Glutamate Toxicity in Young Neurons
1. Use young primary neurons (3DIV) grown on small coverslips in a 6-well plate
for this experiment
2. Prepare media for glutamate toxicity assay
a. Prepare 100ml MEM/BSA/Hepes
i. 97.5ml MEM (Gibco, Cat # 51200)
ii. BSA (0.01%; Sigma Cat # A1470)
iii. Hepes Buffer (25mM; Sigma Cat # H0887)
b. Prepare 3mM glutamate toxicity media
i. 10ml MEM/BSA/Hepes
ii. 10µl Glycine (Sigma Cat #7126)
iii. 60µl Glutamate (Sigma Cat # G1251)
3. Warm media in the 37ºC water bath
4. Add 500µl control (MEM/BSA/Hepes) media or 500µl glutamate toxicity media
to respective wells in a 24-well plate (2-3 wells per condition)
5. Obtain plated neurons from incubator and transfer 1 small coverslip into each of
the media-containing wells of the 24-well plate using sterile foreceps
6.
Return cells to the incubator for appropriate duration of time
7. If cells are used for viability assays, incubate cells for 24hr after which an LDH
assay for the determination of cell viability will be performed (See LDH assay
protocol for experimental details)
8. If cells are used for Immunocytochemistry to determine the activation of cleavedcaspase-3, allow neurons to incubate for 9hr, after which cells are fixed and
processed by following the steps in our Immunocytochemistry protocol.
JNS 11.14.09