Adi 900 067

Pre-iBAs were differentiated to mature iBAs and cAMP assays were performed during days 6-8 of their differentiation. In brief, cells were washed with DMEM (25 mM glucose) without FBS and serum-depleted with DMEM (25 mM glucose) without FBS, 2 hours prior to isoproterenol stimulation and/or compound treatment. Whenever pre-treatment was required, this was performed during the 2 h serum-depletion period. Stimulation with isoproterenol or forskolin and treatment with compounds was acutely performed for 5 min – 30 min. After the stimulation period, cells were rapidly washed with PBS and subsequently processed according to manufacturer’s instructions. The Enzo cAMP ELISA kit was utilized to process cells after stimulation and to measure intracellular cAMP concentrations (Enzo, ADI-900-067). An aliquot of the cell lysate was used for BCA protein assay, for protein quantification and normalization of the cAMP concentrations. Biochemical colorimetric assays were detected by SynergyMix plate reader (BioTek). Colorimetric assays were analyzed by Gen5 v3.08 (BioTek).

The human GLP-1 receptor gene was introduced into CHO-K1 cells via transfection, following the methodology described in a recent study [35 (link)]. The effective introduction of genetic material was verified the next day using the quantitative polymerase chain reaction (qPCR) technique outlined below. Cells expressing GLP-1 receptors were then exposed to SA at the specified doses for 1 h. The intracellular cAMP levels were then assessed with a commercially available ELISA kit (ADI-900-067, Enzo Life Sciences, Farmingdale, NY, USA). The specified samples were subjected to triplicate assays. A control group of CHO-K1 cells that had not been transfected with the GLP-1 receptor gene was used. The cAMP concentrations of different groups were compared.

Total anti-TSHR binding antibodies, stimulating anti-TSHR antibodies and total T4 levels were analyzed in sera as previously described (23 (link)). Briefly, to determine total anti-TSHR binding antibody titers, 25 μl serum was combined with 75 μl human control serum and the antibody concentration was measured using a commercial TSH receptor antibody concentration (TRAK) kit following manufacturer´s instructions (Thermo Fisher Scientific Cat# 701.1, RRID : AB_2927546.). CHO cells stably transfected with mouse TSHR, kindly provided by Sandra McLachlan, were used to perform a bioassay in order to measure stimulating anti-TSHR antibodies. cAMP production of the cells was measured by ELISA (Enzo Life Sciences Cat# ADI-900-067, RRID : AB_2814712). Total T4 concentrations were determined via ELISA following the instructions of the vendor (DRG International Cat# EIA-1781, RRID : AB_2927536).