Preparation of High Serum for A1 cells

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