Fig. 3. 1 is selective and can be used to visualise A_2AR in live cells.

a Live HEK293 cells expressing SNAP-A_2AR were labelled with SNAP-surface-AF488 and then treated with 250 nM 1 in the absence or presence of ZM241385 for 2 h prior to the capture of single equatorial confocal images. Cells treated with 250 nM 1 were then washed multiple times over 1 h and then imaged. Left hand column represents Cy5 fluorescence, middle column AF488 fluorescence and right hand column the merged image. Fluorescent intensity values (8-bit greyscale) were determined for Cy5 (b) and AF488 (c) in membrane regions of interest from the images obtained as described in a and also in cells treated with 10 μM ZM241385 for 1 h after 2 h incubation with 250 nM 1 (images shown in Supplementary Fig. 4). Each point represents the values obtained from one cell. Images were obtained in four independent experiments, error bars represent mean ± SD. d HEK293 cells expressing SNAP tagged versions of one of the four adenosine receptor subtypes (A₁, A_2A, A_2B or A₃) were labelled with SNAP-surface-AF488 prior to the addition of 250 nM 1 for 2 h prior to the capture of single equatorial images. Images shown in a and d are representative of images taken in four (a) or three (d) independent experiments, with all image sets taken using identical settings for laser power, gain, and offset in both channels. Scale bar shown represents 10 µm.