Insulin Preparation

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Media for passaging A1 cells.

  • For 400 mls
  • FCS 40 ml
  • Eagle's MEM 252 ml
  • DdH2O 100 ml
  • 100xPen/Strep 2 ml
  • 100x Glutamine 2 ml
  • Insulin (stock 1 mg/ml) 2 ml

Insulin Materials:

  • 250 mg insulin (sigma I 5500)
  • 0.1 M HCL
  • 2x 400 ml tissue culture bottles
  • 225 ml APBS (4 parts PBS, 1 part ddH2O)
  • steri top 200ml
  • 6-7 50 ml conical tubes

Execution

  1. put the insulin powder into the 400 ml tissue culture bottle
  2. add 25 mls of 0.1 M HCL and swirl until dissolved
  3. while swirling slowly add 225 mls APBS ( final solution should be clear )
  4. filter sterilize the solution ( use the steri top )
  5. aliquot 4 ml per tube and store in freezer
  6. label the tubes with insulin (red)


Gelatin Materials:

  • Gelatin (Sigma, Porcine skin, 300 bloom)—in chemical room
  • 500 ml ddH2O
  • 2X500 ml Cell culture bottles
  • Water bath
  • Steritop
  • 12-13 50 ml conicals

Execution:

  1. Weigh 3.75 g gelatin and put into the bottle
  2. Add 500 ml ddH2O
  3. Mix and heat to 65 C for about 15 minutes
  4. Check that is all dissolved
  5. Under the tissue culture hood, while the gelatin is still hot, vacuum filter the gelatin into a new 500 ml bottle
  6. Aliquot the gelatin, 40 ml/conical tube
  7. Store at 4 C.