Freezing of C2C12 cells: Difference between revisions
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==Materials | == Materials == | ||
* 15 ml FALCON tube | * 15 ml FALCON tube | ||
* PBS w/o Ca2+ and Mg2+ | * PBS w/o Ca2+ and Mg2+ | ||
| Line 10: | Line 10: | ||
* freezing box | * freezing box | ||
==Procedure | == Procedure == | ||
* wash the cells with 5 ml PBS | * wash the cells with 5 ml PBS | ||
* remove the PBS and add 2 ml TE | * remove the PBS and add 2 ml TE | ||
Revision as of 08:36, 27 March 2015
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