Amplite fluorimetric acetylcholinesterase assay kit

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 thiocholine produced from the hydrolysis of acetylthiocholine by the endogenous AchE enzyme in each sample was quantified by a fluorescence colorimetric assay to examine the effect of ILA on AchE activity, which is a biochemical marker for neuronal differentiation in PC12 cells [28 (link)]. In brief, PC12 cells were grown and treated for five consecutive days, as mentioned. The cells were then lysed with ice-cold NP-40 cell lysis buffer containing 150 mM NaCl, 50 mM Tris (pH 8.0), 2 mM EDTA (pH 8.0) and 1% (v/v) NP-40. AchE activity of the cell lysates was determined by the Amplite Fluorimetric Acetylcholinesterase Assay Kit (AAT Bioquest, Sunnyvale, CA, USA) according to the manufacturer’s instructions. Assay signals were read with a fluorescence absorbance microplate reader (SH-9000, Corona Electric, Ibaraki, Japan). at Ex/Em = 490/520 nm. The AchE activity was determined from the standard curve and normalized with the protein concentration in each sample. The protein concentration was determined by Pierce bicinchoninic acid (BCA) protein assay kit (Invitrogen, Paisley, UK) with bovine serum albumin as a standard.

PC12 cells (10,000 cells/mL per well) were cultured in collagen type IV-coated 24-well culture plates, as mentioned. The cells were then exposed to the antagonists of AhR receptor (ANF and CH223191) at a final concentration of 1 µM for 1 h prior to the treatment with NGF (25 ng/mL) and the test compounds (reference control, ILA or tryptophan at a final concentration of 100 nM) for five consecutive days. Control cells without ANF and CH223191 were also grown and treated under the same conditions. The medium and test compounds were replaced after three days. Following treatment, the cells were lysed with ice-cold NP-40 cell lysis buffer. AchE activity of the cell lysates was then determined by the Amplite Fluorimetric Acetylcholinesterase Assay Kit (AAT Bioquest, Sunnyvale, CA, USA) according to the manufacturer’s instructions. The AchE activity was determined as described above.