cAMP accumulation assay

PTH and PTHrP stimulated cAMP accumulation was measured by a LANCE Ultra cAMP kit from PerkinElmer (Boston, MA, USA). After 24 h culture, the transfected cells were seeded into 384-well microtiter plates at a density of 3000 cells per well in HBSS supplemented with 5 mM HEPES, 0.1% (w/v) BSA or 0.1% (w/v) casein and 0.5 mM 3-isobutyl-1-methylxanthine. The cells were stimulated with different concentrations of peptide agonists for 40 min at RT. Eu-cAMP tracer and ULight^TM-anti-cAMP were then diluted by cAMP detection buffer and added to the plates separately to terminate the reaction. Plates were incubated at RT for 1 h and the fluorescence intensity was measured at 620 nm and 665 nm by an EnVision multilabel plate reader from PerkinElmer (Boston, MA, USA).