Optiplate microplates

Cells were transferred to serum-free medium 2 h prior to assay. Ligands and controls (10 µl of 10× concentrates) were added to 90 µl medium and incubated for 5 min at 37 °C. The final concentration of DMSO was 0.2%. The reaction was stopped by aspiration of the medium and addition of 50 µl lysis buffer (PerkinElmer Life Sciences). To measure ERK1/2 phosphorylation, the AlphaScreen^®SureFire ERK 1/2 assay kit (PerkinElmer Life Sciences) was used with the high sensitivity protocol. Phosphorylated ERK1/2 was measured in 384-well white OptiPlate microplates (PerkinElmer Life Sciences) with the Fusion AlphaScreen multilabel reader (PerkinElmer Life Sciences).

Equilibration of five islets was performed in KRB containing 2.8 mM glucose for 30 min at 37 °C in 96-well plates. If not mentioned otherwise, islets were stimulated with peptide solution or respective controls in KRB for 30 min at 37 °C. Islets were lysed with acid ethanol and supernatant as well as lysates kept frozen at −20 °C until measurement. To measure insulin the AlphaLISA technology (Perkin Elmer) was used according to the manufacturer’s protocol in 384-well OptiPlate microplates (Perkin Elmer) with the EnVision Multilabel Reader (Perkin Elmer). Somatostatin amounts were determined using either AlphaLISA (Perkin Elmer) or ELISA (Phoenix Pharmaceuticals). For reliable measurement protocol adaptions were necessary. For AlphaLISA these were as follows: increasing sample volumes to 35 µl/well, reduced bead volume of 5 µl/well, and an extended incubation period with donor beads of 90 min. For the ELISA measurement the sample incubation time was extended to over-night and the biotinylated peptide was diluted 1:5 to achieve reliable results.

For cAMP assays, cells were split into 96-well plates (1.5 × 10⁴ cells/well), whereby the plates for the HEK293T cells had been coated with 0.0002% poly-L-lysine (Merck) in PBS for 30 min at 37 °C prior to use. Cells were transfected with Lipofectamine2000 (ThermoFisher) according to manufacturer’s protocol 24 h later. For basal activity measurements, between 100 ng and 250 ng of DNA per well were transfected and for peptide activation detection, 100 ng DNA per well. 48 h post transfection, cells were washed with serum- and phenol red-free DMEM containing 1 mM 3-isobutyl-methyl-xanthine (IBMX) for 5 min. IBMX inhibits phosphodiesterases and subsequently blocks the degradation of cAMP. To analyze the effects of agonistic peptides (Peptides and Elephants GmbH), transfected cells were treated with 1 mM peptide in DMEM with 1 mM IBMX for 1 h at 37 °C.
To stop the incubation, the media was removed and the cells were lysed with LI buffer (PerkinElmer Life Sciences) and frozen at −20 °C. To measure cAMP concentrations, samples were thawed and the AlphaScreen cAMP assay kit (PerkinElmer Life Sciences) was applied following the manufacturer’s instructions in 384-well white OptiPlate microplates (PerkinElmer Life Sciences) utilizing the Spark Cyto multimode plate reader (Tecan).