Difference between revisions of "Preparation of High Serum for A1 cells"
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| − | ==== For 400 ml | + | == Material == |
| − | + | ''For 400 ml'' | |
* FCS 40 ml | * FCS 40 ml | ||
* Eagle’s MEM 252 ml | * Eagle’s MEM 252 ml | ||
| Line 8: | Line 8: | ||
* Insulin (stock: 1mg/ ml) 4 ml | * Insulin (stock: 1mg/ ml) 4 ml | ||
| + | == Procedure == | ||
# grow at 25ºC in 2% CO2 | # grow at 25ºC in 2% CO2 | ||
# if you want to grow without CO2, then substitute L-15 for the MEM | # if you want to grow without CO2, then substitute L-15 for the MEM | ||
| − | + | ==== passage once per week 1:3 ==== | |
| − | + | # wash with APBS (100 ml PBS + 25 ml ddH2O) | |
| − | + | # trypsinize with TE | |
| − | + | # stop with media | |
| − | + | # centrifuge cells at 100 rpm 3 minutes | |
==== to make myotubes: ==== | ==== to make myotubes: ==== | ||
| − | + | # wash cells 1x with APBS | |
| − | + | # put cells in 0.5% FCSmedia | |
| − | + | # wait 3 days | |
Latest revision as of 14:28, 8 June 2015
Material
For 400 ml
- FCS 40 ml
- Eagle’s MEM 252 ml
- DdH2O 100 ml
- Pen/ Strep 4 ml
- Glutamin 4 ml
- Insulin (stock: 1mg/ ml) 4 ml
Procedure
- grow at 25ºC in 2% CO2
- if you want to grow without CO2, then substitute L-15 for the MEM
passage once per week 1:3
- wash with APBS (100 ml PBS + 25 ml ddH2O)
- trypsinize with TE
- stop with media
- centrifuge cells at 100 rpm 3 minutes
to make myotubes:
- wash cells 1x with APBS
- put cells in 0.5% FCSmedia
- wait 3 days