Primary Rat Mesencephalic Neuron–Glia, Neuron-Enriched, Microglia-Enriched, and Astroglia-Enriched Cultures - Drugs of Abuse: Neurological Reviews and Protocols - page 382

3. Resuspend the pellet in 10 mL of prewarmed glia maintenance medium (37°C).
For seeding into 150-cm 2 fl asks, seed a volume of cell suspension equivalent to
two brains to each poly- D -lysine-coated fl ask preloaded with glia maintenance
medium for a total of 30 mL/fl ask.

4. For seeding into 24-well culture plates, fi rst determine the density of cell suspen-
sion with trypan blue and a hemocytometer. Adjust the cell density to 1

10 6 /mL

×

with glia maintenance medium.

5. With a repeat pipet, add 0.5 mL of the cell suspension to each well of the
24-well plates.

6. Four days after initial seeding, completely remove the spent medium and add
prewarmed fresh glia maintenance medium at 30 mL/fl ask or 0.5 mL/well of
24-well plates.

7. Cultures are usually ready for use 14 d after initial seeding.

3.4. Method IV: Microglia-Enriched Cultures
Microglial cells in the primary mixed glial cultures have the unique charac-
teristic of growing loosely attached to a confl uent monolayer of astroglial
cells that grow very tightly adhered to the poly- D -lysine-coated plastic surface.
Therefore, a nearly pure preparation of microglia can be obtained simply by
dislodging the microglia off the astroglia layer by a gentle shaking the cultures
for a suffi cient period of time.

1. When the mixed glial cultures in the 150-cm 2 culture fl asks grow to confl uence
(14 d after initial seeding), tightly cap the 150-cm 2 culture fl asks and seal the
cap area with Parafi lm.

2. Stack the fl asks on a fl at platform shaker and shake the fl asks at 180 rpm for
5 h at room temperature.

3. In a cell culture hood, collect the medium into 50-mL conical tubes and pellet
the cells by centrifugation for 8 min at 200 g at room temperature. Add 30 mL of
the mixed glia culture maintenance medium to each 150-cm2 fl ask and put the
fl ask back to the cell culture incubator. A second harvest of microglia may be
possible after an additional 7 d in culture ( see Note 7 ).

4. Resuspend the cell pellet in an appropriate volume of microglia/astroglia
maintenance medium. Determine the cell density with trypan blue solution and
a hemocytometer. Adjust the cell density to 1 × 10 6 /mL.

5. Seed 0.1 mL, 0.5 mL, and 2 mL of the cell suspension to wells of 96-, 24-, and
6-well plates, respectively. Maintain the culture in 5% CO 2 -95% air at 37°C in a
humidifi ed environment. The cultures are ready for treatment the next day. The
purity of the enriched microglial cultures can be determined by immunostaining
with microglia- and astroglia-specifi c markers. Usually, the cultures are at least
95% pure for microglia.