-
Prepare solutions : Enzyme (E), heavy inhibitor (HI),
light inhibitor (LI),
culture media. Place at 37 C.
-
Prepare for dissection : Spray dissection area with
70% EtOH. Sterilize
dissection instruments by immersion in 70% EtOH for
10 minutes. Air dry dissection instruments by propping
on petri dish covers. Measure out HBSS in two 50
ml tubes. One tube should have approx 40 mls; the other about
15 mls. Place on ice.
- Dissection :
(i) Embryonic: Euthanize pregnant rat with
CO
2 (3-5 mins). Remove uterus and place in 15 ml
HBSS in 100 mm petridish.
Move to dissection scope.
Newborn: Euthanize by decapitation. Place
in 15 ml HBSS and
move to dissection scope.
(ii) Use fine scissors or forceps to remove
bone (cranium)
overlying brain.
(iii) Transfer brains to new HBSS in 35 or
60 mm plates.
(iv) Remove pia, starting at the bottom of
the brain.
(v) Cut brain along midline. Pinch off thalamus
at the ventral
medial surface.
(vi) With the medial surface of the brain
facing you, remove
the hippocampus by pinching with
a pair of forceps.
(vii) Pinch off the basal ganglia and infratemporal
cortex
(roughly the ventral 1/3 of the brain).
You should now be left with a bowl which is the
neocortex.
(viii) Transfer the cortex to new HBSS and
cut it into small
pieces (roughly 1/2 mm)
(ix) Use a cut/flamed pasteur pipet to transfer
pieces of cortex
to 1/2 of the enzyme soln (E) in a 15
ml tube.
- Dissociation:
(i) Leave tissue in 'E' at 37 C for 20 mins.
Add remaining 1/2 of enzyme soln., and leave
cells at 3l C for an additional 20 mins. Rock
the tube occasionally. At the end of this period
move
'E' with tissue, HI, LI, media to TC hood.
[during the enzymatic step clean up dissection area, and
wash dissection instruments very well with water]
(ii) Remove 'E', leaving tissue at the bottom of the tube.
(iii) Rinse cells 1X with LI.
(iv) Remove LI, add HI, leave tissue in HI for 2 mins at 37
C.
(v) Remove HI, rinse tissue 1X with 5 mls media.
(vi) Remove media, add 5 mls new media.
(vii) Triturate tissue gently about 10-20 times with 5 ml pipet.
This shouls result in virtually complete dissociation. Allow
any debris to settle, and transfer media with cells to new tube.
(viii) Take a 30 µl aliquot of cells
and count using hemacytometer.
- Plating :
(i) Dilute cells in media as appropriate and plate as follows:
24 well plates: 2-3X10 5 per well
60 mm plates: 3X10 6 per well
(ii) Rock plate back and forth, and sideways gently to ensure
even cell distribution.
Place in 5% CO 2 incubator.
(iii) 2-4 hrs later replace media with fresh warm media (500µl
per well for 24 well plates, 3 ml per 60 mm plate).
