Protocol for Fibrin Gels

1.  Prepare the fibrinogen solution to a [FINAL] = 20mg/ml

a.    Dilute 4.5g Fibronogen (Sigma F-8360) in 255ml serum/antibiotic-free media

b.    Dissolve slowly at 37 degrees for 4 hours

i.       Swirl gently every 30 minutes

c.     When dissolved, filter through a 0.2Ám filter

d.    Store frozen in 25ml aliquots

2.  Prepare Thrombin solution to a [FINAL] = 200U/ml

a.    Add 0.1% BSA to 50ml serum/antibiotic-free media and mix

b.    Add the 50ml of BSA media to the 10,000 Units of thrombin (Sigma T-3399)

c.   Mix well

d.   When dissolved, filter through a 0.2Ám filter

3.  Sterilize the fibrin plate by spraying 70% ethanol 4 times into the cap of the plate and allowing the ethanol to cover the plate

4.  Incubate with the lid facing up in the hood for a minimum of 20 minutes

5.  Aspirate the remaining ethanol

6.  Prepare the fibrin gel

a.    Coat the plates with 0.5 ml of growth media containing 10U/ml thrombin

b.    Add 200ul of 20mg/ml fibrinogen solution drop wise

c.     Mix twice in each direction

d.    Incubate 10 minutes at room temperature

e.    When the gels have formed, continue to next step