SCF can be replaced by either Flt-3L or M-CSF to act in conjunction with IL-3 and SDF-1α to stimulate EC tube formation and hematopoietic cytokines facilitate pericyte recruitment to EC tubes and vascular basement membrane matrix assembly in 3D collagen matrices. (A) M-CSF and Flt3L were added to replace SCF and were compared with SCF/IL-3/SDF-1α by adding them to IL-3/SDF-1α. In addition, ECs were either primed with FGF alone, VEGF-A/FGF-2, or control and then cultured for 72 hours. After fixation, total EC tube area was determined (n ≥ 10; P ≤ .01). *Denotes significance over control ECs; ⁺denotes significance over the FGF-2–only primed ECs. (B) ECs were left untreated or were primed with VEGF-A/FGF-2 and then incorporated with GFP pericytes and hematopoietic cytokines (SCF/IL-3/SDF-1α) into 3D collagen matrices. Assays were allowed to develop over 5 days and average EC vessel area measured (n ≥ 10; P ≤ .01). *Denotes significance over control. (C,D) Basement membrane matrix deposition was assessed in the presence or absence of EC priming with VEGF-A/FGF-2. Cultures were fixed and stained with antibodies to laminin, collagen type IV, and CD31. Fluorescent images of this staining were acquired, quantitated (C), or overlaid with images of GFP pericytes (D). Bar equals 20 μm.