - Human topoisomerase 1

KW - Microscopic slides

KW - Rolling circle amplification

KW - Single cell detection

UR - http://www.scopus.com/inward/record.url?scp=85071296425&partnerID=8YFLogxK

U2 - 10.1016/bs.mie.2019.07.003

DO - 10.1016/bs.mie.2019.07.003

M3 - Book chapter

C2 - 31668235

AN - SCOPUS:85071296425

SN - 9780128170908

T3 - Methods in Enzymology

SP - 43

EP - 57

BT - Enzyme Activity in Single Cells

A2 - Allbritton, Nancy L.

A2 - Kovarik, Michelle L.

PB - Elsevier

ER -