Revised 10/27/03
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Maintance of Primary Cultures
Primary cultures are maintained at 37oC & 5% CO2.
Media is partially replaced every 2 – 3 days.
For Neuronal Cultures: Glial cell proliferation is stopped by adding 1-2µM cytosine arabinoside to the
cultures two days after dissociation. On the third day, the media is changed from Plating Media to
Neurobasal. All Neuronal experiments are conducted 3 weeks after dissociation.
For Mixed Cultures: Glial cells are inhibited after two weeks in culture with 1-2 M cytosine
arabinoside, after which the cultures were maintained in growth medium containing 2% serum and
without F12-nutrients. All experiments are conducted one week following mitotic inhibition (25-29 days
in vitro).
Revised 10/27/03
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Preconditioning with chemical ischemia
Cortical cultures were exposed to 3 mM KCN prepared in sterile, glucose-free balanced salt
solution (150 mM NaCl, 2.8 mM KCl, 1 mM CaCl2 and 10 mM HEPES; pH 7.2) for 90 min at 37oC in
5% CO2. Cyanide treatment was terminated by rinsing cells (200:1) and replacing wash solution with
maintenance medium (2% serum, no F12).
Twenty-four hours after the preconditioning stimulus, cells were treated with the glutamate receptor
agonist NMDA. Immediately prior to agonist treatment, cells were rinsed (200:1) in MEM with Earle’s
salt (0.01% bovine serum albumin and 25 mM HEPES, without phenol red). Cells were then exposed for
60 min to 100 M NMDA in the presence of 10 M glycine at 37oC and 5% CO2. Treatment was halted
by serial dilution (200:1) in MEM. One ml of MEM was then added per well and the cultures returned to
the incubator. Neuronal viability was determined 18-20 h later by measuring lactate dehydrogenase
(LDH) release with an in vitro toxicology assay kit (Sigma, St. Louis, MO). Forty l samples of medium
were assayed spectrophotometrically (490:630) according to the manufacturers protocol, to obtain a
measure of cytoplasmic LDH released from dead and dying neurons (Hartnett et al., 1997b). LDH results
were confirmed qualitatively by visual inspection of the cells and, in several instances, quantitatively by
cell counts by the method of Rosenberg and Aizenman (1989).
At various times after KCN treatment, cultures were harvested to assess the extent protein
induction. Cells were placed on ice, washed twice with ice cold phosphate buffered saline (PBS; 4.3 mM
Na2HPO4. 7 H20, 1.4 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl; pH 7.4scraped from the dish using a
rubber policeman in 500 L of TNEB (50 mM Tris-Cl, pH 7.8, 2 mM EDTA, 100 mM NaCl and 1% NP40). Of this, 200 L was saved for protein determination and the remaining 300 L was resuspended in
an equal volume of Laemmali buffer, heated to 95C for 5 min and stored at -20C.
Protein
concentrations were determined spectrophotometrically by using a microprotein assay kit (BioRad).