Jul 12, 2022
Striatal dopamine measurement through HPLC
- Pranay Srivastava1,
- Waijiao Cai2,
- Xiqun Chen1
- 1MassGeneral Institute for Neurodegenerative Disease, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, USA Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, Towson, MD, USA;
- 2MassGeneral Institute for Neurodegenerative Disease, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, USA Department of Integrative Medicine, HuaShan Hospital, Institutes of Integrative Medicine, Fudan University, Shanghai, China
Protocol Citation: Pranay Srivastava, Waijiao Cai, Xiqun Chen 2022. Striatal dopamine measurement through HPLC. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpbjdplzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: February 18, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 58413
Keywords: ASAPCRN
Abstract
Protocol for striatal dopamine measurement in mouse brain
Materials
PE Solution Recipe: (250 mL):
Polished ddH2O- 249.06 mL
0.1mM EDTA (From 0.5 mM stock)- 50 uL
1uM DHBA (From 10mM stock)- 25 uL
50mM Phosphoric Acid (From 85% Fisher #A260 HPLC grade)- 855.3 uL
Catecholamine Standard Prep:
Prepare 10 mM Stock in water or PE solution. (May be frozen as aliquots at –80C)
-Dopamine = 18.96 mg to 10 mL
(Dopamine hydrochloride, Sigma #H8502, MW 189.64)
-DOPAC = 16.82 mg to 10 mL
(3,4 Dihyroxyphenylacetic Acid, Acros #22560050, MW 168.15)
-HVA = 18.22 mg to 10 mL
(Homovanillic acid, MilliporeSigma #69673-25MG, MW 182.17)
-DHBA= 22.01 mg to 10 mL
(3,4 Dihydroxybenzlamine Hydrobromide, Sigma #858781, MW 220.06)
***(INTERNAL STANDARD IN PE SOLUTION ONLY)
Costar® Spin-X® Centrifuge Tube Filters, 0.22 µm Pore CA Membrane, Nonsterile - Cat#8161
Weigh the striatal tissue
Note - make sure to keep it frozen – can keep it on dry ice for the process
Add 20x PE buffer supplemented with an internal standard solution to tissue.
(eg. 10 mg sample tissue would receive 200 uL of PE solution)
Use a Teflon pestle to homogenize the tissue into the solution by crushing it against the walls of the tube.
Note - Do not sonicate as it may oxidize our analyte)
Note - Keep homogenized samples on ice until centrifugation
Note - If using a hand homogenizer, note that the machine produces heat during prolonged use which can degrade the analytes. Allow time to cool between samples.
Alternatively, can use Dounce homogenizers
Centrifuge at 14000 rpm, 4°C, 00:15:00
15m
Remove supernatant and transfer to Costar Spinx (#8161) 0.22 - MCA filter tube
Centrifuge for 00:05:00 at 14.000 rpm, 4°C, 00:05:00 or until all liquid has passed through the filter.
Note - Filters have a max volume of 500uL, and may have to perform multiple spins/samples if the supernatant volume is >500ul.
10m
The resulting solution can be run on HPLC or stored at -80 °C .
Running a standard - HPLC
Perform serial dilution of the stocks as follows to create the standard curve.
Load at least 50 uL into each vial to run the standard curve in triplicate (on HPLC).
Run the samples