Difference between revisions of "Passaging A1 cell"
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| Line 8: | Line 8: | ||
* 3x 175 cm2 flasks | * 3x 175 cm2 flasks | ||
| − | Procedure: | + | '''Procedure:''' |
| − | + | * put 10 ml Gelatin into the new 175 cm2 flasks ( transfer the gelatine from the first to the second and from the second into the third → remove the remaining liquid) | |
| − | + | * 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 5 ml HS – media | |
| − | + | * centrifuge at 1000 rpm, 3 min at 4ºC | |
| − | + | * aspirate the supernatant | |
| − | + | * dissolve the pellet into 3 ml fresh media | |
| − | + | * fill in the new flasks 25 ml HS for A1 cells | |
| − | + | * add into each flask 1 ml of the cell suspention | |
The cells grow at 25ºC | The cells grow at 25ºC | ||
Revision as of 08:07, 26 September 2014
Materials:
- APBS (100 ml PBS + 25 ml ddwater)
- TE in APBS (36 ml APBS + 4 ml TE)
- Gelatin (warmed to 37ºC)
- HS for A1 cells
- 1x 15 ml FALCON tubes
- Pipettes
- 3x 175 cm2 flasks
Procedure:
- put 10 ml Gelatin into the new 175 cm2 flasks ( transfer the gelatine from the first to the second and from the second into the third → remove the remaining liquid)
- 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 5 ml HS – media
- centrifuge at 1000 rpm, 3 min at 4ºC
- aspirate the supernatant
- dissolve the pellet into 3 ml fresh media
- fill in the new flasks 25 ml HS for A1 cells
- add into each flask 1 ml of the cell suspention
The cells grow at 25ºC