Preparation of High Serum for A1 cells: Difference between revisions
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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) | # wash with APBS (100 ml PBS + 25 ml ddH2O) | ||
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==== 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