Measuring Adenosine Receptor Activity in CHO Cells

Plasmids encoding wild-type or variant human A_2A adenosine receptors were transfected into CHO-K1 cells (ATCC product CCL-61) using lipofectamine 2000. The cell line was authenticated by the manufacturer and also determined to be free from mycoplasma via a PCR-based assay, agar culture method, and Hoechst DNA stain method. 24 h after transfection, cells were detached and grown in 96-well plates in medium containing equal volume of DMEM and F12 supplemented with 10% fetal bovine serum, 100 Units/ml penicillin, 100 µg/ml streptomycin, and 2 µmol/ml glutamine. After growing for 24 h, culture medium was removed and cells were washed twice with PBS. Cells were then treated with assay buffer containing rolipram (10 µM) and adenosine deaminase (3 units/ml) for 30 min followed by addition of agonist and incubated for 20 min. The reaction was terminated upon removal of the supernatant, and addition of 100 µl Tween-20 (0.3%). Intracellular cAMP levels were measured with an ALPHAScreen cAMP assay kit (PerkinElmer, Catalog Number: 6760635D) following the manufacture’s protocol.