Serum free dmem

MMCT was performed as previously described (Ohira et al. 2019a (link), b (link)). Briefly, donor cells were incubated with 0.05 μg/ml colcemid (Sigma) in F12 (Invitrogen) containing 20% FBS (JRH Biosciences) for 72 h. Micronuclei were harvested by treatment with 10 μg/ml cytochalasin B (Sigma) and centrifugation and sequentially filtered through 8, 5, and 3 μm polycarbonate filters (Whatman Nuclepore, Kent, UK). The fusion was mediated by 47% polyethylene glycol 1000 (Wako), followed by extensive washing with serum-free DMEM (Sigma). After incubation for 24 h in the culture medium, cells with chromosome transfer were selected with G418 (Sigma) or blasticidin S hydrochloride (Wako).

MMCT was performed as previously described^{23 (link)}. Briefly, the donor cells were incubated with 0.05 μg/ml colcemid (Sigma, St. Louis, MO, USA) in F12 (Invitrogen) containing 20% FBS (JRH Biosciences) for 72 hr. Micronuclei were harvested by treatment with 10 μg/ml cytochalasin B (Sigma) and centrifugation, and were sequentially filtered through 8, 5, and 3 μm polycarbonate filters (Whatman Nuclepore, Kent, UK). Fusion was mediated by 47% polyethylene-glycol 1000 (Wako), followed by extensive washing with serum-free DMEM (Sigma). After incubation for 24 hr in F12 supplemented with 10% FBS, cells with chromosome transfer were selected with G418 (Sigma). CHO DG44 cells were cultured with 300 μg/ml G418. CHO K1 cells were cultured with 800 μg/ml G418.

The effects of oxyntomodulin analogues and native GLP-1, glucagon and oxyntomodulin on cyclic adenosine monophosphate (cAMP) accumulation were assessed in Chinese hamster ovary (CHO-K1) cells over-expressing the rat glucagon receptor (cDNA from Origene, Maryland, USA) and Chinese hamster lung (CHL) cells over-expressing the rat GLP-1 receptor (a kind gift from Professor Bernard Thorens, University of Lausanne, Switzerland). For cAMP studies, cells were seeded at a density of 150000 cells/ml in a 48 well plate (Nunc, VWR International Inc., Chicago, USA) and incubated for 24 h. Cells were serum starved for 1 h and then incubated for 30 mins at room temperature with a range of peptide concentrations diluted in serum free DMEM (Sigma–Aldrich, Dorset, UK) in the presence of 1 mM IBMX (3-isobutyl-1-methylxanthine, Sigma–Aldrich). After incubation, medium was removed and cells lysed with 0.1 M HCl +0.5% Triton-X. cAMP levels were measured using an ELISA kit according to manufacturer’s instructions (ADI-900-066, Enzo Life Sciences, New York, USA), optical density was read at 405 nm on a Biotek ELx808 (Wolf Laboratories, York, UK). EC₅₀’s were calculated using Prism v5 (GraphPad Software Inc., San Diego, CA, USA). Concentrations were applied in duplicate or triplicate per experiment and each experiment repeated a minimum of 3 times.