Biovision assay kit

The activity of β-glucuronidase was measured using BioVision Assay Kit (BioVision Incorporated, Milpitas, CA, USA). The assay was performed following manufacturer’s protocol. Briefly, cells were trypsinized, centrifuged and lyzed in Assay Buffer. Lysates were centrifuged (10,000×g, 5 min, 4 °C) and supernatants were collected. 5 µl of supernatants, along with the positive and negative control, as well as standards, were transferred into 96-well plates. The Assay Buffer and the Substrate were added and fluorescence was measured immediately (Ex/Em = 330/450 nm) and then every 5 min for 1 h at 37 °C using an Infinite M1000 PRO microplate reader (Tecan). Activity of β-glucuronidase was calculated according to equation:
β-glucuronidase activity [µU/ml] = B/(Δt × V)
where, B—amount of the reaction product obtained in Δt, Δt—reaction time (when the linear increase of the product was observed), V—sample volume added to the reaction.
The results were normalized according to the number of harvested cells.

Serum H₂O₂ concentration levels were measured by BioVision assay kit (BioVision, Inc., CA, USA) according to manufacturer's instructions. The principles based on the present of horse radish peroxidase, the OxiRed probe, react with H₂O₂ to produce a product with color that can be measured spectrophotometrically.

Blood samples were collected on ice, heparinized and spun at room temperature before storage of plasma at 4°C or allowed to clot and spun at 4°C before flash-freezing of serum in liquid nitrogen. Serum metabolites were measured by small-scale enzymatic assay for glucose (1070; Stanbio Laboratory, Boerne, TX) or using a standard automated glucose monitor. Serum insulin levels were determined by an ultrasensitive mouse-ELISA (Crystal Chem, Downers Grove, IL). Plasma levels for insulin like growth factor -1 (IGF-1) was determined using Quantikine ELISA kit for mouse/rat IGF-1(MG100; R&D Systems, MN, USA). Radioimmunoassay kit for glucagon estimation (GL-32K; Millipore, Billerica, MA) was used. For glucagon, blood samples were collected in tubes containing aprotinin (1 g/ml, Bayer Healthcare, Berkeley, CA), and the serum was stored at -80°C before radio-immunoassay. Serum amylase levels were analyzed using Biovision assay kit (K711-100; Biovision, Milpitas, CA).