Ultrasonic homogenizer vp 050n

For acetylcholinesterase (AChE) activity assay, mice lungs were rapidly dissected under deep anaesthetisation and frozen in liquid nitrogen. The frozen lung samples were homogenised in ice-cold phosphate buffer (50 mM, pH 7.4) using an Ultrasonic Homogenizer VP-050N (TAITEC, Tokyo, Japan). The lung homogenates were centrifuged at 11,000×g for 20 min, and the supernatants were aliquoted and stored at − 80 °C until assay. The concentration of total protein was determined using the Quick Start Bradford Protein Assay reagents (Bio-Rad, Tokyo, Japan) and bovine serum albumin standard (Bio-Rad). AChE activity was measured by duplicate analysis per sample using the Amplite Fluorimetric Acetylcholinesterase Assay Kit (AAT Bioquest, Sunnyvale, CA, USA) in accordance with the manufacturer’s instructions. The AChE activity of each sample was determined by the standard curve, and enzymatic activity was calculated as mU/mg protein.

The left testicle was homogenized in physiological solution by sonication for 40 s (output 40%, 20 kHz; Ultrasonic Homogenizer VP-050N, Taitec Corp., Koshigaya City, Saitama, Japan). Testicle homogenates were centrifuged at 10,000 rpm (1619 Rotor Universal 320 Centrifuge, Hettich^®, Tuttlingen, DE) for 10 min at 4 °C and the supernatants were collected to evaluate lipoperoxidation and the enzymatic activity levels of SOD and GPX. Before determination, the total protein concentrations in supernatants were calculated by Bradford assay. Lipoperoxidation was determined by malondialdehyde (MDA) identification using thiobarbituric acid as proposed by Buege and Aust [26 ]. SOD and GPX activity levels were evaluated using RANSOD and RANSEL kits, respectively, following the manufacturers’ instructions.