Stimulation of C2C12 Cells with Neural Agrin
From SDMRC
Contents
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
