I. Aorta Ring Assay:

1. Samples:
   1. Reference BME-Aorta ring: No serum
   2. Reference BME-Aorta ring: 5% FBS
   3. Test BME-Aorta ring: No serum
   4. Test BME-Aorta ring: 5% FBS
2. Add 200 µl of BME to 4 wells of a 48-well plate.
3. Cover plate and incubate at 37°C for 30 minutes.
4. Add a slice of Rat aorta in the middle of each well.
5. Cover aorta slice with 100 µl of BME.
6. Cover plate, and incubate at 37°C for 30 minutes.
7. Add 0.5 ml of RPMI (containing pen-strep) to each well.
8. Add 50 µl of FBS to samples B and D.
9. Incubate plate at 37°C for 6 days. Change media every day.
10. New capillaries should form in the samples with FBS, but should not form in the samples that do not contain FBS.

II. Tube Assay:

1. Samples:
   1. SVEC4-10 Cells 5% FBS-No BME
   2. SVEC4-10 Cells NO FBS-Reference BME
   3. SVEC4-10 Cells 5% FBS-Reference BME
   4. SVEC 4-10 Cells NO FBS- Test BME
   5. SVEC 4-10 Cells 5% FBS-Test BME
2. Add 300 µl of BME to 4 wells of a 48-well plate.
3. Cover plate and incubate at 37°C for 30 minutes.
4. Add 26,000 SVEC4-10 cells to each well in 200 µl of DMEM.
5. Add 20µl of FBS to samples A, C and E.
6. Incubate plate at 37°C for 24 hours.
7. Capillary like structures will form in all the samples within 4 hours of the assay. After 24 hours, the tube formation in the samples that do not contain FBS will disappear. The tube formations will only remain if FBS is present.

III. PC-12 Cell Neurite Outgrowth:

1. Samples:
   1. PC-12 Cells NO Nerve Growth Factor (NGF)- Reference BME
   2. PC-12 Cells 0.4 nM NGF- Reference BME
   3. PC-12 Cells NO NGF-Test BME
   4. PC-12 Cells 0.4 nM NGF-Test BME
2. Add 300 µl of BME to 4 wells of a 48-well plate.
3. Cover plate and incubate at 37°C for 30 minutes.
4. Add 26,000 PC-12 cells (previously treated with Trypsin to make a single cell suspension) to each well in 200 µl of RPMI 1640 medium containing 10% Horse serum, and 5% FBS.
5. Add 20 µl of 40 nM NGF to samples B and D.
6. Incubate plate at 37°C for 24 hours to 1 week.
7. Neurite Outgrowth can be seen in samples B and D within 24 hours. The best nerve growth can be seen in a week. No outgrowth should appear in samples A and C.