2.7. Flow Cytometry Analysis of Endothelial Cell Surface Markers

Changes in surface markers due to hypoxia were analyzed by flow cytometry. Endothelial cells were plated in (Keith et al., 2011)well plates (1 mL of media containing 2 × 10⁵ cells) and incubated under hypoxia. Cells were collected, washed twice with PBS/BSA, incubated and labeled with APC anti‐human CD31 antibody (Biolegend), APC anti‐human CD34 antibody (Biolegend), PE anti‐human VEGFR1 antibody (Biolegend) or APC anti‐human VEGFR2 antibody (Biolegend) for 45 minutes, washed twice with PBS/BSA and analyzed by Flow Cytometry (FACScan using CellQuest software, BD Bioscience). Isotype controls for APC and PE (Biolegend) were used. Data were expressed as a percentage of positive cells to target molecules.