Difference between revisions of "Freezing of A1 cells"
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(Created page with "''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 ...") |
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| Line 2: | Line 2: | ||
Materials: | 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: | 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 | |
| − | |||
Revision as of 08:40, 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