Butyrylcholinesterase assay kit

rhBChE-overexpressing GFFs and WT GFFs were cultured in DMEM/F12 with 15% FBS until they were up to 70–90% confluent, then the medium was changed with Opti-MEMTM (Gibco, Brooklyn, NY, USA) for 1 day. The rhBChE in the supernatant was detected using the Butyrylcholinesterase assay kit (Nanjing Jiancheng Bioengineering Institute). After the addition of organophosphorus pesticides, GFFs were seeded in 96-well plates to evaluate the MID (median lethal dose) of Dichlorvos in acetone (GBW 081320). Then, rhBChE and WT GFFs were seeded separately in 96-well plates and cultured in DMEM/F12 with 15% FBS adding the MID of Dichlorvos for 24 h. Then, cell viability was detected by an MTT Cell Proliferation and Cytotoxicity Assay Kit (Solarbio, Tongzhou, Beijing, China) in accordance with the manufacturer’s instructions.

Serum BChE activity was detected using Butyrylcholinesterase assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), following the manufacturer’s instructions. The enzymatic reaction rate of BChE was measured using the Ellman method [26 (link)]. In a 96-well plate, 10 μL of serum was added into phosphate buffer (215 μL, pH 8.0, 100 mmol/L) and incubated at 37 °C for 5 min. Following that, 25 μL of 37 °C preheated MIX Buffer (5,5′-dithiobis-2-nitrobenzoic acid, DTNB, 3.4 mmol/L; butyrylthiocholine iodide, BTCh, 2.5 mmol/L) was added and mixed well. The mixed solution was immediately placed in a 37 °C microplate reader (Berthold Technologies, TriStar2S LB942, Bad Wildbad, Germany) and measured at 412 nm every 2–3 min (starting at 5 min and ending at 30 min).