HEK293 cells, stably expressing GloSensor-22F cAMP probe (HEK-Gs), had been generated in-house and characterized earlier 36 (link). In order to obtain double-stable cell lines, overexpressing GloSensor-22F cAMP probe and a desired SSTR subtype, HEK-Gs cells were transfected with SSTR2_HA-P2A-mCherry, SSTR3_Myc-P2A-mCherry or SSTR5_Flag-P2A-mCherry plasmids. Transfections were carried out with Xfect polymer (Clontech Laboratories, Cat#631317), according to the manufacturer`s instructions. After 4-6 weeks of continuous selection with 500 μg/ml geneticin (G418; Roche, Cat#04727878001), the evolved stable clones were further sorted at least twice with FACSAria IIu cell sorter (Beckton Dickinson; provided by Cell Imaging Core of Turku Centre for Biotechnology) to collect the brightest fraction of mCherry-positive cells. As mCherry and SSTRs are transcriptionally coupled via P2A linker 46 , both proteins are expected to accumulate in proportional amounts in the cells with the plasmid expression, which makes a rationale for the above FC-aided enrichment approach. The expression of target SSTR subtypes in the procured cultures was eventually validated with indirect immunolabelling in flow cytometry analysis, as described below. BON1 cells, stably expressing GloSensor-22F cAMP probe (BON-Gs), were derived and characterized in a similar fashion.