Freezing of C2C12 cells

Material

• 15 ml FALCON tube
• PBS w/o Ca2+ and Mg2+
• TE in PBS
• Freezing media (10% DMSO + 40% serum + 50% DMEM)
• Pipettes and tips
• Glas pipetts
• cryo tube
• ice
• freezing box

Procedure

1. wash the cells with 5 ml PBS
2. remove the PBS and add 2 ml TE
3. let it incubate for 3 – 5 min
4. stop the TE with 5 ml HS – media and resuspend the cells
5. pipette the cell suspension back in the FALCON tube
6. centrifuge the cells (1000 xg, 3 min at 4 ºC)
7. remove the old media
8. resuspend the cells 1 ml freezing media
9. quickly pipette the liquid in the cryo tube
10. put the cells in the freezing box and store this for one day at –80 ºC
11. place the cells one day later into the liquid nitrogen