Biomedical Engineering Reference
In-Depth Information
to 8 mL with dissection medium. Slowly pass the tissues through the orifi ce of
the 10-mL pipet three to fi ve times without foaming. Fit a 1-mL pipet tip to the
10-mL pipet and again slowly pass the tissues through three to fi ve times. Repeat
the process with a 200-
µ
L pipet tip fi tted to the 10-mL pipet (
see
Note 4
).
2. Cap the tube and centrifuge at 200
g
for 10 min at room temperature.
3. In a tissue culture hood, discard the supernatant, and resuspend the pellet in
5-10 mL of prewarmed neuron-glia maintenance medium (37°C). Transfer 30
µ
L
of the cell suspension to a microtube.
4. Add 270
µ
L of Trypan blue to the microtube. Invert the tube to mix and load 10
µ
L
of the mixture to a hemocytometer to estimate the density of cells.
10
6
/mL with warm
maintenance medium. Gently invert the conical tube to ensure a good mix of
the cell suspension. With a repeat pipet fi tted with a 12.5-mL tip, gently but
precisely add 0.5 mL of the cell suspension to each well of the poly-
D
-lysine-
coated 24-well plates. Gently move the plate a few times in a left-to-right and
back-and-forth fashion to ensure even plating of the cells in the wells.
5. In a culture hood, adjust the density of viable cells to 1
×
6. Place the plate in the cell culture incubator.
7. Three days later, carefully add, along the side of the wells, 0.5 mL of the
maintenance medium (prewarmed to 37°C) to each well of the 24-well plates.
8. For neuron-glia cultures, cultures are usually ready for treatment 7 d after the
initial seeding.
9. The relative abundance of the major types of cells of interest, that is, astrocytes,
microglia, neurons, and dopaminergic neurons in particular, can be determined
following immunocytochemical staining with cell type specifi c markers (
see
Note 5
). At the time of treatment, the cultures are usually made up of 50%
astrocytes, 10% microglia, 40% neurons in general, and up to 1% dopaminergic
neurons.
3.2. Method II: Mesencephalic Neuron-Enriched Cultures
The protocol that we routinely use to prepare neuron-enriched cultures
involves the use of cytosine
-
D
-arabinofuranoside (ara-C) to suppress the
growth of glial cells in a neuron-glia culture.
1. Neuron-enriched cultures are prepared by fi rst establishing the neuron-glia
cultures following the exact procedure described in the preceding.
2. At 48 h after initial seeding, an aliquot of ara-C stock solution was added to the
culture medium to achieve a fi nal concentration of 5-15
M
in a fi nal volume of
1 mL/well (
see
Note 6
). Two to three days later, the ara-C-containing medium
was removed and prewarmed midbrain neuron-glia medium was added back
(1 mL/well). Cultures are ready for use 7 d after initial seeding.
µ
3. The composition of the neuron-enriched cultures can again be determined
following immunostaining for cell type-specifi c markers. In general the cultures
contain 85-95% of neurons. The rest of the cells are primarily astrocytes with
very few contaminating microglial cells.
Search WWH ::
Custom Search