Figure 6.
Analysis of Angiogenic Potential of mRNA-Engineered EPCs by Tube Formation Assay
1 × 105 murine EPCs were cultivated overnight, and then they were transfected with 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α mRNA or with an mRNA cocktail containing 1.6 μg ANG-1, 0.8 μg VEGF-A, and 0.5 μg SDF-1α mRNA. After 24 hr, EPCs were detached and 1 × 104 EPCs were seeded on Matrigel-coated angiogenesis slides. After 4 hr of incubation at 37°C, the formation of tubes was examined by phase-contrast microscopy. Microscopic images were analyzed using NIH ImageJ software with Angiogenesis Analyzer plugin, and segments are shown in magenta, master segments in orange, branches in green, and meshes in blue. The numbers (Nb) of nodes, segments, and master segments and the total (Tot.) segment length, total mesh area, and branching interval were quantified and compared to the medium control. The unit of area and length is pixel (px). Scale bars represent 100 μm. Results are shown as mean + SD (n = 3). Statistical differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test (*p < 0.05, ***p < 0.001, and ****p < 0.0001).