Table 1.
Angiogenesis Assay Using Geltrex in Induced Pluripotent Stem Cell–Endothelial Cells and Human Umbilical Vein Endothelial Cells
| Step | Parameter | Value | Description |
|---|---|---|---|
| 1 | Dispense Geltrex™ | 10 μL | 4°C |
| 2 | Incubation time | 1 h | 37°C |
| 3 | Library compounds | 25 μL | 2 × concentration stock |
| 4 | Plate cells | 25 μL | 7.5 × 103 |
| iCell endothelial cells | |||
| 3.5 × 103 | |||
| HUVECs | |||
| 5 | Incubation time | 16 h | 37°C, 5% CO2 |
| 6 | Dispense 3 × staining solution | 25 μL | Calcein AM (6 μmol/L) in medium |
| 7 | Incubation time | 15 min | 37°C, 5% CO2 |
| 8 | Acquire images | 4 × Objective | With FITC filter |
Step Notes
1. Plate: Black clear bottom 384-well plate. Dispense Geltrex on ice.
3, 4, 6. For the assay in iCell endothelial cells, each solution and cell suspension were prepared by VascuLife® Basal Medium containing 4 mM l-glutamine LifeFactor and 0.1% iCell Endothelial Cells Medium Supplement.
For the assay in HUVECs, cell suspension was prepared by “2 × Assay Medium” and each solution was prepared by Medium 199. “2 × Assay Medium” consisted of Medium199 containing the EGM™-2 BulletKits™ at 2 × concentration, also the VEGF component was replaced with 12.5 ng/mL VEGF from R&D Systems.
4. Final DMSO concentration on assay plate: 0.5%.
8. ImageXpress was used for image acquisition.
DMSO, dimethyl sulfoxide; EC, endothelial cell; HUVEC, human umbilical vein endothelial cell; VEGF, vascular endothelial growth factor.