Cell Biology

We designed a fluorescence resonance energy transfer (FRET)-based approach to study the ligand binding constants of the adenosine A_2A receptor (A_2AR). Our assay is based in the interaction of a fluorescent A_2AR agonist ligand (MRS5424) with an A_2AR tagged with the cyan fluorescent protein (CFP) at the N-terminus (i.e. A_2AR^CFP) and expressed in living cells. Thus, upon fast superfusion of the A_2AR^CFP expressing cells with MRS5424, the ligand-receptor interaction is determined by single-cell FRET in a real-time mode. Accordingly, our approach allowed immediate ‘real-time’ readout of the ligand-receptor interaction, thus allowing kinetic binding experiments, a feature impossible to achieve using conventional radioisotope-labelled ligands. In addition, since our assay permitted the visual confirmation of receptor localization it also allowed localized saturation binding experiments.

Transient gene expression via biolistic particle delivery is a widely used technique for gene functional analysis in plants. In this protocol we describe a modified single-cell transient expression assay through transformation with a particle inflow gun of the model PDS-1000/He system (Bio-Rad). This assay was originally optimized for analyzing cell death activity and disease resistance function of the barley MLA (mildew locus A) disease resistance proteins against the powdery mildew fungus, which can be further adopted for other purposes for other types of plant proteins and in some other plant species, including Arabidopsis thaliana.