hAChE, hBuChE, Acetylcholinesterase Activity Assay Kit and Butyrylcholinesterase Activity Assay Kit were purchased from Sigma Aldrich. And tacrine hydrochloride were purchased from MedChemExpress. The capacity of all the target compounds to inhibit hAChE and hBuChE activities were assessed by Ellman’s method. The concentration of compound producing 50% of enzyme activity inhibition (IC50) was calculated by nonlinear regression analysis of the response-concentration (log) curve, using the Graph-Pad Prism program package (Graph Pad Software; San Diego, CA). Results are expressed as the mean ± SD of at least three different experiments performed in triplicate.
Acetylcholinesterase activity assay kit
Manufactured by Merck Group
Sourced in United States
The Acetylcholinesterase Activity Assay Kit is a laboratory tool designed to measure the activity of the enzyme acetylcholinesterase. Acetylcholinesterase is responsible for the breakdown of the neurotransmitter acetylcholine, which is involved in various physiological processes. The kit provides the necessary reagents and protocols to quantify the enzymatic activity of acetylcholinesterase in a range of sample types.
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10 protocols using acetylcholinesterase activity assay kit
1
Cholinesterase Inhibition Assay
2
Hippocampal Acetylcholinesterase Activity Assay
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Acetylcholinesterase activity assay kit (Sigma-Aldrich, USA) was used based on an optimized version of Ellman’s method. Tissue total protein concentration was determined by Bradford protein assay. Activity was presented as unit per gram total of protein in hippocampal tissue.
3
Planarian Acetylcholinesterase Activity Assay
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After exposure, the planarians were washed 3X with IO water and then homogenized in 200 µL 1% Triton X-100 in PBS as described in (Hagstrom et al, 2017 (link); 2018 (link)). Briefly, after sitting on ice for about 15–20 min, the homogenate was centrifuged at 20,817 x g at 4°C for 30 min. The supernatant (clarified homogenate) was transferred to a clean, chilled tube and subsequently used. An Ellman assay (Ellman et al, 1961 (link)) was performed on the clarified homogenate using an Acetylcholinesterase Activity Assay kit (Sigma-Aldrich). Absorbance was read at 412 nm every minute for 10 min using a SpectraMax ABS Plus (Molecular Devices, San Jose, CA) spectrophotometer. AChE activity was calculated as the rate of change of absorbance per minute during the linear portion of the reaction. AChE activity was normalized by protein concentration as determined by a Coomassie (Bradford) protein assay kit (Thermo Scientific, Waltham, MA) and compared to the average respective solvent control samples (set at 100% activity) tested on the same day. Activity measurements were performed with 3 technical replicates per condition and 4 independent experiments (biological replicates).
4
Quantifying Acetylcholinesterase Activity in Mice
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Acetylcholine esterase activity was measured in mouse skeletal muscle (hindlimb) and serum using Acetylcholinesterase Activity Assay Kit (Sigma-Aldrich Cat# MAK119). Skeletal muscle samples were homogenized in 0.1 M phosphate buffer (pH 7.5), centrifuged at 12,100×g for 5 min, and the supernatant was obtained. Diluted serum samples or supernatant from skeletal muscle samples was utilized as per the manufacturer’s instruction. AChE activity was then normalized to protein content in the homogenates determined using the PierceTM BCA Protein Assay Kit. The assay utilizes the Ellman method in which thiocholine reacts with 5,5-dithiobis (2-nitrobenzoic acid) to produce a colorimetric product indicative of AChE activity.
5
Acetylcholinesterase Activity in Rat Brain
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In this study, transfluthrin-1.6% (TNSF) and EO-MRC were exposed to different rat groups (n = 6) for 21 days. Control animals received no exposure. After completion of 24 h from the last exposure, all animals were humanely sacrificed by cervical dislocation. Brain tissue samples were collected and homogenized using 0.1 M phosphate buffer, pH 7.5, followed by centrifugation at 14,000 rpm for 5 min. Cleared supernatants were used for assay, as per the procedure described in the technical bulletin of Acetylcholinesterase activity assay kit (Sigma-Aldrich, St. Louis, MO 63103 USA; Catalog Number: MAK119).
