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