Suc ala ala phe mca

NEP activity measurements were performed on primary neurons after 15–28 days of in vitro (DIV15–28) culture as previously described [29 (link)]. Somatostatin (Peptide institute 4023), TT232 (Tocris 3493), recombinant ENSA (abcam ab92932), recombinant NSG-1 (Creative BioMart NSG1–332H), recombinant NUCKS-1 (Creative BioMart NUCKS1–10956M) and diazoxide (Wako 364-98-7) were added as appropriate concentrations, and cells were incubated for a further 24 h. Neurons were then incubated with substrate mixture 50 µM suc-Ala-Ala-Phe-MCA (Sigma S8758), 10 nM benzyloxycarbonyl Z-Leu-Leu-Leucinal (Peptide institute 3175-V) and cOmplete EDTA-Free-Protease inhibitor (Roche Diagnostics 4693132) in 0.2 M MES buffer (pH6.5) with or without Thiorphan (Sigma T6031) for 1 h at 37 ˚C. Following this, 0.1 mM phosphoramidon (Peptide Institute 4082) and 0.1 mg/ml leucine aminopeptidase (Sigma L-5006) were added, and the reaction mixture was incubated at 37 ˚C for a further 30 min. 7-Amino-4-methylcoumarin fluorescence was measured at excitation and emission wavelengths of 380 nm and 460 nm, respectively. Centrifugal 10 and 30 kDa filters (Merck UFC503096, 501096) were used to separate conditioned media obtained from cortical/hippocampal neurons.

Cells, grown in black 96‐well plates with a clear bottom, were washed with 0.1 M MES (pH = 6.5) and incubated with substrate mix (0.1 M MES (pH = 6.5), 1× complete protease inhibitor (#786‐331; G‐Biosciences, Maryland Heights, MO, USA), 1 μM Z‐Leu‐Leu‐Leu‐H (aldehyde) (#3175; Peptide Institute, Osaka, Japan), 0.5 mM Suc‐Ala‐Ala‐Phe‐MCA (#S8758; Sigma‐Aldrich, St. Louis, MO, USA) with or without 10 μM Thiorphan (#T6031; Sigma‐Aldrich) for 1 hr at 37°C. The cells were then incubated with 15 μM Phosphoramidon (#4082; Peptide Institute) and 0.14 μl of LAPase (#L5006; Sigma‐Aldrich) in a final volume of 100 μl. Reaction was stopped with 10 nM of EDTA and fluorescence was measured in a plate reader (λ_Ex = 355 nm, λ_Em = 460 nm). Differences between substrate mix with and without Thiorphan represent neprilysin activity.