Anti cd47

HUVEC were purchased from (Lonza) and were maintained using EGM2 medium (Lonza). HUVEC were used between passages 4 and 5. Jurkat T cells (E6.1, ATCC) and the corresponding CD47-deficient Jurkat mutant JinB8 were provided by Dr. Eric Brown, Genentech (Reinhold et al., 1999 (link)) and were maintained at 2.0 × 10⁵ cells per ml in RPMI 1640 medium (Life Technologies) supplemented with 10% FBS, glutamine, penicillin, and streptomycin. Jurkat cells were maintained for a maximum of 4 weeks for experiments. Human anti-CD3 and anti-CD47-FITC (BD Biosciences), anti-CD69 (R&D systems), anti-actin, anti-tubulin, anti-VEGFR2, anti-VEGFR2 Y¹¹⁷⁵ (Cell Signaling), anti-CD47 (B6H12, Abcam) and azide-free functional grade anti-CD47 (e-Biosciences) were purchased from the indicated vendors.

Cells were washed with PBS and stained with a fixable viability dye. After washing, cells were labeled at 4℃ for 30 min with anti-CD3, -CD4, -CD8, and -CD25 antibodies conjugated with fluorescent dye (eBioscience, San Diego, CA, USA), anti-CD47 (eBioscience), and PerCP-Cy5.5 anti-rat secondary antibody. For intracellular labeling, cells were fixed and permeabilized with cytofix/cytoperm buffer (eBioscience) and labeled with anti-human FOXP3 antibody (eBioscience) conjugated with FITC. Labeled cells were quantified using a BD FACSVerse flow cytometer, and the data were analyzed using FlowJo Software (BD Bioscience, San Jose, CA, USA).