Primary Striatal Neuronal Culture - Drugs of Abuse: Neurological Reviews and Protocols - page 373

slide (120,000 cells/0.4 mL/0.7 cm 2 growth area/chamber). Add 2 mL to a
24-well plastic dish. Cell density can be adjusted to comply with objectives
of experiment.
12. Place the slide or dish into a culture incubator at 37°C in 5% CO 2 and 100%
humidity.

3.4. Cell Culturing

1. After overnight incubation, the culture medium is replaced with a fresh mainte-
nance mixture of 70% DMEM and 30% NB.

2. On d 3-4 after plating, 5 M Ara-c, a mitotic inhibitor that inhibits the growth

of dividing glial cells, is added to reduce the population of glial cells ( see
Note 5 ).

µ

3. Cells will grow for 5-14 d, depending on objectives of experiment, before use.
Exchange the medium every 5-7 d ( see Note 6 ).

3.5. Culture Characteristics
Using the cell preparation procedures outlined in the preceeding, the follow-
ing characteristics can be anticipated (for details, see ref. 2 ; see Note 7 ):

1. Newly dissociated cells immediately start to adhere to the surface of the slides
after plating. The adherence usually fi nishes within the fi rst hour ( Fig. 1A ).

2. The very fi rst type of cells to grow during the fi rst few days is the fl at cell. These
cells form a monolayer. Neuronal cells are seen in the cultures soon after the
monolayer is formed. They extend their processes on the top of the monolayer
(5) and build synapse-like connections among each other by the end of fi rst
week. During the next week, neurons increase slightly in size and greatly in the
number, length, thickness, and complexity of their processes.

3. Soma and processes of individual cells are very distinctive throughout course of
cell growth in culture. Glial cells, consisting of 5-10% of a whole population,
include mainly astrocytes, microglia, and oligodendrocytes. Astrocytes are the
major type of glial cells. They are large and fl at with phase-dark, large pale
nuclei (25-35 m) and widely spread cytoplasm (monolayer) that covers the

large surface. Microglia have large cell bodies (>25

µ

m) ranging from compact
amoeboid cells to bipolar rod-shaped or occasionally ramifi ed cells with variation
in the length and complexity of branches. Under a light microscope, microglia
show a large pale nucleus surrounded by a unique granular cytoplasm. Because
they are phagocytic, phase-bright debris-fi lled microglia can sometimes be seen
in the early culture. A few small oligodendrocyte-like cells are identifi ed by their
small, round, phase-bright soma (8-15

µ

m), which have a few thick processes
emanating from it. These processes could branch extensively to form a dense,
weblike halo around the cell body. Neuronal cells are a predominant cell type in
cultures (>90% of the total cells), which are small (8-12

µ

µ m) or medium-sized

(13-19 µ m) cells with extended processes that are often long or branched or both