Measurement of Dopamine Uptake in Neuronal Cells and Tissues - Drugs of Abuse: Neurological Reviews and Protocols - page 457

7. Count the radioactivity in collected lysate and subsequent washes with 10 mL
of scintillation fl uid.

3.2. [ 3 H]DA Uptake in Striatal Tissues
3.2.1. Preparation of Crude P 2 Synaptosomal Fraction
Measuring [ 3 H]DA uptake in striatal tissues depends on the amount of tissue
collected ( see Note 3 ). To prepare a crude P 2 synaptosomal fraction, striata
obtained from one rat (or striata from fi ve mice) would be required (18) .

1. Homogenize the tissue in 1.0 mL of tissue homogenization buffer with an
electronic tissue homogenizer (Tekmar Tissumizer or Brinkmann Polytron), fi ve
times for 5-s bursts with ice cooling in between each burst.

2. Centrifuge at 1000 g for 20 min at 4°C.

3. Discard pellet and centrifuge the supernatant at 27,000 g for 20 min at 4°C.

4. Resuspend the P 2 pellet in 1.0 mL of tissue homogenization buffer for assay use.

3.2.2. Preparation of Striatal Homogenate
If tissue is limited, for example, to measure [ 3 H]DA uptake in a treated
mouse where tissue cannot be pooled, striatal homogenate is therefore used.
In this situation, the mouse striatum is homogenized in 500 µ L of tissue

homogenization buffer and directly used for the assay.

3.2.3. [ 3 H]DA Uptake Assay Procedure

1. Set up the assay tubes (16 100 mm) on ice as suggested below. Pipet all the

necessary ingredients, except the P 2 synaptosomes or tissue homogenate. All
ingredients should be made in the assay buffer and kept on ice. A typical DA
uptake assay can be set up as follows (volumes in

×

µ L):

Uptake assay Pargyline Mazindol [ 3 H]DA Membrane

Tube buffer (100 µ M ) (100 µ M ) (0.5 µ M ) protein

Total uptake 300 50 1 0 50 100

Nonspecifi c uptake 250 50 50 50 100

2. Initiate the assay by adding 100 L of P 2 membrane protein (or homogenate) to

each tube, vortex-mix, and incubate at 37°C for 6 min.

µ

3. Terminate the reaction by adding 5 mL of ice-cold uptake assay buffer to the
assay tube and immediately pour the sample over a buffer-saturated Whatman
GF/B (2.4 cm) fi lter on a fi lter manifold apparatus under vacuum.

4. Rinse the fi lter three times with 5 mL of the assay buffer. Dry fi lter in oven (50°C)
overnight before counting for activity with 10 mL of scintillation fl uid.