Total circulating DPP4 was assessed by a specific enzyme-linked immunosorbent assay (ELISA) (R&D Systems, DuoSet, lot P185646), using a 1:1 dilution (1 part serum to 1 part saline) as sample and performed according to the manufacturer's instructions.
DPP4 serum enzymatic activity was determined as the rate of cleavage of 7-amino-4-methylcoumarin (AMC) from the fluorogenic substrate Glycyl-Prolyl-AMC (Gly-Pro-AMC; Sigma, USA) in a 96-well microplate in SpectraMax® M5 ROM v3.0.22 Molecular Devices spectrofluorometer (USA) at 360 nm excitation and 460 nm emission for 15 min and in duplicate. The rate of liberated fluorescence was calculated per minute (U/min). All enzymatic reactions were performed with 10 µL of serum sample in activity buffer (25 mM HEPES, pH 7.5) in the presence of 20 µM of the substrate in a final volume of 100 µL (30 (link)). To confirm the specificity of DPP4 activity in these assays, a highly selective DPP4 inhibitor (Sitagliptin; Sigma) was incubated with some serum samples for 15 min before proceeding to hydrolysis of the substrate, in triplicate, with Sitagliptin at a final concentration of 1 µM.
DPP4 serum enzymatic activity was determined as the rate of cleavage of 7-amino-4-methylcoumarin (AMC) from the fluorogenic substrate Glycyl-Prolyl-AMC (Gly-Pro-AMC; Sigma, USA) in a 96-well microplate in SpectraMax® M5 ROM v3.0.22 Molecular Devices spectrofluorometer (USA) at 360 nm excitation and 460 nm emission for 15 min and in duplicate. The rate of liberated fluorescence was calculated per minute (U/min). All enzymatic reactions were performed with 10 µL of serum sample in activity buffer (25 mM HEPES, pH 7.5) in the presence of 20 µM of the substrate in a final volume of 100 µL (30 (link)). To confirm the specificity of DPP4 activity in these assays, a highly selective DPP4 inhibitor (Sitagliptin; Sigma) was incubated with some serum samples for 15 min before proceeding to hydrolysis of the substrate, in triplicate, with Sitagliptin at a final concentration of 1 µM.
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