Difference between revisions of "Stimulation of C2C12 Cells with Neural Agrin"
From SDMRC
(Created page with "==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...") |
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| − | == | + | ==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 | Make sure cells were able to differentiate for five days (no longer than seven) you should see myotubes formed | ||
| − | Creation of stimulation solution | + | ===Creation of stimulation solution=== |
| − | Warm solutions needed (PBS, diff media) | + | # Warm solutions needed (PBS, diff media) |
| − | Collect the agrin in a tube rack | + | # 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 | |
| − | 18 ml (20 ml to be safe) of diff media | + | # 20 ul of PBS or agrin |
| − | 20 ul of PBS or agrin | + | # Mix solutions and vortex |
| − | vortex | + | |
| − | Creating Stimulation Plates (three experiments plates, the 4th is before stimulation) | + | ===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 | + | # 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 | + | # *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 | + | # Add 2 ml per well of corresponding solution |
| − | Place in incubator | + | # Place in incubator |
| − | The experiment starts here so collect proteins or RNA 6hrs from this point | + | # The experiment starts here so collect proteins or RNA 6hrs from this point |
Latest revision as of 20:13, 19 July 2018
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
