Isolation of Spinal cord neuron from neonatal rat
Complete culture medium: Leibowitz’s L15 medium (Invitrogen) supplemented with
7.5% Sodium Bicarbonate (Sigma), 7.2% glucose, Australia), 1%
penicillin/streptomycin or 20µg/ml Gentamycin, 0.1% progesterone (Sigma),
2% horse serum , 1% Insulin (Invitrogen), 1% putrescene (ICN Biochemicals
Inc.), 1% conalbumin (Sigma), 0.1% sodium selenite (Sigma) and containing
1ng/ml BDNF , 100 pg/ml GDNF and 10 ng/ml CNTF (Peprotech, USA).
Isolation medium: complete culture medium supplement with 0.1 ml of 4% bovine serum
albumin (BSA; CSL) in PBS–0.16 g/l EDTA and 0.1 ml DNase (1 mg/ml stock;
Sigma Pharmaceuticals).
1) P1 to P3 rats were rendered unconscious by ice-induced hypothermia and killed
by decapitation. The thoracic cage was opened and the heart snipped with a pair
of scissors and as much of the blood was drained as possible by blotting with
tissue paper. The animals were kept cold on ice, and the vertebral column
removed quickly using curved jeweller’s forceps. Either the lumbar cord or the
entire spinal cord spanning from the spino-medullary junction to the sacral levels
was exposed.
2) The spinal cord was then lifted out and placed in ice-cold phosphate buffered
saline (PBS) containing 0.6% (w/v) glucose (PBS-G). The isolation of an entire
spinal cord took about 10 min from the time the animal was killed to its
placement in PBS-G.
3) The cord was then cut into 1 mm slices using a pair of iridectomy scissors and put
into 1 ml of 0.05% trypsin in PBS-G before being incubated at 37 ºC for 15 min
with frequent agitation.
4) Then 0.8 ml of isolation medium was added to each spinal cord and the tube was
agitated by hand for 3 min. The fragments were allowed to settle and the
supernatant was removed and kept on ice.
5) A further 0.8 ml of complete medium, 0.1 ml 4% BSA and 0.1 ml DNase was
added to each tube containing the fragments and the tissue was gently triturated
through a fire flamed glass pipette to obtain an even cell suspension. Overvigorous dissociation at this stage dramatically reduced the yield of neurons.
6) The supernatant was then centrifuged through a layer of 4% BSA for 10 min at
300xg. The pellet was re-suspended in 2 ml of complete medium then layered
onto 5 ml of 6.7% metrizamide and centrifuged for 15 min at 700xg. The interface
was collected in 2 ml of isolation medium and diluted 3-fold with isolation
medium before being centrifuged through a final 4% BSA cushion.
7) The resultant pellet was re-suspended in complete culture medium and plated at a
density of one spinal cord per well (equivalent to 2x104 cells) of a six-well plate.
The wells or coverslips were coated with Laminin or polymer.
8) Medium is changed once every three days.
References:
1).Anderson, K.N. et al. Isolation and culture of motor neurons from the newborn mouse
spinal cord. Brain Research Protocols 12 (2004) 132– 136.
2).Camu, W and Henderson, C.E. Purification of embryonic rat motoneurons by panning
on a monoclonal antibody to the low-affinity NGF receptor. Journal of Neuroscience
Methods, 44 (1992) 59-70.