TITLE: Quantifying Acetylcholinesterase activity in striatal brain tissue with DTNB assay
AUTHORS: Stefania Vietti-Michelina, Stephanie J Cragg

[DESCRIPTION]
This protocol describes the steps to measure acetylcholinesterase (AChE) activity in mouse striatal brain tissue, including how to store tissue samples, extract AChE and obtain Michaelis-Menten like plots to measure the rate of AChE activity. This protocol can be used to quantify AChE activity across different brain regions or genotypes. Reagents and materials from Abcam kit ab138871 are used. This kit uses DTNB to measure the thiocholine (TCh) resulting from the hydrolysis of the substrate acetylthiocholine (ATCh) by AChE. 

[STEPS]
SECTION: Preparation of ex vivo  mouse brain slices
1. Harvest dorsal and ventral striatal tissue punches (1.2 mm diameter) were  from 300 μm thick acute coronal striatal tissue slices, prepared with a vibratome.
SECTION: Preparation of ex vivo  mouse brain slices
2. Equilibrate slices at room temperature for at least 1-hour prior collection of the punches in bicarbonate-buffer based artificial cerebrospinal fluid (aCSF) (See Materials.)
SECTION: Preparation of ex vivo  mouse brain slices
3. Dry single tissue punches and place in 1.5 mL tubes. Snap freeze samples at -80°C and store at -80°C until needed for the assay.
SECTION: Preparation of the DTNB Assay
4. Thaw all the kit components at RT before starting and prepare stock solutions as instructed in assay kit manual.
 

SECTION: Preparation of the DTNB Assay
5. After preparing all stock solutions, divide into single use aliquots and store at -20°C
 

SECTION: Preparation of the DTNB Assay
6. Prepare the AChE standards. From the AChE stock solution (50 U/mL) prepare the 1000 mU/mL solution and then use it to perform serial dilutions of 300, 100, 30, 10, 3, 1, 0.1 mU/mL. 
 

SECTION: Preparation of the DTNB Assay
7. Prepare the lysis buffer,  0.5% IGEPAL CA-630 in 1x PBS. 
 

SECTION: Preparation of the DTNB Assay
8. Prepare the tissue. Incubate each tissue punch in lysis buffer using 200 uL of lysis buffer for each sample. Incubate for 10 minutes on ice. 
SECTION: Preparation of the DTNB Assay
9. Sonicate for 10 - 15 seconds, 2-3 times with a hand-held sonicator.
SECTION: Preparation of reaction mix
11. Add 20x DTNB stock and 20x ATCh stock into assay buffer.
 

SECTION: Preparation of reaction mix
12. Prepare two different reaction mix, one with the substrate ATCh and one without, with final volumes informed by the number of wells in use.For the ATCh mix add 20x DTNB stock and 20x ATCh stock to assay buffer, while for the substrate-free mix add 20x DTNB stock into assay buffer
SECTION: DTNB Assay
13. Put 50 uL of AChE standards in the standard wells (300-0-1 mU/mL in duplicates). Add 50 uL of assay buffer in the blank control wells (also in duplicates).
SECTION: DTNB Assay
14. Add 50 uL of samples in each sample well (in duplicates). 
 

SECTION: DTNB Assay
15. Incubate for 15 minutes at RT in the dark.
SECTION: DTNB Assay
16. Measure changes in absorbance in a microplate reader at OD=410 nm
SECTION: Analysis
17. Subtract the blank values and average all duplicates. 
SECTION: Analysis
18. Calculate AChE activity as mU/mL for each sample from the standard curve.
SECTION: Analysis
19. When comparing genotypes or brain regions normalise by tissue volume. 
SECTION: Preparation of the DTNB Assay
10. Centrifuge each sample tube at 650 rcf for 5 minutes at 4°C. Then, collect the supernatant into a new tube ensuring no pellet is transferred.