Insulin Preparation

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.