cAMP reporter gene assay

Cell lines were transiently transfected with CRE-luc reporter plasmid (Stratagene, Germany) using PolyFect transfection reagent (Qiagen, Germany), following the transient transfection protocol. Briefly, cells were seeded at a density of 4 × 10⁵ cells per well in 6-well plates (Sarstedt, Germany). Cell lines were incubated for 24 h in an incubator. The formation of transfection complex containing CRE-luc reporter plasmid and PolyFect transfection reagent in RPMI1640 without FBS-supplemented medium was performed at room temperature for 10 min. During the time of transfection complex formation, the cells were washed with PBS 1× once, and new complete RPMI1640 was added to the cells. The transfection complex was diluted in complete RPMI1640 medium and transferred to the cells in each well, and the cells were incubated for 24 h to allow reporter gene expression. Then, the cells were harvested and seeded at a density of 6 × 10⁴ cells per well in white, clear-bottom 96-well plates (Greiner, Germany). After 48 h, the cells were exposed to various concentrations of adenosine for 3 h (agonist activity study). In case of antagonist activity study, selective antagonists were incubated 30 min prior to adenosine administration. After 3 h of adenosine incubation, cells were lysed using lysis reagent (8 mM Tricine, 1 mM DTT, 2 mM EDTA, and 5% w/v Triton X-100) for 10 to 20 min at 4 to 8 °C. Luminescence was measured after addition of luciferase assay reagent (30 mM Tricine, 10 mM MgSO₄, 0.5 mM EDTA, 10 mM DTT, 0.5 mM ATP, 0.5 mM coenzyme A, and 0.05 mM D-luciferin) by a LUMIstar Galaxy microplate reader (BMG Labtech, Germany).