Difference between revisions of "Preparation of High Serum for A1 cells"
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| Line 1: | Line 1: | ||
| − | + | ==== For 400 ml ==== | |
FCS 40 ml | FCS 40 ml | ||
| Line 8: | Line 8: | ||
Insulin (stock: 1mg/ ml) 4 ml | Insulin (stock: 1mg/ ml) 4 ml | ||
| − | + | * 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: | + | ==== to make myotubes: ==== |
| − | + | * wash cells 1x with APBS | |
| − | + | * put cells in 0.5% FCSmedia | |
| − | + | * wait 3 days | |
Revision as of 10:15, 13 February 2015
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
- 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