Passaging C2C12 cells: Difference between revisions
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== | == Material == | ||
* 1x 15ml FALCON tube | * 1x 15ml FALCON tube | ||
* PBS w/o Ca2+ and Mg2+ | * PBS w/o Ca2+ and Mg2+ | ||
| Line 6: | Line 6: | ||
* pipettes | * pipettes | ||
* 75 cm2 flask | * 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 | |||
Latest revision as of 14:31, 8 June 2015
Material
- 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