Amplite® Fluorimetric Acetylcholinesterase Assay Kit *Red Fluorescence*

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.

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

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 Amplite^TM 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
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

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.

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).