Dear all,

I'm working on a model of glutamate induced excitotoxicity using Rat E-18 Cortical neuron culture. Here's my protocol in brief:

1. Primary cortical neurons were kept for 10 days in Neural Basal Medium (Gibco) with B-27 supplement, 5% serum and 10 uM Ara-c
2. In day 10, medium was replaced with drug/vehicle-containing Serum-free medium (SFM)
3. And then treated with SFM medium containing 1-5 mM glutamate for 30 min
4. Replaced with SFM and incubated for 2 hrs.
5. SFM was replaced by MTT (5 mg/ml in HBSS with sodium pyruvate and HEPES-> incubated for 8 hrs.
6. DMSO was added to dissolve the crystal. -> OD540 measurement


Unfortunately, result coming out from the above procedure has 2 problems:

1. The glutamate treatment (1-5 mM) cannot produce a very significant toxicity. Viability of neurons only reduced to around 80% of the untreated control. From those paper I read, 1 mM treatment is sufficient to produce 50% viability.
2. The OD540 reading of MTT is quite low (around 0.2-0.3). I don't know if it is good to increase the MTT incubate time. Would 8 hrs produce adverse effect to the cell?

Anyone have experience on this model? Please kindly give me some suggestion.

I have used a solution called locke's solution for glutamate toxicity. I dont have the recipe now but you could search for it on pubmed. You will also need to add glycine. And usually its done in hippocampal neurons. 1-3 hrs is sufficient. But you have to optimize it as to what suits your experiments.

Good Luck !!!