HUVEC or human artery endothelial cells (HUAECs) and human brain-derived vascular pericytes or bovine retinal pericytes are trypsinized and seeded within 3D collagen matrices at a density of 2 × 106 cells/ml for EC and 4 × 105 cells/ml for pericytes. Both types of EC or pericytes work very well in this assay system. FGF-2 and stromal-derived factor-1α (SDF-1α) are each added at 200 ng/ml into the collagen gel mix (2.5 mg/ml of rat collagen type I). Twenty-five μl of gel is added into each individual 96 well (A/2 plates from Costar). After 30 min of equilibration in a CO2 incubator, culture medium is added (100 μl per well) which is Medium 199 (100 μl per well supplemented with 1:250 dilution of Reduced Serum Supplement II) [160], and 40 ng/ml each of FGF-2, stem cell factor (SCF), interleukin-3 (IL-3), and 50 μg/ml of ascorbic acid are added. Cultures are then placed in a CO2 incubator for 72–120 h. After this time, cultures are fixed with 3% paraformaldehyde in PBS (120 μl per well). Cultures can then be imaged under fluorescence to quantify EC tube area and pericyte recruitment under the various conditions of treatment. Individual cultures can be immunostained with various antibodies directed to EC or pericyte cell surfaces and/or ECM antigens (i.e., basement membrane proteins such as fibronectin, laminin, and collagen type IV) [153]. Alternatively, cultures can be used to make lysates using SDS sample buffer or used to isolate total RNA to perform gene expression experiments [153]. Real-time movies can be performed to examine pericyte recruitment and measure pericyte motility during capillary assembly and maturation [154]. In the latter case, nuclear GFP-labeled pericytes can be utilized to track pericyte motility
HUVEC or human artery endothelial cells (HUAECs) and human brain-derived vascular <br>pericytes or bovine retinal pericytes are trypsinized and seeded within 3D collagen matrices <br>at a density of 2 × 106<br> cells/ml for EC and 4 × 105<br> cells/ml for pericytes. Both types of EC <br>or pericytes work very well in this assay system. FGF-2 and stromal-derived factor-1α <br>(SDF-1α) are each added at 200 ng/ml into the collagen gel mix (2.5 mg/ml of rat collagen <br>type I). Twenty-five μl of gel is added into each individual 96 well (A/2 plates from Costar). <br>After 30 min of equilibration in a CO2 incubator, culture medium is added (100 μl per well) <br>which is Medium 199 (100 μl per well supplemented with 1:250 dilution of Reduced Serum <br>Supplement II) [160], and 40 ng/ml each of FGF-2, stem cell factor (SCF), interleukin-3 <br>(IL-3), and 50 μg/ml of ascorbic acid are added. Cultures are then placed in a CO2 incubator <br>for 72–120 h. After this time, cultures are fixed with 3% paraformaldehyde in PBS (120 μl <br>per well). Cultures can then be imaged under fluorescence to quantify EC tube area and <br>pericyte recruitment under the various conditions of treatment. Individual cultures can be <br>immunostained with various antibodies directed to EC or pericyte cell surfaces and/or ECM <br>antigens (i.e., basement membrane proteins such as fibronectin, laminin, and collagen type <br>IV) [153]. Alternatively, cultures can be used to make lysates using SDS sample buffer or <br>used to isolate total RNA to perform gene expression experiments [153]. Real-time movies <br>can be performed to examine pericyte recruitment and measure pericyte motility during <br>capillary assembly and maturation [154]. In the latter case, nuclear GFP-labeled pericytes <br>can be utilized to track pericyte motility
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