Cell Line Generation. CHO-K1 cells were BacMam transfected, for 24 hr, with vector containing human A 2A (5% v/v) or A 2B (5% v/v). CHO A 2A and CHO A 2B cells were maintained in Dulbecco's modified Eagle's medium/Ham's F-12 medium (Sigma) supplemented with 10% FBS. Once transfected, cells were harvested and frozen at -150 °C until required for assay. cAMP Assays. CHO A 2A and CHO A 2B cells were thawed, and seeded at 500 and 1000 cells per well, respectively, in white 384-well plates in assay buffer (Hank's balanced salt solution; Lonza, Basel, Switzerland) supplemented with 100 µM rolipram and 1 U/mL adenosine deaminase, pH 7.4) in the absence or presence of antagonists, for 1 hour at 37°C. EC 80 ( A 2A 6 nM/ A 2B 19 nM) concentration of NECA, were added and plates further incubated for 30 minutes. cAMP was detected using cAMP Gs Dynamic kit (Cisbio, Codolet, France) according to the kit instructions. Plates were read on a PHERAstar FS microplate reader (BMG LabTech,Offenburg, Germany) using standard homogeneous time resolved fluorescence settings. Homogeneous time resolved fluorescence ratios were determined by dividing emissions at 665 nm by emissions at 620 nm and multiplying by 10,000. Data analysis. Percentage agonist response was normalized to 10 µM NECA (100%) and DMSO (0%).