Sh 9000lab

Neprilysin activity of PC12 cells was measured using a commercially available SensoLyte Neprilysin Fluorimetric Activity Assay Kit (AS-72223, AnaSpec, Inc.). At the end of cultivation, PC12 cell extracts (with the same concentration of protein) were diluted to bring the expected concentration within the range of the standard curve and reacted with 5-FAM/QXL™-520 Neprilysin substrate according to the manufacture’s instruction. After 60 minutes of incubation, fluorescence intensity was measured at excitation/emission = 490 nm/520 nm using a microplate reader (SH‐9000Lab, Hitachi, Japan).

We also performed colorimetric assay to determine cholinesterase activities according to the report by Ellman et al. (1961). Briefly, 10 µL enzyme (or enzyme-sample mixture) solution was mixed with 180 µL Ellman’s reagent [0.5 mM 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB) in 0.1 M phosphate buffer, pH 7.4] and incubated for 30 minutes at room temperature. Then 10 µL substrate [acetylthiocholine iodide (20 mM in double distilled water)] was added into the mixed solution and further incubated for another 10–30 minutes at room temperature. The absorbance was measured at 405 nm using a microplate reader (SH‐9000Lab, Hitachi).

Oxygen radical absorbance capacity (ORAC) assay kit was purchased from Cell Biolabs Inc., USA. The experiments were performed according to the manufacturer’s protocol with some modifications. The water-soluble vitamin E analog Trolox™ was used as the antioxidant standard. Briefly, samples (25 μL) were first added into the wells of a microplate (black 96-well plates, Nunc™ black microwell, Thermo Fisher, Scientific, Tokyo, Japan). Fluorescein (150 μL; 70 nM final concentration) and 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) (25 μL; 12 mM final concentration) solutions were quickly mixed in a pipetting reservoir and added to the wells of samples rapidly by using a multi-channel pipette. The plate was immediately placed in a fluorescence microplate reader (SH-9000Lab, Hitachi, Tokyo, Japan), and the fluorescence intensities were recorded every 5 minutes for 60 minutes at 37°C with excitation/emission = 480 nm/520 nm. Seven calibration solutions using Trolox (0, 2.5, 5, 10, 25, 50, and 100 μM final concentrations) as the standard antioxidant were also carried out in the same run. All reaction mixtures were prepared in triplicate, and at least three independent runs were performed for each sample (Xiao and Miwa, 2017).

Cell viability in PC12 cells were measured by PrestoBlue® Assay according to the manufacturer’s protocol. At the end of cultivation, PC12 cells were incubated for 3 hours at 37°C with fresh medium supplemented with 10% PrestoBlue^® (v/v; Thermo Fisher Scientific, Tokyo, Japan; A13261). The PrestoBlue^® reduction by the cells expressed as fluorescence intensity units was measured on a microplate reader (SH‐9000Lab, Hitachi) with excitation/emission = 560 nm/590 nm.