Freezing of A1 cells: Difference between revisions
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| Line 1: | Line 1: | ||
''in one 175 cm2 flask'' | ''in one 175 cm2 flask'' | ||
Materials: | === Materials: === | ||
* APBS (100 ml PBS + 25 ml diwater) | * APBS (100 ml PBS + 25 ml diwater) | ||
* TE in APBS (36 ml APBS + 4 ml TE) | * TE in APBS (36 ml APBS + 4 ml TE) | ||
| Line 9: | Line 9: | ||
* freezing media | * freezing media | ||
Procedure: | === Procedure: === | ||
# aspirate the old media from the cells | # aspirate the old media from the cells | ||
# wash the cells 1 times with 5 ml APBS | # wash the cells 1 times with 5 ml APBS | ||
Revision as of 13:00, 12 January 2015
in one 175 cm2 flask
Materials:
- APBS (100 ml PBS + 25 ml diwater)
- TE in APBS (36 ml APBS + 4 ml TE)
- 1x 15 ml FALCON tubes
- Pipettes
- 1 cryo tube
- freezing media
Procedure:
- aspirate the old media from the cells
- wash the cells 1 times with 5 ml APBS
- remove the APBS
- add 5 ml TE
- wait till the cells are detached
- stop the TE with HS – media
- centrifuge at 1000 rpm, 3 min at 4ºC
- aspirate the supernatant
- dissolve the pellet into 1 ml freezing media
- transfer the cells into the cryo tube and label the tube with the cell name, passage number and date
- store the tube for one day at –80ºC
- store the vial in liquid N2