TY - JOUR
T1 - Using a qPCR device to screen for modulators of ABC transporter activity: A step-by-step protocol
AU - Möhle, Luisa
AU - Schwarzová, Barbora
AU - Krohn, Markus
AU - Stefan, Sven Marcel
AU - Pahnke, Jens
N1 - Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.
PY - 2020/6/1
Y1 - 2020/6/1
N2 - Introduction: Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are transmembrane proteins which actively transport a large variety of substrates across biological membranes. ABC transporter overexpression can be the underlying cause of multidrug resistance in oncology. Moreover, it has been revealed that increased ABCC1 transporter activity can ameliorate behavioural changes and Aβ pathology in a rodent model of Alzheimer's disease and it is currently tested in AD patients. Methods: Finding substances that modulate ABC transporter activity (inhibitors and activators) is of high relevance and thus, different methods have been developed to screen for potential modulators. For this purpose, we have developed a cell-based assay to measure the kinetics of ABCC1-mediated efflux of a fluorescent dye using a common qPCR device (Agilent AriaMx). Results: We validated the specificity of our method with vanadate and benzbromarone controls. Furthermore, we provide a step-by-step protocol including statistical analysis of the resulting data and suggestions how to modify the protocol specifically to screen for activators of ABCC1. Discussion: Our approach is biologically more relevant than cell-free assays. The continuous detection of kinetics allows for a more precise quantification compared with assays with single end-point measurements.
AB - Introduction: Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are transmembrane proteins which actively transport a large variety of substrates across biological membranes. ABC transporter overexpression can be the underlying cause of multidrug resistance in oncology. Moreover, it has been revealed that increased ABCC1 transporter activity can ameliorate behavioural changes and Aβ pathology in a rodent model of Alzheimer's disease and it is currently tested in AD patients. Methods: Finding substances that modulate ABC transporter activity (inhibitors and activators) is of high relevance and thus, different methods have been developed to screen for potential modulators. For this purpose, we have developed a cell-based assay to measure the kinetics of ABCC1-mediated efflux of a fluorescent dye using a common qPCR device (Agilent AriaMx). Results: We validated the specificity of our method with vanadate and benzbromarone controls. Furthermore, we provide a step-by-step protocol including statistical analysis of the resulting data and suggestions how to modify the protocol specifically to screen for activators of ABCC1. Discussion: Our approach is biologically more relevant than cell-free assays. The continuous detection of kinetics allows for a more precise quantification compared with assays with single end-point measurements.
UR - http://www.scopus.com/inward/record.url?scp=85085958234&partnerID=8YFLogxK
U2 - 10.1016/j.vascn.2020.106882
DO - 10.1016/j.vascn.2020.106882
M3 - Journal articles
C2 - 32474136
AN - SCOPUS:85085958234
SN - 1056-8719
VL - 104
SP - 106882
JO - Journal of Pharmacological and Toxicological Methods
JF - Journal of Pharmacological and Toxicological Methods
M1 - 106882
ER -
