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 0.2m 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 0.2m 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