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HEK293 cells transfected with HA-tagged WT ghrelin receptor or mutants were seeded at a density of 4 10 4 cells per well into 96-well culture plates and incubated for 24 h at 37 C in 5% CO 2 .The cells were then incubated with 2 mol/L Fluo-4 AM in HBSS (5.4 mmol/L KCl, 0.3 mmol/L Na 2 HPO 4 , 0.4 mmol/L KH 2 PO 4 , 4.2 mmol/L NaHCO 3 , 1.3 mmol/L CaCl 2 , 0.5 mmol/L MgCl 2 , 0.6 mmol/L MgSO 4 , 137 mmol/L NaCl, 5.6 mmol/L D-glucose and 250 mol/L sulfinpyrazone, pH 7.4) at 37 C for 40 min