(Modified from Tranquilo)

à Make Under Sterile Conditions ß

Fibrinogen (Fluka #46312 > 70% protein content)

Thrombin (Sigma #T4648 70 NIH units/mg)

Fibrinogen

• 1. Warm 108 mL PBS to 37^oC.

  2. Add 1.08 (g) of fibrinogen (slowly sprinkle on top)

(gently agitate, do not vortex or stir)

• 1. Filter through a 0.20 mm syringe filter

  2. Distribute into 3.0 ml aliquots.

  3. Store at –80^oC.

Thrombin

1. Dissolve 0.0064g of thrombin powder (450 NIH units) in 18 mL of PBS to yield a solution of 25 NIH units/ml.

2. Filter through a 0.20-um syringe filter

3. Distribute into 0.5 ml aliquots.

4. Store at –80^oC.

Another Modified Protocol for making Bovine Fibrin (Sigma)

Source

Fibrinogen (Fluka #46312 > 70% protein content)

Thrombin (Sigma #T4648 52 NIH units/mg)

Need:

Aprotinin: 225 KIU/ml

Fibrinogen: 3 mg/ml

Thrombin: 1.25 NIH Units/ml

CaCl₂ 20 mmol

Stock Solutions:

Aprotinin: 2250 KIU/ml à Already Made

Fibrinogen: 10 mg/ml à Add 0.45 g in 45 ml

Thrombin: 150 NIH/ml à Add 14.4225 mg (750 NIH units) in 5 ml

CaCl2: 0.000680180 mol/ml à Add 0.5 g in 5 ml (water)

Aprotinin: 2250 KIU/ml à 0.6ml X ?

Fibrinogen: 10 mg/ml à 1.8 ml X 25 (Aliquots)

Thrombin: 300 NIH/ml à 0.2 ml X 25 (Aliquots)

CaCl2: 0.000680180 mol/ml à 0.2 ml X 25 (Aliquots)

For a Final Volume of 0.6 ml (Per Well) Add:

Aprotinin: 135 KIU à 0.06 ml

Fibrinogen: 1.8 mg à 0.18 ml

Thrombin: 0.75 NIH Units à 0.005 ml

CaCl2: 11.6 mmol à 0.017 ml

Stock Solution Preparation

Aprotinin

• 1. Take out of freezer and thaw.

  2. Place 0.6 ml in each tube

Fibrinogen

• 1. Warm 45 mL PBS to 37^oC.

  2. Add fibrinogen (slowly sprinkle on top)

(gently agitate, do not vortex or stir)

• 1. Filter through a 0.20 mm syringe filter

  2. Aliquot

  3. Store at –80^oC.

Thrombin

5. Dissolve thrombin powder in PBS

6. Filter through a 0.20-um syringe filter

7. Aliquot

8. Store at –80^oC.

CaCl₂

1. Dissolve CaCl₂ in distilled water

2. Filter through a 0.20-um syringe filter

3. Aliquot

4. Store at –80^oC