DPPIV enzyme activity of the STB-EVs was determined using a DPPIV-Glo Protease Assay (Promega, Southampton, UK). Briefly, the luminescent DPPIV-Glo Reagent was incubated at R/T for 30 min with either clinical samples (STB-EVs isolated from perfused placentae from both GDM and normal pregnancy), Tris-BSA (blanks) or a purified DPPIV enzyme in Tris-BSA as standard (Recombinant human CD26 protein, Abcam, Cambridge, UK). Luminescence was measured using a FLUOstar Omega apparatus (BMG Labtech, Aylesbury, UK).
For the DPPIV enzymatic inhibition studies, MEDIUM/LARGE STB-EVs and SMALL STB-EVs were pre-treated with vildagliptin (DPPIV-specific inhibitor) (Sigma Aldrich, Gillingham, UK) (0–100 nM) for 1 h, and DPPIV residual enzymatic activity was determined.
STB-EV-bound DPPIV ability to break GLP-1 was measured using a GLP-1 active assay according to manufacture instructions (Cisbio, Codolet, France). MEDIUM/LARGE STB-EV and SMALL STB-EV pools of three GDM patients in series dilution were mixed with 1400 pg/ml of GLP-1, respectively, and incubated with anti-Active GLP-1-Tb3+Cryptate Antibody and with anti-Active GLP-1-d2 antibody overnight at RT. Recombinant DPPIV (Recombinant human CD26 protein, Abcam, Cambridge, UK) was used as positive control, and PBS was used as negative control. HTRF measurement was performed using a CLARIOstar apparatus (BMG Labtech, Aylesbury, UK).
For the DPPIV enzymatic inhibition studies, MEDIUM/LARGE STB-EVs and SMALL STB-EVs were pre-treated with vildagliptin (DPPIV-specific inhibitor) (Sigma Aldrich, Gillingham, UK) (0–100 nM) for 1 h, and DPPIV residual enzymatic activity was determined.
STB-EV-bound DPPIV ability to break GLP-1 was measured using a GLP-1 active assay according to manufacture instructions (Cisbio, Codolet, France). MEDIUM/LARGE STB-EV and SMALL STB-EV pools of three GDM patients in series dilution were mixed with 1400 pg/ml of GLP-1, respectively, and incubated with anti-Active GLP-1-Tb3+Cryptate Antibody and with anti-Active GLP-1-d2 antibody overnight at RT. Recombinant DPPIV (Recombinant human CD26 protein, Abcam, Cambridge, UK) was used as positive control, and PBS was used as negative control. HTRF measurement was performed using a CLARIOstar apparatus (BMG Labtech, Aylesbury, UK).