Materials and Reagents

C2C12 myoblasts
DMSO (Sigma-Aldrich, catalog number: 472301 )
Fetal bovine serum (FBS)
Horse serum
DMEM (high glucose) (Life Technologies, Invitrogen™, catalog number: 11965142 )
P/S solution
Fugene HD (FHD) (Roche Diagnostics, catalog number: 04709691001 )
Growth media (see Recipes)
Transfection mix (see Recipes)
Freezing media (see Recipes)
Differentiation media (see Recipes)

Equipment

Standard tabletop centrifuges
Water bath
CO₂ incubator
100 mm culture dishes
Eppendorf tube

Procedure

Grow cells from frozen stock
1. Briefly thaw cells in a 37 °C pre-warmed water bath.
2. Once cells are thawed, pipette into Eppendorf tube and spin for 5 min at 1,000 rpm. Aspirate media. Resuspend cells in 10 ml growth media and plate in 100 mm dish.
3. Split the cells when they grow to 80% confluency.
4. Refreeze the cells: freeze the cells in freezing media.
Passage cells
1. Once cells reach 80% confluency, split as 1:40 to a new dish. 3-4 days later, it will be 80% confluency again.
2. Never let cells grow confluency. They will differentiate.
Transfection
1. Day 0: seed cells (low density, < 50%).
2. Day 1: transfection: (optimum 20% FBS, NO P/S).
3. Day 2: change media (growth media for regular growth or differentiation media for differentiation purpose).
4. 4-5 days for complete differentiation under confluency.
5. 3-4 days for complete differentiation under starvation media, but growth is restricted.
Example:
1. For 6-well plate: Seed 100,000-150,000 cells total in all 6 plates (10-15,000/cm²).
2. Transfect 1 μg DNA/6-wells (total).

Recipes

Freezing media
50% FBS
10% DMSO
40% Growth media
Growth media for C2C12 cells
DMEM
20% FBS
1% P/S
Transfection mix
FHD (1 μg DNA: 4 μl FHD)
Differentiation media
DMEM
1% horse serum
1% P/S

Acknowledgments

This protocol was developed in the Michael Granato Lab at University of Pennsylvania, Philadelphia, USA, and this work was supported by NIH grant R01HD037975.

References

Jing, L., Lefebvre, J. L., Gordon, L. R. and Granato, M. (2009). Wnt signals organize synaptic prepattern and axon guidance through the zebrafish unplugged/MuSK receptor. Neuron 61(5): 721-733.
Lefebvre, J. L., Jing, L., Becaficco, S., Franzini-Armstrong, C. and Granato, M. (2007). Differential requirement for MuSK and dystroglycan in generating patterns of neuromuscular innervation. Proc Natl Acad Sci U S A 104(7): 2483-2488.