HPLC CD73 Enzymatic Activity Assay

To determine the AMPase activity by high performance liquid chromatography (HPLC), 0.2 × 10⁶ CD8⁺ T cells or peritoneal macrophages were incubated with 1 µM 1,N⁶-etheno-AMP (eAMP, Biolog) for 30 min at 37°C. After the incubation, cells were removed (450 × g, 5 min, 4°C) and all samples were passed through 10 kDa size exclusion filters (10,000 × g, 10 min, 4°C, Pall Corporation) and stored at -20°C until analyses. The analyses was performed on reversed-phase HPLC system (Agilent Technologies) with a 250 mm × 4.6 mm C8 Luna column (5 µm particle size, Phenomenex) as stationary phase. The mobile phase consisted of different compositions of HPLC buffer A (20 mM KH₂PO₄, pH 6.0) and B (50% buffer A, 50% methanol), and elution of the nucleotides from the column resulted from an increasing methanol content in the mobile phase [0.0 min (0.0% buffer B), 5.0 min (0.0% buffer B), 27.5 min (100.0% buffer B), 30.0 min (100.0% buffer B), 32.0 min (0.0% buffer B), 43.0 min (0.0% buffer B)]. The signals in both systems were detected by fluorescence detection (230 nm excitation wavelength, 410 nm emission wavelength). Different amounts of etheno-nucleotides (Biolog) were measured to quantify eAMP and the degradation product etheno-adenosine (eADO).