Neurite Rescue

Supplement Experiment Protocol   

1. Plate 120,000-150,000 cells per well in 6-well plates

2. 24 hours later, differentiate cells using differentiation reagent.

3. Wait 48+ hours to allow neurite outgrowth and run experiment

4. Prepare MGO (500µL of sterile DECP-Treated water + 8.9 µL [5.6M] MGO)

5. Prepare Differentiation Media (ex: 20 mL media + 10 µL NGF)

6. Serial dilutions of supplement:

   Make 40 mM: add 5 µL to 2mL of differentiation media to make 100 µM dose concentration + 2.5 µL MGO

   Make 4 mM: add 5 µL to 2mL of differentiation media to make      10 µM dose concentration + 2.5 µL MGO

   Make 400 µM: add 5 µL to 2mL of differentiation media to make   1 µM dose concentration + 2.5 µL MGO

   Make 40 µM: add 5 µL to 2mL of differentiation media to make 100 nM dose concentration + 2.5 µL MGO

   Make 4 µM: add 5 µL to 2mL of differentiation media to make       10 nM dose concentration + 2.5 µL MGO

   Make 400 nM: add 5 µL to 2mL of differentiation media to make      1 nM dose concentration + 2.5 µL MGO 

7. Make controls:

   Con (+): 2 mL of differentiation media

   Con (-) MGO: 2 mL of differentiation media + 2.5 µL MGO

   Con (-) 100 µ of supplement: 2mL of differentiation media + 5 µL of 40 mM solution of supplement. 

8. Take cells out of incubator and vacuum off media.

9. Add each dose concentration and controls to individual wells.

10. Take pictures at 0, 3, 6, 12 and 24 hour.