Anti human vegfr 2 kdr apc

Confluent monolayers of HUVECs were prepared into 8-well chamber-glass slides (Lab-Tek II CC² Chamber Slide system, Nunc, USA) at a density of 5.6×10⁴ cells/well and incubated for 48 hours before used in the experiment. DV-specific CD8⁺ T cell lines (2×10⁴ cells/well), HLA-matched BLCLs (2×10⁴ cells/well), and HLA-A11 restricted DV NS3 peptides (5 μg/ml of DV1, DV2, or DV3/4), or DMSO (0.5%) were added to HUVECs monolayers cultures. After 48 hours supernatants and T cells were removed and HUVECs monolayers were stained with fluorescence conjugated antibodies to CD31 (platelet endothelial cell adhesion molecule-1, PECAM-1) 1:25 in 1% BSA in PBS (Anti-CD31 -FITC, eBioscience, USA), and VEGFR2 1:25 in 1% BSA in PBS (anti-human VEGFR-2/KDR-APC, R&D System, USA) at 4 °C for 30 minutes, washed twice with 1% BSA in PBS. Then, the HUVECs were incubated with DAPI (0.5 μg/ml, Sigma-Aldrich, USA) for 5 minutes at room temperature, and fixed with 1% paraformaldehye. The slides were mounted with VectorShield^® mounting solution and inspected with confocal fluorescence microscopy (AxioVision LE software, Zeiss, Germany).