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Amplite® Fluorimetric Acetylcholinesterase Assay Kit *Red Fluorescence*

Acetylcholinesterase dose response was measured in a solid black 96-well plate with Amplite® Fluorimetric Acetylcholinesterase Assay Kit using a Gemini fluorescence microplate reader (Molecular devices).
Acetylcholinesterase dose response was measured in a solid black 96-well plate with Amplite® Fluorimetric Acetylcholinesterase Assay Kit using a Gemini fluorescence microplate reader (Molecular devices).
Acetylcholinesterase dose response was measured in a solid black 96-well plate with Amplite® Fluorimetric Acetylcholinesterase Assay Kit 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)
Acetylcholinesterase, also known as AChE, is an enzyme that degrades (through its hydrolytic activity) the neurotransmitter acetylcholine, producing choline and an acetate group. It is mainly found at neuromuscular junctions and cholinergic synapses in the central nervous system, where its activity serves to terminate synaptic transmission. AChE has a very high catalytic activity- each molecule of AChE degrades about 5000 molecules of acetylcholine per second. Acetylcholinesterase is also found on the red blood cell membranes, where it constitutes the Yt blood group antigen. Acetylcholinesterase exists in multiple molecular forms, which possess similar catalytic properties, but differ in their oligomeric assembly and mode of attachment to the cell surface. This Amplite® Fluorimetric Acetylcholinesterase Assay Kit provides one of the most sensitive methods for the detecting AChE activity. The kit uses Amplite® Red to quantify the choline produced from the hydrolysis of acetylcholine by AChE through choline oxidase-mediated enzyme coupling reactions. The fluorescence intensity of Amplite® Red™ is proportional to the formation of choline, thus the AChE activity.


Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black


Example protocol


Protocol summary

  1. Prepare AChE working solution (50 µL)
  2. Add AChE standards or AChE test samples (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 notes
Thaw all the kit components at room temperature before starting the experiment.


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. AmpliteTM Red stock solution (250X):
Add 40 µL of DMSO (Component G) into the vial of AmpliteTM Red (Component A) to make 250X AmpliteTM Red stock solution.  Note: Avoid exposure to light. 

2. Acetylcholinesterase standard solution (50 units/mL):
Add 100 µL of Assay Buffer (Component E) into the vial of Acetylcholinesterase Standard (Component D) to make 50 Units/mL Acetylcholinesterase standard solution. 

3. Acetylcholine stock solution (1000X):
Add 100 µL of Assay Buffer (Component E) into the vial of Acetylcholine (Component C) to make a 1000X Acetylcholine stock solution.


Acetylcholinesterase standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11402

Add 20 µL of 50 Units/mL Acetylcholinesterase standard solution to 980 µL of Dilution Buffer (Component F) to generate 1000 mU/mL Acetylcholinesterase standard solution. Take 1000 mU/mL Acetylcholinesterase standard and perform 1:10 in Dilution Buffer (Component F) to get 100 mU/mL Acetylcholinesterase standard (AS7). Then take 100 mU/mL Acetylcholinesterase standard (AS7) and perform 1:3 serial dilutions to get serially diluted Acetylcholinesterase standard (AS6 - AS1) with Dilution Buffer (Component F). Note: Diluted Acetylcholinesterase standard solution is unstable and should be used within 4 hours.


1. Add 5 mL of Assay Buffer (Component E) to the bottle of Acetylcholinesterase Probe (Component B) and mix well.

2. Add 5 μL of 1000X Acetylcholine stock solution into the bottle of Acetylcholinesterase Probe mixture and mix well.

3. Add 20 μL of 250X AmpliteTM Red stock into this bottle of Acetylcholinesterase Probe mixture to make Acetylcholinesterase (AChE) working solution.  Note: This Acetylcholinesterase (AChE) working solution should be used promptly and kept from light.  The assay background would increase with longer storage time.

For guidelines on cell sample preparation, please visit


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


Table 2. Reagent composition for each well.

AS1 - AS750 µLSerial Dilutions (0.14 to 100 mU/mL)
BL50 µLDilution Buffer (Component F)
TS50 µLtest sample
  1. Prepare Acetylcholinesterase 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 AChE working solution to each well of Acetylcholinesterase standard, blank control, and test samples to make the total Acetylcholinesterase assay volume of 100 µL/well. For a 384-well plate, add 25 µL of AChE 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

Product Family



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