Passaging A1 cell: Difference between revisions
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== Material == | |||
* APBS (100 ml PBS + 25 ml ddwater) | * APBS (100 ml PBS + 25 ml ddwater) | ||
* TE in APBS (36 ml APBS + 4 ml TE) | * TE in APBS (36 ml APBS + 4 ml TE) | ||
| Line 8: | Line 8: | ||
* 3x 175 cm2 flasks | * 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 | ''The cells grow at 25ºC'' | ||
Latest revision as of 14:23, 8 June 2015
Material
- 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