Acetylcholinesterase Fluorescent Activity Kit, 2 Plate

Sample values: A variety of serum and plasma samples were tested in the assay, including chicken, mouse, rat, dog, monkey, pig and human samples.
Values averaged 19,188 mU/mL.
RBC ghost samples ranged from 6,216 to 18,552 mU/mL with an average of 13,158 mU/mL

Allow the kit reagents to come to room temperature for 30 minutes.
We recommend that all stan- dards and samples be run in duplicate to allow the end user to accurately determine AChE activ- ity.
Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit.
Assay Buffer Prepare the Assay Buffer by diluting one part of the 10x Assay Buffer Concentrate with nine parts deionized water for a 1:10 dilution.
It is stable for up to 3 months when stored at 4 °C. ® www.ArborAssays.com 7 WEB INSERT 150728 reagent preparatiOn cOntinued ThioStar® Detection Reagent Allow the desiccator to warm to room temperature prior to opening.
Remove a vial of ThioStar Reagent from the desiccator and add 700 μL of the provided DMSO to the vial and vortex thor- oughly.
Store any unused reconstituted Detection Reagent at 4 °C in the desiccator and use within 2 months.
Acetylcholinesterase Substrate Add 700 μL of the provided DMSO to the AChE Substrate vial and vortex thoroughly.
This is a 10x concentrate of the substrate.
Store any unused reconstituted AChE Substrate at room tempera- ture and use within 2 months.
Reaction Mix Dilution Table 1/2 Plate Full Plate 10X AChE Substrate Concentrate 300 μL 550 μL 10X ThioStar® Concentrate 300 μL 550 μL DMSO 2.4 mL 4.4 mL Standard Preparation AChE Standards are prepared by labeling five test tubes as #1 through #5.
Briefly spin vial of stan- dard in a microcentrifuge to ensure contents are at bottom of vial.
Pipet 450 μL of Assay Buffer into tube #1 and 250 μL into tubes #2 to #5.
Carefully add 50 μL of the AChE Standard to tube #1 and vortex completely.
Take 250 μL of the AChE solution in tube #1 and add it to tube #2 and vortex completely.
Repeat these serial dilutions for tubes #3 through #5.
The activity of AChE in tubes 1 through 5 will be 100, 50, 25, 12.5, and 6.25 mU/mL.
Use all Standards within 2 hours of preparation.

Serum & Plasma Store separated serum or plasma on ice until assaying or freeze in aliquots for later use. Samples must be diluted in Assay Buffer prior to running in the kit. Any samples with AChE activity outside the standard curve range should be diluted further with Assay Buffer to obtain readings within the standard curve. Serum and plasma typically have to be diluted ≥ 1:300 to read in the assay. Erythrocytes (RBCs) Blood is collected in the presence of heparin or EDTA. The sample is then centrifuged and the plasma and white cell layer are removed from the RBC layer. The RBCs are suspended and gently washed twice with three volumes of isotonic saline (0.9 % ), separating the cells by centrifugation at 600 x g for 10 minutes and discarding the saline after each step. To lyse the RBCs, four volumes of cold deionized water are added to the RBCs. The cells are then vortexed and incubated for 10 minutes at 4°C, or allowed to undergo a freeze/thaw. Samples are centrifuged at 14,000 rpm for 10 minutes at 4°C and the supernatant discarded. Further wash the membrane pellet with two or three volumes of isotonic saline, with centrifuga- tion in between, until it is only slightly pink. The smaller dark red pellet on the bottom is non-lysed RBCs and should be avoided. Solubilize the white membrane ghost pellet with Triton X-100. Final assay dilution of the solubilized RBC ghost sample must be sufficient that the assay sample con- tains ≤ 0.01 % Triton X-100. Use all samples within 2 hours of dilution.

1. Use the plate layout sheet on the back page of the insert to aid in proper sample and standard identification. Set plate parameters for a 96-well Corning Costar 3915 plate. See: http://www.ArborAssays.com/resources/lit.asp for plate dimension data.
   2. Pipet 100 μL of samples or standards into duplicate wells in the plate.
   3. Pipet 100 μL of Assay Buffer into duplicate wells as a Zero standard.
   4. Add 50 μL of the prepared Reaction Mix to each of the wells using a repeater pipet.
   5. Gently tap the sides of the plate to ensure adequate mixing of the reagents.
   6. Incubate at room temperature for 20 minutes.
   7. Read the fluorescent emission at 510 nm with excitation at 370-410 nm. Please contact your plate reader manufacturer for suitable filter sets.

Average the duplicate FLU readings for each standard and sample.
Create a standard curve by reducing the data using the 4PLC fitting routine on the plate reader, after subtracting the mean FLUs for the zero standard.
The sample activity obtained should be multiplied by the dilution fac- tor to obtain neat sample values.
Or use the online tool from http://www.myassays.com/arbor-assays-acetylcholinesterase-fluorescent-activity-kit.assay to calculate the data. The MyAssays logo is a registered trademark of MyAssays Ltd.