Vegf a

For analyzing of the antagonistic response to VEGF, tube formation, scratch wound migration, and cell proliferation assays were performed after the exposure of HUVECs to 50 ng/mL VEGF-A (R&D Systems, Minneapolis, MN), which induced a significant angiogenic response. For the cell proliferation assays, cells were incubated overnight in endothelial basal media (EBM; Lonza, Walkersville, MD, USA) containing 0.5% FBS or supplemented with VEGF-A and/or VEGF inhibitors. The cells were washed with PBS and counted in 4 random microscope fields. The tube formation assay was performed by seeding cells on Matrigel-coated plates (BD Bioscience, Bedford, MA, USA) and incubating in EBM containing 0.5% FBS or supplemented with VEGF-A and/or VEGF inhibitors. After overnight incubation, tubule networks were quantified by measuring the tubule length in 4 random microscope fields. For the analysis of scratch wound migration, confluent cell monolayers grown on 6-well plates were scratched using a micropipette tip. After the plates were washed with PBS to remove dislodged cells and media, they were incubated with EBM containing 0.5% FBS or supplemented with VEGF and/or VEGF inhibitors for 8 hours. Cell migration was observed by optical microscopy and quantified by measuring the number of cells that had migrated from the wound edges.

B cells collected from the transwell of co-cultured systems were washed and re-suspended in FACS buffer (PBS, 0.5% BSA, and 2 mM EDTA). Cells were stained with antibodies to the surface markers CD19, CD27, and CD38 (BD Biosciences) for 30 min on ice, followed by intracellular staining for Ki67 and VEGFA (BD Biosciences) using a BD Cytofix/Cytoperm kit. Antibodies were diluted FACS buffer for surface staining and in BD Perm/Wash buffer for intracellular staining. Cells were analyzed on a NovoCyte Quanteon and the data were analyzed using FlowJo Software.