Figure 3

Detection of cAMP generated by donor cells using sensor cells. (A) Preincubation of co-cultures of GS-293 (50,000 cells) with FSHR-293 (50,000 cells) for 45 minutes with assay medium either in the presence or absence of IBMX and monitoring luminescence after addition of rFSH (200 mIU/ml). GS-293 cells could detect cAMP production upon stimulation of FSHR-293 cells with rFSH only in the presence of IBMX. In the absence of IBMX, no luminescent signal upon FSHR-293 stimulation could be detected. Data represented as mean of triplicates for one representative experiment ± standard error of the mean (SEM). (B) EPAC-293 and FSHR-293 co-cultures (50,000 cells each) were stimulated with rFSH (200 mIU/ml) in the presence or absence of IBMX. A change in FRET ratio (480/528 nm) was observed after rFSH stimulation. EPAC-293 cells could detect cAMP production from co-cultured FSHR-293 cells only in the presence of IBMX. Data represented as mean of triplicates for one representative experiment (± SEM). (C) Co-culture of GS-293 (25,000 cells) with KK-1 (75,000 cells) and monitoring luminescence after addition of rLH (100 ng/ml). Data represented as mean of triplicates for one representative experiment (± SEM). (D and E) Absence of LHCGR and FSHR expression in GS-293 cells is shown by luminescence values upon rLH (100 ng/ml) and rFSH (200 mIU/ml) stimulation being similar to unstimulated GS-293 control. Stimulated co-culture of GS-293 cells with either KK-1 or FSHR-293 cells was used as positive control for LHCGR and FSHR expression, respectively. Unstimulated co-cultures of GS-293 with either KK-1 or FSHR-293 were used as negative control. Data represented as mean of duplicates for one representative experiment [± standard deviation (SD)]. All the experiments have been independently repeated at least three times. (Relative Light Unit: RLU).