Biology Reference
In-Depth Information
7. Unroll the cortex and remove the hippocampus and striatum.
Pinch off the cortex from the rest of the brain and remove the
meninges (see Note 3). Then transfer the cortex to ice-cold
HBSS.
8. When all the cortices have been collected, transfer the tissue to
a 15 ml tube and incubate tissue in 0.125% Trypsin-EDTA at
37°C for 25-30 min.
9. Add 1 ml of bovine calf serum to stop the reaction and centri-
fuge at 2,500 ´
g
for 2 min.
10. Aspirate out the trypsin and FBS and replace with 2 ml of
medium (Dulbecco's Modified Eagle Medium: Nutrient
Mixture F-12 (DMEM/F12), 10% heat-inactivated bovine calf
serum (see Note 4), 2 mM
L
-glutamine, 25 mM HEPES,
25 IU/mL penicillin, 25
g/ml streptomycin).
11. Using a fire polished glass Pasteur pipet,
gently
triturate up and
down 20 (max. 30) times avoiding bubbles (see Note 5).
12. Filter the cells through a sterile 40
μ
m cell strainer into a 50 ml
tube and further dilute the cell suspension with 20-30 ml of
media.
13. Count the number of cells using Trypan Blue exclusion.
14. Plate cells at approximately 100,000 cells/cm
2
and keep in a
humidified incubator with 5% CO
2
at 37°C.
15. Four to 5 days after plating, replace 50% of the media with
fresh media. Then add 25% fresh media to the cells every 4-5
days.
μ
To test the neuronal viability in mixed cultures we first devel-
oped a method to distinguish between neurons and astrocytes. For
this purpose we have chosen the cell specific markers for each cell
type. NeuN is a neuronal protein that is almost exclusively expressed
in neuronal nuclei (
30
). Astrocytes abundantly express glial
fibrillary acidic protein (GFAP), which is widely used as a specific
marker for these glial cells. By using two different fluorochromes
for NeuN and GFAP, respectively, we were able to determine the
percentage of neurons in normal cultures and distinguish between
these two cell types. Furthermore, we added TUNEL staining as a
third “color” to visualize DNA fragmentation, a hallmark of apop-
tosis, in either cell type. Nuclei of cells in normal conditions are
negative for TUNEL, whereas DNA fragmentation and associated
TUNEL staining is abundant in apoptotic cells (
31
). Cells under
pathological conditions undergoing apoptosis display pycnotic
nuclei, that are condensed or fragmented, which are displayed by
Hoechst 33342 staining as dense dots with significantly smaller
nuclei from normal cells (
32
). The Hoechst dye is excited at
350 nm (ultraviolet light) and emits a blue/cyan fluorescent light
at 461 nm (Hoechst AG, Germany, manufacturer's specification).
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