Difference between revisions of "Passaging C2C12 cells"
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| Line 7: | Line 7: | ||
* 75 cm2 flask | * 75 cm2 flask | ||
== procedure == | == procedure == | ||
| − | + | # remove 5 ml of the old media in the 15 ml FALCON tube | |
| − | + | # throw the rest of the old media away | |
| − | + | # wash the cells with 5 ml PBS | |
| − | + | # remove the PBS | |
| − | + | # add 2 ml of the TE in the dish | |
| − | + | # let this incubate for ~4min at 37ºC | |
| − | + | # stop the TE with the old media and resuspend the cells in this | |
| − | + | # pipette the cell suspension in the FALCON tube back | |
| − | + | # centrifuge the suspension for 1000 xg for 3 min at 4 ºC | |
| − | + | # remove the old media | |
| − | + | # resuspend the cells in an appropriate volume of HS medium (f.e.: 10ml) | |
| − | + | # fill in the new 75 cm2 flask 14 ml of new media | |
| − | + | # pipette an appropriate volume of the cell suspension in he new flask and resuspend it in the new flask (f.e.: if you want to dilute your cell 1: 20 put in the new flask 0.5 ml of the resuspended cells in the FALCON tube) | |
| − | + | # close the flask and put it in the 37 ºC | |
Revision as of 08:41, 27 March 2015
materials
- 1x 15ml FALCON tube
- PBS w/o Ca2+ and Mg2+
- TE in PBS (4 ml TE + 36 ml PBS)
- HS for C2C12 cells
- pipettes
- 75 cm2 flask
procedure
- remove 5 ml of the old media in the 15 ml FALCON tube
- throw the rest of the old media away
- wash the cells with 5 ml PBS
- remove the PBS
- add 2 ml of the TE in the dish
- let this incubate for ~4min at 37ºC
- stop the TE with the old media and resuspend the cells in this
- pipette the cell suspension in the FALCON tube back
- centrifuge the suspension for 1000 xg for 3 min at 4 ºC
- remove the old media
- resuspend the cells in an appropriate volume of HS medium (f.e.: 10ml)
- fill in the new 75 cm2 flask 14 ml of new media
- pipette an appropriate volume of the cell suspension in he new flask and resuspend it in the new flask (f.e.: if you want to dilute your cell 1: 20 put in the new flask 0.5 ml of the resuspended cells in the FALCON tube)
- close the flask and put it in the 37 ºC