Fitc conjugated streptavidin

HUVECs were seeded in a 24-well plate and treated with sFlt-1 (1 µg/mL) for 4–5 h to analyze adhesion molecule expression. Cells were harvested with Accutase (Sigma-Aldrich, St. Louis, MO, USA), collected by centrifugation (200× g, 5 min), and resuspended in 100 µL FACS buffer (PBS with CaCl₂ and MgCl₂ containing 0.5% FCS and 0.5% NaN₃) containing the following antibodies (1:50 dilution): biotin-conjugated anti-human intercellular adhesion molecule-1 (anti-ICAM-1, BioLegend, San Diego, CA, USA), PE-conjugated anti-human CD62E (anti-E-selectin, eBioscience, San Diego, CA, USA), PerCP/Cy5.5-conjugated anti-human CD62P (anti-P-selectin, BioLegend, San Diego, CA, USA), and APC-conjugated anti-human vascular cell adhesion molecule-1 (anti-VCAM-1, BioLegend, San Diego, CA, USA). Cells were incubated for 20 min at 4 °C, washed, and incubated with FITC-conjugated Streptavidin (1:50 dilution, BD Bioscience, CA, USA) for 20 min at 4 °C. Isotype-matched antibodies served as negative controls, and capture beads (Ultracomp eBeads, Invitrogen, Waltham, MA, USA) were used to set compensation. After washing, the cells were fixed in 1% PFA in FACS buffer and analyzed using the FACSCalibur flow cytometer (BD Bioscience, San Jose, CA, USA). Acquired data were processed with the FlowJo 10.6.2 Software (BD Bioscience, San Jose, CA, USA).