Quantifying HUVEC Surface Protein Expression

Human umbilical vein endothelial cells (HUVECs) were grown in 8-well chamber slides (Ibidi, USA) for 3 days prior to incubat with 20 μg/ml of anti-NS1 mAbs or irrelevant anti-E mAb (4G2) for 1 h on ice. Rabbit anti-human CD31 or anti-PECAM-1 (1:10 dilution, Santa Cruz Biotechnology, USA) was used as positive control of endothelial cell surface staining^{11 (link),13 (link)}. After washing three times with DMEM containing 2% FBS, cells were incubated for 30 min on ice with goat anti-mouse IgG or anti-rabbit IgG conjugated with Alexa Fluor 488 (1:500 dilution, Thermo Fisher Scientific) and Hoechst 33,342 (1:1000 dilution, Thermo Fisher Scientific) for staining of nuclei. Stained cells were visualized and imaged with a Carl Zeiss LSM800 with Airyscan confocal microscope. The fluorescence intensity of staining (Alexa Fluor 488) on the cell surface for each clone was determined using Zeiss microscopy Zen imaging software. The fluorescence intensity of the specific protein signal was normalized by nuclear staining intensity from the entire area of each captured image.