DeCicco-Skinner Katie L, Henry Gervaise H, Cataisson Christophe, Tabib Tracy, Gwilliam J Curtis, Watson Nicholas J, Bullwinkle Erica M, Falkenburg Lauren, O'Neill Rebecca C, Morin Adam, Wiest Jonathan S
Department of Biology, American University;
Department of Biology, American University.
J Vis Exp. 2014 Sep 1(91):e51312. doi: 10.3791/51312.
Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1 hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.
血管生成是正常组织发育和伤口愈合的重要过程，但也与多种病理状况相关。使用本方案，可在体外以快速、可量化的方式测量血管生成。将原代或永生化内皮细胞与条件培养基混合，接种于基底膜基质上。内皮细胞会响应条件培养基中发现的血管生成信号形成毛细血管样结构。管腔形成迅速，内皮细胞在1小时内开始排列，含管腔的小管在2小时内开始出现。可使用相差倒置显微镜观察管腔，或者在测定前用钙黄绿素AM处理细胞，通过荧光或共聚焦显微镜观察管腔。形成的分支点/节点、环/网的数量或管腔的数量或长度可轻松量化，作为体外血管生成的指标。总之，该测定法可用于以快速、可重复和定量的方式鉴定参与促进或抑制血管生成的基因和途径。
