Freezing of C2C12 cells

Materials:

• 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:

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