Amplite® Fluorimetric Acetylcholine Assay Kit Red Fluorescence

Acetylcholine dose response was measured in a 96-well solid black plate with Amplite® Fluorimetric Acetylcholine Assay Kit (Cat. # 11403) using a Gemini fluorescence microplate reader (Molecular devices).

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
Quotation	Request
International	See distributors
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Spectral properties

Excitation (nm)	571
Emission (nm)	584

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12171501

Overview SDS Protocol

Excitation (nm)

571

Emission (nm)

584

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.

Platform

Fluorescence microplate reader

Excitation	540 nm
Emission	590 nm
Cutoff	570 nm
Recommended plate	Solid black

Components

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

Spectral properties

Excitation (nm)	571
Emission (nm)	584

Images

Figure 1. Acetylcholine dose response was measured in a 96-well solid black plate with Amplite® Fluorimetric Acetylcholine Assay Kit (Cat. # 11403) using a Gemini fluorescence microplate reader (Molecular devices).

Citations

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Authors: Nishio, Takahiro and Taura, Kojiro and Iwaisako, Keiko and Koyama, Yukinori and Tanabe, Kazutaka and Yamamoto, Gen and Okuda, Yukihiro and Ikeno, Yoshinobu and Yoshino, Kenji and Kasai, Yosuke and others, undefined
Journal: Journal of Gastroenterology (2017): 1--12

Protective Effect of α-Lipoic Acid against α-Cypermethrin-Induced Changes in Rat Cerebellum
Authors: Elsawy, H and Al-Omair, MA and Sedky, A and Al-Otaibi, L
Journal: Journal of Chemical Neuroanatomy (2017)

Spirulina maxima Extract Ameliorates Learning and Memory Impairments via Inhibiting GSK-3β Phosphorylation Induced by Intracerebroventricular Injection of Amyloid-β 1--42 in Mice
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Journal: International Journal of Molecular Sciences (2017): 2401