Passaging A1 cell: Difference between revisions
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'''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 | |||
Revision as of 08:06, 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