1. Twenty-four mL venous blood was collected at each time point into Vacutainer CPT Mononuclear Cell Preparation Tubes.
2. MNCs recovered by density-gradient centrifugation in these tubes were washed twice with PBS and once in EPC growth media consisting of medium199 supplemented with 20% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL).
3. Cells were resuspended in media, plated at a density of 5x10⁶ per well on dishes coated with human fibronectin, and incubated at 37°C in humidified 5% CO₂.
4. After 48 hours, nonadherent cells suspended in the growth media were replated onto fibronectin-coated 24-well plates at a density of 10⁶ per well.
5. Media was changed every 3 days.

 EPC colony-forming units after 7 days in culture (Figure A).
Cell clusters alone without emerging spindle cells (Figure B).

Reference
Tiffany M. Powell, Jonathan D. Paul, Jonathan M. Hill, Michael Thompson, Moshe Benjamin, Maria Rodrigo, J. Philip McCoy, Elizabeth J. Read, Hanh M. Khuu, Susan F. Leitman, Toren Finkel, Richard O. Cannon III. Granulocyte Colony-Stimulating Factor Mobilizes Functional Endothelial Progenitor Cells in Patients With Coronary Artery Disease. Arteriosclerosis, Thrombosis, and Vascular Biology. 2005; 25: 296-301