View source for Stimulation of C2C12 Cells with Neural Agrin

==Summary==
We stimulate the myotubes ''in vitro'' with neural agrin to observe defects in Acetylcholine clustering. A defect in clustering can possibly result in a defect of the neuromuscular junction development. 

===Experiment===
* PBS is the control (found in the freezer, bottom shelf, agrin box, PBS 0.1% BSA 1000x)
* Neural agrin (x1000) is the treatment (found in the freezer, bottom shelf, agrin box, mc 3,4,8)
* Four time points: before, 6hrs, 12hrs, 24 hrs of stimulation

Make sure cells were able to differentiate for five days (no longer than seven) you should see myotubes formed

===Creation of stimulation solution===
# Warm solutions needed (PBS, diff media) 
# Collect the agrin and PBS (from the freezer) in a tube rack 
# Add 18 ml (20 ml to be safe) of diff media to two separate 50 ml tubes
# 20 ul of PBS or agrin 
# Mix solutions and vortex

===Creating Stimulation Plates (three experiments plates, the 4th is before stimulation)===
# Label plates PBS and Agrin (top three wells and bottom three wells) and the time points
# *do one treatment at a time so cells aren’t out of solution long* remove old cell media with pipette tips and vacuum
# Add 2 ml per well of corresponding solution
# Place in incubator 
# The experiment starts here so collect proteins or RNA 6hrs from this point