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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).
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).
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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Spectral properties
Excitation (nm)571
Emission (nm)584
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22


Excitation (nm)
Emission (nm)
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.


Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black


Example protocol


Protocol Summary
  1. Prepare ACh standards or ACh test samples (50 µL)
  2. Add ACh working solution (50 µL)
  3. Incubate at room temperature for 10 - 30 minutes
  4. 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.


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


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.


For convenience, use the Serial Dilution Planner:

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.


  1. Add 5 mL of Assay Buffer (Component D) to the bottle of Acetylcholine Probe(Component B) and mix well.
  2. 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. 


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.
Table 2. Reagent composition for each well.
AS1 - AS750 µLSerial Dilutions (0.14 to 100 µM)
BL50 µLAssay Buffer
TS50 µLtest sample
  1. 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.
  2. 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.
  3. Incubate the reaction at room temperature for 10 to 30 minutes, protected from light.
  4. Monitor the fluorescence increase with a fluorescence microplate reader at Ex/Em = 540/590 nm (Cutoff = 570 nm). 


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Spectral properties

Excitation (nm)571
Emission (nm)584



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