Protocols for Measuring Glutamate Uptake: Dose-Response and Kinetic Assays in In Vitro and Ex Vivo Systems

Abstract

This article presents detailed descriptions of procedures and troubleshooting tips for basic in vitro and ex vivo uptake assays for the functional characterization of glutamate transporters and the assessment of the effect of compounds that modulate their activity. Assays are performed in cell lines that transiently or stably express a particular transporter under investigation, in primary cultures of astrocytes, or in ex vivo synaptosomal preparations that endogenously express these transporters. Two main assays are described, including dose-response assays to measure potencies of test compounds for stimulation or inhibition of function (EC 50 or IC 50 values, respectively) and kinetic functional assays to calculate apparent affinity (K M ) and maximal velocity (V max ) of radiolabeled substrate uptake into the cells. The methods described use glutamate transporters as an example; however, the protocols can be adapted to other neurotransmitter transporters using their respective substrates (e.g., GABA uptake through GATs, serotonin uptake through SERTs, among others). © 2018 by John Wiley & Sons, Inc.