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