Stimulation of C2C12 Cells with Neural Agrin

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Stimulation of C2C12 cells with Agrin

As mentioned before: -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 in a tube rack Mix solutions in a 50 ml tube 18 ml (20 ml to be safe) of diff media 20 ul of PBS or agrin 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

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