Measurement of Dopamine Uptake in Neuronal Cells and Tissues - Drugs of Abuse: Neurological Reviews and Protocols - page 456

of 1 N HCl, then adjust the fi nal volume to 10 mL with H 2 O. Store the 1 m M
mazindol solution in 100- L aliquots at -20°C. Before each assay the frozen

1 m M mazindol aliquot is thawed and diluted 10-fold with either cell preincubation
buffer or tissue DA uptake assay buffer to yield a concentration of 100

µ

µ M . This

drug solution in the amount of one tenth (50 µ L) of the fi nal assay volume (500 µ L)

will be injected; therefore, the fi nal assay drug concentration is 10 µ M . The

remaining diluted drug solution should be discarded after each use.

3. Pargyline: Pargyline concentration before assay: 100 M . To prepare 1 m M

pargyline solution, dissolve 1.96 mg of pargyline hydrochloride (mol wt 195.7)
in 10 mL of H 2 O. Store the 1 m M pargyline solution in 100-

µ

L aliquots at -20°C.
Before each assay the frozen 1 m M pargyline aliquot is thawed and diluted
10-fold with either cell preincubation buffer or tissue DA uptake assay buffer to
yield a concentration of 100

µ

µ M . This drug solution in the amount of one tenth

(50 µ L) of the fi nal assay volume (500 µ L) will be injected; therefore the fi nal

assay drug concentration is 10 µ M . The remaining diluted drug solution should

be discarded after each use.

3. Methods
3.1. [ 3 H]DA Uptake in Cultured Neuronal Cells
[ 3 H]DA uptake in primary cultures of rat mesencephalic cells is measured
according to the method previously described by Prochiantz et al. (16) with
modifi cations (17) .

1. Wash cultured cells (1 × 10 -5 cells per 16-mm well) twice with cell preincubation

buffer.

2. Incubate the cells in 500 µ L of cell preincubation buffer, containing 10 µ M

pargyline, 0.05 M mazindol at 37°C for 15 min

( see Note 2 ). A typical DA uptake assay can be set up as follows (volumes in

µ M [ 3 H]DA with or without 10 µ

µ L):

Cell preincubation Pargyline Mazindol [ 3 H]DA

Cell culture well buffer (100 µ M ) (100 µ M ) (0.5 µ M )

Total uptake 400 50 1 0 50

Nonspecifi c uptake 350 50 50 50

3. Terminate the uptake assay by placing the plates on ice and carefully aspirate the
incubation medium into a radioactive waste container.

4. Rapidly wash the cells three times with 1 mL of ice-cold PBS. Aspirate the
washes into a radioactive waste container.

5. Lyse the cells with 300 L of 0.5 N NaOH for 1 h at room temperature. Collect

the lysate into a scintillation vial.

µ

6. Wash the well with 500 L of acidifi ed

ethanol. Collect these washes and combine them with the lysate in the scintil-
lation vial.

µ L of distilled water, followed by 500 µ