—Protocol—

C2 Mouse Myoblast Culture

(C2C12 or C2F3)

From the Laboratory of Helen M. Blau, Stanford University
S. Hughes, 12/90; B. Blakely, 10/96

1. Thaw frozen C2 cells rapidly in 37°C bath and dilute into 10 ml of C2GM in a 100 mm Falcon tissue culture dish (#3003).

2. Incubate cells at 37°C in 10% CO₂.

3. Change medium after all cells have adhered.

4. Routinely split the cells 1:20 (no more than 1:50) when they get dense, usually every 2 days. Do not allow the cells to reach confluence or they will begin to differentiate, even in growth media. Cells are usually grown to about "70% - 80%" confluence. Refeed cells on second day if they are not split.

5. To differentiate, allow cells to grow to about "90%" confluence. Switch dense cells into FM and feed every day. Ideally, the cells should be plated on collagen coated dishes before differentiating (for better adherence of the myotubes). In a healthy culture, you should see some spontaneous twitching of the myotubes.

6. You will not get 100% differentiation in FM. Cells can be treated with AraC (10 mM) when switched into FM to eliminate dividing cells. This keeps the myoblasts from crowding out the myotubes, and allows a myotube culture to be maintained for about 2 weeks.

 

C2 Growth Media (C2GM):

DME (Gibco) + 20% FBS, -OR- 5% FBS + 15% "defined, supplemented" calf serum (Hyclone, iron supplemented), to save money on FBS.

Fusion Media (FM):

DMEM + 2% horse serum (Hyclone).

Notes:

C2C12 were subcloned in Helen Blau's lab around 1980 from the C2 line of David Yaffe. C2F3 were subcloned in Helen Blau's lab around 1990. C2F3 cells do not differentiate quite as rapidly as C2C12, though after a couple of days in differentiation conditions, they have fused just as well as C2C12. C2C12 are more likely to exhibit some differentiation in growth conditions than C2F3, and therefore they require more careful attention. If they are not cultured carefully, or even after many passages of continuous culture, the cultures may not differentiate as well and will need to be re-cloned. C2C12 cells are available from the ATCC (product number CRL-1772). However, ATCC C2C12 cells differentiate more slowly than original C2C12 cells and in fact behave more like C2F3 cells.

C2 cells do not grow and differentiate well in 5% CO₂. We have heard that they behave reasonably well in 8% CO₂.