Supplement Experiment Protocol
Plate 120,000-150,000 cells per well in 6-well plates
24 hours later, differentiate cells using differentiation reagent.
Wait 48+ hours to allow neurite outgrowth and run experiment
Prepare MGO (500µL of sterile DECP-Treated water + 8.9 µL [5.6M] MGO)
Prepare Differentiation Media (ex: 20 mL media + 10 µL NGF)
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
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.
Take cells out of incubator and vacuum off media.
Add each dose concentration and controls to individual wells.
Take pictures at 0, 3, 6, 12 and 24 hour.