Difference between revisions of "Passaging A1 cell"
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| (4 intermediate revisions by the same user not shown) | |||
| Line 1: | Line 1: | ||
| − | + | == 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 | + | == 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