NMDA Toxicity
Toxicity protocol developed for primary neuronal cultures from E17 forebrain that are grown in culture for at least 3 weeks and
plated in 24 well dishes. In addition to the glutamate (NMDA) binding site, there are also multiple binding sites on the
NMDA receptor for modulatory compounds. Efficient NMDA receptor activation requires not only NMDA but also a coagonist, glycine. Activation can also be modulated by the binding of polyamines. Immature neurons do not express functional
NMDA receptors and should not be used. To study oxidative injury in young neurons, expose them to glutamate for depletion
of antioxidants.
Before proceeding with the experiment:
a) check to see if cells have been fed. Always feed cells the morning of exposure. Changes in
glucose metabolism will alter responsiveness to receptor activation.
b) check if these ingredients are prepared and in stock, or prepare them as
followed:
10 mM NMDA stock made in ddH20 (Sigma Cat M3262)
10 mM Glycine stock made in ddH20
These stocks can be aliquoted and frozen at -20*C indefinitely.
MEM/.01% BSA/Hepes
20mg BSA (Sigma cat#: A7888)
5 ml Hepes (1M)
185 ml MEM (Gibco BRL cat#: 51200-038)
1) Obtain NMDA and Glycine aliquots
2) Obtain two 50ml conical tubes label one Glycine, and the other NMDA
3) Estimate amount of MEM/.01% BSA/Hepes solution that will be needed and place into
both conical tubes. (For exposure and washes, plan on preparing 1ml NMDA and 3ml MEM/.01%
BSA/Hepes for each well of a 24 well dish)
4) Within proper labeled conical tubes dilute Glycine and NMDA in MEM/0.01%
BSA/Hepes solution.(Glycine is always a 1/1000 dilution of a 10mM stock, NMDA is always a
1/100 dilution of a 10mM stock for final concentrations of 10uM Glycine and 100uM NMDA)
**Remember both NMDA and Glycine are required to active the NMDA receptor**
e.g.: Place 15ml MEM/.01% BSA/Hepes solution into each conical tube.
Then place 15ul Glycine in the Glycine labeled conical tube and 150ul
NMDA+15ulGlycine into the NMDA labeled conical tube
For dose response curves of toxicity, expose cells to 3, 10, 30, 100, 300 uM NMDA. Use 10
uM Glycine with all concentrations of NMDA.
5) Obtain designated plates from the incubator, and check coverslips to insure equal
viability across conditions before proceeding.
6) Obtain 10ml pipette and remove media from each well
7) Using a 10ml pipette place 2ml MEM/.01% BSA/Hepes into each well to wash cells
8) Using same pipette remove the MEM/.01% BSA/Hepes media from each well
9) Using a 5ml pipette place 1ml NMDA per desired wells. For controls, expose cells only
to glycine.
10) Label plates on both the top & sides
11) Incubate for 60 minutes at 37*C
12) Use a 10ml pipette to remove media
13) Using a 10ml pipette wash each well with 2ml MEM/.01% BSA/Hepes
14) Using same 10ml pipette remove media from each well
15) Using 5ml pipette add 500ul of MEM/.01% BSA/Hepes to all wells
16) Incubate overnight, keep any remaining MEM/.01% BSA/Hepes for Day 2 LDH assay
Day 2 LDH Assay – or other viability assay
Protocol relies on TOX007 kit from Sigma an absorbance based LDH assay
1) Obtain control media (MEM/.01% BSA/Hepes)
2) Obtain LDH dye and substrate out of tissue culture fridge
3) Obtain LDH enzyme (800ul or 400ul) from -20º tissue culture fridge
4) Obtain a Nun96 clear bottom plate, and a glass dish
5) Within the glass dish place 800ul or 400ul of enzyme (amount is dependent on quantity
of enzyme received from -20º fridge)
6) Add enzyme proportional amounts (800ul or 400ul) of LDH Dye and Substrate into
same glass dish.
7) Obtain neuronal plates from incubator
8) Pipette 40ul of control media and place into rows A1-F1
***NEVER
USE ROW G WITH LDH ASSAYS***
9) Pipette 40ul from each well, do each step in duplicates,
e.g.: 40ul of KCN NMDA in <A2,A3> <B2,B3>…
10) Pipette 40ul from each well, and do in duplicates
e.g. KCN Glycine in < A4, A5> <B4,B5>…
11) Pipette 40ul from each well and do in duplicates
e.g. D2C NMDA in <A6,A7> <B6,B7>…
12) Pipette 40ul from each well and do in duplicates
e.g. D2C Glycine in <A8,A9> <B8,B9>…
**Remember for each sample a new column needs to be used, also keep track of the
order of samples**
13) Use repeater to add 20ul of Enzyme/Dye/Substrate mixture to each well
14) Take plate to plate reader and set to : Absorbance, 492, shake 5 seconds, Nun96
15) Save as LDH assay, date, and then print results
BMcL 7/06