6
Spectrophotometric Assay for Acetylcholinesterase
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Inhibition of acetylcholinesterase activity was determined using Ellman’s colorimetric method as modified by Eldeen et al. [49 (link)]. The activity was determined by using an acetylcholinesterase activity assay kit (Sigma Aldrich) according to the manufacturer’s instructions. Briefly, the working reagent was prepared freshly as 2 mg/200 µL of assay buffer provided in the kit and 190 µL per well was transferred to a 96-well plate. Each concentration of the saponin (10 µL) was then added to the respective wells of the plate and was mixed thoroughly. Wells containing only distilled water (200 µL) were used as the blank. The calibrator (200 µL) was added to separate wells. Plates were then incubated at room temperature. After 2 min, the initial measurement was done by measuring the absorbance at 412 nm and after 10 min the final absorbance was measured at 412 nm. Inhibition and activity were calculated according to the formulas provided by the manufacturer.
7
Acetylcholinesterase Activity Assay
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The cholinesterase activity was assayed with a commercial kit (Acetylcholinesterase Activity Assay Kit, Sigma, USA) according to the manufacturer’s instruction.
8
Acetylcholinesterase Inhibition Assay
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For the analysis of AChE activity, differentiated SH-SY5Y cells were treated with different concentrations of TA8Amino (0.1, 1, 5, and 10 µM), donepezil (10 µM), and tacrine (10 µM). After 24 h of treatment, the analysis was performed with Acetylcholinesterase Activity Assay Kit (Sigma-Aldrich) according to the manufacturer’s protocol.
9
Antioxidants and Neuroprotective Factors
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Acetylcholinesterase activity was measured using an acetylcholinesterase activity assay kit (Sigma-Aldrich, St. Louis, USA) based on Ellman’s method. Total antioxidant capacity (TAC) was measured using a complete antioxidant capacity test kit (Bio Diagnostic Co. in Cairo, Egypt) based on the method described by Koracevic et al. [17 (link)]. Brain-derived neurotrophic factor levels were measured using a Human BDNF ELISA kit from Boster Biological Technology (Encyclopedia Cir., Fermont, CA), according to the manufacturer’s instructions. Brain BDNF levels were expressed as pg/g of tissue [18 (link)]. Activity levels of the endogenous antioxidant enzyme superoxide dismutase (SOD) were measured using SOD assay kit (Caymanv Chemicals Company, Ann Arbor, MI, USA) following the manufacturer’s instructions. Superoxide dismutase activity was expressed as U/mg of protein. Caspase 3 activity was measured using the Caspase-3 Colorimetric Activity Assay Kit (Chemicon, Temecula, USA) [19 (link)].
10
Dopaminergic Neuron Differentiation Assay
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L-dopa and acetylcholinesterase activity assay kit (Cat# CS0003) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Folin–Ciocalteu phenol reagent and aluminum chloride were obtained from Loba Chemie (Mumbai, India). Quercetin (98% purity) was purchased from Chanjao Longevity Co., Ltd. (Bangkok, Thailand). P19 cell line ATCC CRL-1857 was obtained from American Type Culture Collection, USA. Alpha minimal essential medium (α-MEM), fetal bovine serum (FBS), newborn calf serum (NCS), and antibiotics-antimycotic solution were purchased from Gibco, USA. All trans-retinoic acid, cytosine-1-β-D-arabinoside, 1:250 porcine trypsin, poly-L-lysine (MW > 300,000), XTT (2,3-bis(2-methoxy-4-nitro-5-sulphonyl)-2H-tetrazolium-5-carboxanilide sodium), and phenazine methosulfate (PMS) were obtained from Sigma, USA. Dimethylsulfoxide (DMSO) and methanol analytical grade were purchased from Merck, Germany. A total of 96-well plates were purchased from Corning, USA. A 100-mm Bacteriological culture dish was obtained from Hycon, USA.
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