96 well white half area microplate

CHO cells transfected with hAR subtypes were washed with phosphate-buffered saline, detached with trypsin, and centrifuged for 10 min at 200× g. Cells were seeded in a 96-well white half-area microplate (Perkin Elmer, Boston, USA) in a stimulation buffer composed of Hank Balanced Salt solution, 5 mM HEPES, 0.5 mM Ro 20-1724, 0.1% BSA, 1 IU/mL adenosine deaminase. cAMP levels were then quantified by using the AlphaScreencAMP detection kit (Perkin Elmer, Waltham, MA, USA) following the manufacturer’s instructions [47 (link)]. At the end of the experiments, plates were read with the Perkin Elmer EnSight Multimode Plate Reader.

CHO cells transfected with hAR subtypes were washed with phosphate-buffered saline, detached with tripsine and centrifuged for 10 min at 200× g. Cells were seeded in a 96-well white half-area microplate (Perkin Elmer, Boston, USA) in a stimulation buffer composed of Hank Balanced Salt Solution, 5 mM HEPES, 0.5 mM Ro 20–1724, 0.1% BSA. The cAMP levels were then quantified by using the AlphaScreen cAMP Detection Kit (Perkin Elmer, Boston, MA, USA), following the manufacturer’s instructions [54 (link)]. At the end of the experiments, the plates were read with a Perkin Elmer EnSight Multimode Plate Reader.

In lymphocytes, the potency of the well-known A_2A and A₃ adenosine agonists CGS 21680 and Cl-IB-MECA (0.1 nM–1 μM) was investigated. Cells were seeded in 96-well white half-area microplate (Perkin Elmer, Boston, MA, USA) in a stimulation buffer composed of Hank Balanced Salt Solution, 5 mM HEPES, 0.5 mM Ro 20-1724, 0.1% BSA. Forskolin (1 μM) was used to stimulate adenylyl cyclase activity after the addition of Cl-IB-MECA. cAMP levels were then quantified by using the AlphaScreen cAMP Detection Kit (Perkin Elmer) following the manufacturer’s instructions. At the end of the experiments, plates were read with the Perkin Elmer EnSight Multimode Plate Reader.