Preparation of High Serum for A1 cells

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

1. grow at 25ºC in 2% CO2
2. if you want to grow without CO2, then substitute L-15 for the MEM

passage once per week 1:3

1. wash with APBS (100 ml PBS + 25 ml ddH2O)
2. trypsinize with TE
3. stop with media
4. centrifuge cells at 100 rpm 3 minutes

to make myotubes:

1. wash cells 1x with APBS
2. put cells in 0.5% FCSmedia
3. wait 3 days