Freezing C2C12 and Satellite Cells

Freezing C2C12 and Satellite Cells

Freezing C2C12 and Satellite cells

Methods

• Mr. Freeze box with isopentane
• Cryotube (in tissue culture box)
• Warm PBS, basic FGF
• Trypsin
• Gelatin coated dish (cold room, satellite cells only)
• Basic FGF recipe (keep for three days in a 50 ml tube) >>> Use 2.7 ul of basic FGF for satellite cells, the rest is prolif. Media

Freezing C2C12 cells

1. Remove old media
2. 5 ml of PBS to wash for 10 seconds and remove
3. 1 ml of trypsin for 5 minutes until cells detach
4. Pipette 3ml of prolif media into 15 ml tubes (one for each plate of cells being frozen)
5. Once cells are detached add the 1 ml of cells into the 3ml of prolif media
6. Centrifuge the 15 ml tubes (in this example three 15 ml tubes) to collect cells at the bottom [centrifuge at 1.3g for 5 minutes]

Prepare cryotube media

• 900 ul of prolif media
• 100 ul of DMSO (cabinet)

1. Mix solution three times each tube
2. After centrifuge, aspirate the pellet (remove the liquid) tip the 15 ml tube to vacuum out the liquid with a pipette tip, its okay if a little media is left over.
3. Resuspend cells by tapping or dragging the tube
4. Take up the media from the cryotube and place into the 15 ml tube of cells, flush a little, take up everything from the tube and place back into the cryotube
5. Seal and place cryotubes into the Mr. Freeze box. Close the box and put in the -80 freezer for at least 24 hours

Freezing Satellite cells: ???

1. Remove old media
2. 1 ml of trypsin (no PBS wash)
3. Pipette 3ml of basic FGF media in each tube and 8 ml into the gelatin coated dish
4. Flush 5-6 times on dish
5. Take the full volume into the 15 ml tube and pulse in pipette
6. 100 ul goes in the dish (spread the five dots) rest goes in a 15 ml tube
7. Flush petri dish, label (date, name, cell type, passage)
8. Shake dish gently in incubator