Ip one tb assay kit

COS-7 cells were cultured and transiently transfected with plasmids encoding wild-type PTH(1-84) or the [Cys25]PTH(1-84) mutant, as described.^{(7 (link))} Forty-eight hours after transfection, PTH levels were measured in conditioned medium using ntPTH and PTH(1-84) assays. Competition binding assays were performed using membranes prepared from GP-2.3 cells, which are HEK-293 cells stably expressing the wild-type human PTH1R and the Glosensor cAMP reporter,^{(53 )} and ¹²⁵I-rPTH(1-34) radioligand tracer, as described.^{(54 (link))} cAMP assays were performed in SGS-72 cells, which are SaOS-2 cells stably transfected to express the Glosensor cAMP reporter using an Envision (PerkinElmer, Waltham, MA, USA) plate reader to measure intracellular cAMP-dependent, luciferase-based luminescence, as described.^{(55 (link))} Signaling via the PLC/IP₃ pathway was assessed using GP-2.3 cells; the cells were challenged with PTH(1-34) or [Cys25]PTH(1-34) (30 minutes, 37°C), and the resulting intracellular inositol monophosphate (IP₁) levels were measured using the IP-One Tb assay kit (CisBio, Bedford, MA, USA).