Striatal dopamine measurement through HPLC

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 14000rpm,4°C

Remove supernatant and transfer to Costar Spinx (#8161) 0.22 - MCA filter tube

Centrifuge for 0h 5m 0sat 14.000rpm,4°C 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.

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