OptimizedFibrinGelBeadAssayfortheStudyofAngiogenesis

PREPARING CELLS

1. Bring up HUVEC and fibroblasts in M199/10% FBS/Pen-Strep (1:100) 1-2 days before beading.

2. Switch medium to EGM-2 (Clonetics) the day before beading for HUVEC and the day before embedding for fibroblasts.

3. A concentration of ~ 400 HUVEC per bead is needed.

4. 20,000 fibroblasts per well is needed.

COATING THE BEADS WITH HUVEC - DAY -1

1. Trypsinize HUVEC.

2. Allow beads to settle (DO NOT CENTRIFUGE!). Aspirate the supernatant and wash the beads briefly in 1 mL of warm EGM-2 medium.

3. Mix 2500 beads w/ 1X10⁶ HUVEC in 1.5 mL of warm EGM-2 medium in a FACS tube. Place it vertically in the incubator. (This will be enough for ~10 wells. Scale up if needed)

4. Incubate for 4 hours at 37°C, shaking the tube every 20 min. (Good coating is crucial for sprouting.)

5. After 4 hours, transfer the coated beads to a T25 flask in 5mL of EGM-2 and leave O/N.

EMBEDDING COATED BEADS IN FIBRIN GEL - DAY 0

1. Prepare the 2.0 mg/mL fibrinogen solution (See recipe section).

2. Add 0.15 Units/mL of aprotinin to the fibrinogen solution.

3. Transfer coated beads to a 15mL conical tube and let beads settle. Resuspend beads in 1mL of EGM-2 and transfer to a 1.5mL centrifuge tube.

4. Wash the beads 3X with 1mL of EGM-2 by pipeting up and down SLOWLY.

5. Count beads on a coverslip and resuspend in fibrinogen solution at a concentration of ~500 beads/mL.

6. Add 0.625 Units/mL of thrombin to each well.

7. Add 0.5 mL of the fibrinogen/bead suspension to each well of a 24-well plate.
   
   Change the pipette tip for each well !!!
   
   

8. Mix the thrombin and the fibrinogen by going up and down gently with the pipette tip ~ 4 to 5 times. Be careful not to make large bubbles.

9. Leave the plate for 5 min in the hood, then place it in the 37°C-incubator for 10-15 min to generate a clot.

10. While waiting for the clot, trypsinize fibroblasts.

11. Add 1 mL of EGM-2 per well drop wise.

12. Seed fibroblasts on top of fibrin gel at a concentration of 20,000 cells per well.