MATERIALS REQUIRED

1. Decided on membrane assay
   1. BD Matrigel plate preparation. BD BioCoat Angiogenesis System - BD 354149
   2. BD Matrigel, growth factor reduced, phenol-fee - BD 356231
2. EPC cell culture media (EMB2 + bullet pack w/o hydrocortisone)
3. Angiogenesis stimulator (VEGF, SDF, etc.)
4. DPBS - CA2+, MG2+ free - Gibco 13190-144
5. Hank's Balanced Salt Solution (HBSS) - GIBCO 14025-092
6. Cell Dye: Calcein AM - Molecular Probes C3100 (50μg vial) or Dil LDL - Invitrogen L3484
7. DMSO (for Calcein AM)
8. Chamber slides: Lab-Tek II Chamber #1.5 German Coverglass System - Lab-Tek 155382 (coverslip bottoms)

CELL PREPARATIONS

1. Wash cells with PBS
2. Trypsinize cells with 2-3ml trypsin
3. Let cells sit in incubator for 5 minutes
4. Place 10-12ml media into dish
5. Scrape cells with cell scraper
6. Slowly suck up cells and place into 15ml Falcon tube
7. Spin @ 1400rpm for 15 minutes
8. Resuspend cells in 1 ml Media or PBS (depending on needs)

CHEMICAL MODIFICATION (OPTIONAL)

1. Place 1 ml (20-50K) cells into each well of a chamber slide
2. Add 30-100ng of chemical stimulant if needed (chemical factor should be at concentration used in vivo ~ 100ng/ml)
3. Incubate at 37ºC, 5% CO2 for 48hrs
4. Add chemical stimulant to Matrigel before polymerization
5. Add chemical stimulant to cells right before layering them on top of Matrigel

PREPARATION OF MATRIGEL (PLATE)

1. Take Matrigel plate out of -20ºC freezer and let it come to RT for 6-24 hrs
2. Polymerize Plate in 37ºC/5%CO2 incubator for 30 minutes
3. Add cell suspension to plate (15,000 cells per 96 well plate well)
4. Incubate 18-24hrs (HUVEC - may be longer for EPCs) @ 37ºC/5% CO2

PREPARATION OF MATRIGEL (CHAMBER SLIDE)

1. Thaw Matrigel at 4ºC overnight. Place 1ml pipette tips @ 4ºC
2. While on ice or in the refrigerator, pipette 500μl Matrigel into each chamber of a chamber slide with cold tips
3. Polymerize slide in 37ºC/5%CO2 incubator for 30 minutes
4. Add cell suspension (4800 cells/cm2)

MEASUREMENT OF TUBE FORMATION - LABELING WITH CALCEIN AM

1. Warm HBSS to 37ºC
2. Prepare Calcein AM solution (8μg/ml) as follows:
   1. Pipette out 6.25ml HBSS
   2. Add 20μl DMSO to each 50μg vial of Calcein AM
   3. Add 100μl HBSS to the Calcein/DMSO mixture
   4. Pipette the contents of the mixture back into the 6.25ml HBSS
3. Remove media from the plates by gently decanting the medium followed by gentle blotting on a stack of paper towels
4. Wash plate 2 times w/ HBSS - 100μl/well
5. Decant as above
6. Add 50μl/well of 8μg/ml Calcein AM
7. Incubate 30-40 minutes @ 37ºC/5% CO2
8. Decant off Calcein AM
9. Wash 2 times as above
10. Acquire images on Fluorescent scope - plate can be stored for 1-2 hours @ 4ºC with minimum increase in background