Amplite® Fluorimetric Acetylcholine Assay Kit Red Fluorescence

Name: Amplite® Fluorimetric Acetylcholine Assay Kit *Red Fluorescence*
Brand: AAT Bioquest
SKU: 11403
Price: 232 USD
Availability: InStock

Acetylcholine and its metabolites are needed for three main physiological purposes: structural integrity and signaling roles for cell membranes, cholinergic neurotransmission (acetylcholine synthesis), and as a major source for methyl groups via its metabolite, trimethylglycine (betaine) that participates in the S-adenosylmethionine synthesis pathways. It plays an important role in the central nervous system as a precursor for acetylcholine and membrane phosphatidylcholine. This Amplite® Fluorimetric Acetylcholine Assay Kit provides one of the most sensitive methods for the quantifying acetylcholine. The kit uses Amplite® Red™ to quantify acetylcholine through choline oxidase-mediated enzyme coupling reactions. The fluorescence intensity of Amplite® Red™ is proportional to acetylcholine.

Example protocol

AT A GLANCE

Protocol Summary

Prepare ACh standards or ACh test samples (50 µL)
Add ACh working solution (50 µL)
Incubate at room temperature for 10 - 30 minutes
Monitor fluorescence intensity at Ex/Em = 540/590 nm (Cutoff = 570 nm)

Important Thaw all the kit components at room temperature before starting the experiment.

CELL PREPARATION

For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Amplite™ Red stock solution (250X)

Add 40 µL of DMSO (Component E) into the vial of Amplite Red™ (Component A) to make 250X Amplite™ Red stock solution.

2. Acetylcholine standard solution (50 mM)

Add 200 µL of ddH₂O into the vial of Acetylcholine Standard (Component C) to make 50 mM Acetylcholine standard solution.

PREPARATION OF STANDARD SOLUTION

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/11403

Acetylcholine standard

Add 20 µL of 50 mM Acetylcholine standard solution to 980 µL Assay Buffer (Component D) to generate 1000 µM Acetylcholine standard solution. Take 1000 µM Acetylcholine standard and perform 1:10 in Assay Buffer (Component D) to get 100 µM Acetylcholine standard (AS7). Take 100 µM Acetylcholine standard (AS7) and 1:3 serial dilutions to get serially dilited of acetylcholine standard (AS6 - AS1) with Assay Buffer (Component D). Note: Diluted Acetylcholine standard solution is unstable, and should be used within 4 hours.

PREPARATION OF WORKING SOLUTION

Add 5 mL of Assay Buffer (Component D) to the bottle of Acetylcholine Probe(Component B) and mix well.
Add 20 μL of 250X Amplite Red™ stock solution into the bottle of AcetylcholineProbe solution to make Acetylcholine (ACh) working solution.
Note This Acetylcholine (ACh) working solution should be used promptly andkept from light. The assay background would increase with longer storage time.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of Acetylcholine standards and test samples in a solid black 96-well microplate. AS= Acetylcholine Standards (AS1 - AS7, 0.14 to 100 µM); BL=Blank Control; TS=Test Samples.

BL	BL	TS	TS
AS1	AS1	...	...
AS2	AS2	...	...
AS3	AS3
AS4	AS4
AS5	AS5
AS6	AS6
AS7	AS7

Table 2. Reagent composition for each well.

Well	Volume	Reagent
AS1 - AS7	50 µL	Serial Dilutions (0.14 to 100 µM)
BL	50 µL	Assay Buffer
TS	50 µL	test sample

Prepare Acetylcholine standards (AS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
Note Treat cells or tissue samples as desired.
Add 50 µL of Acetylcholine (ACh) working solution to each well of Acetylcholine standard, blank control, and test samples to make the total Acetylcholine assay volume of 100 µL/well. For a 384-well plate, add 25 µL of Acetylcholine (ACh) working solution into each well instead, for a total volume of 50 µL/well.
Incubate the reaction at room temperature for 10 to 30 minutes, protected from light.
Monitor the fluorescence increase with a fluorescence microplate reader at Ex/Em = 540/590 nm (Cutoff = 570 nm).

Spectrum

Open in Advanced Spectrum Viewer

Citations

View all 35 citations: Citation Explorer

Branched-chain amino acids govern the high learning ability phenotype in Tokai high avoider (THA) rats
Authors: Shida, Yukari and Endo, Hitoshi and Owada, Satoshi and Inagaki, Yutaka and Sumiyoshi, Hideaki and Kamiya, Akihide and Eto, Tomoo and Tatemichi, Masayuki
Journal: Scientific reports (2021): 1--16

Adlay hull extracts attenuate $\beta$-amyloid-induced neurotoxicity and oxidative stress in PC12 cells through antioxidative, anti-inflammatory, and antiapoptotic activities
Authors: Tsay, Gregory J and Lin, Yu-Ta and Hsu, Chia-Hong and Tang, Feng-Yao and Kuo, Yueh-Hsiung and Chao, Che-Yi
Journal: Biochemistry and Biophysics Reports (2021): 101020

Fermented rice peptides attenuate scopolamine-induced memory impairment in mice by regulating neurotrophic signaling pathways in the hippocampus
Authors: Corpuz, Henry M and Fujii, Hiroshi and Nakamura, Soichiro and Katayama, Shigeru
Journal: Brain Research (2019): 146322

The Additive Effects of Low Dose Intake of Ferulic Acid, Phosphatidylserine and Curcumin, Not Alone, Improve Cognitive Function in APPswe/PS1dE9 Transgenic Mice
Authors: Okuda, Michiaki and Fujita, Yuki and Sugimoto, Hachiro
Journal: Biological and Pharmaceutical Bulletin (2019): 1694--1706

Oral Administration of Okara Soybean By-Product Attenuates Cognitive Impairment in a Mouse Model of Accelerated Aging
Authors: Corpuz, Henry M and Arimura, Misa and Chawalitpong, Supatta and Miyazaki, Keiko and Sawaguchi, Makoto and Nakamura, Soichiro and Katayama, Shigeru
Journal: Nutrients (2019): 2939

Page updated on May 19, 2025

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