Preparation of High Serum for A1 cells

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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