Mouse anti cd45ra

All human cells were obtained under protocols approved by the Institutional Review Board of Yale University. PBMCs were isolated by density centrifugation of leukopheresis products obtained from anonymized healthy adult volunteers. CD4⁺ or CD8⁺ T cells were isolated from PBMCs using Dynabeads (Invitrogen) magnetic beads, according to manufacturer’s instructions. Activated T cells and monocytes were depleted by incubating the isolated cells with mouse anti-CD14 (BioLegend) and anti–HLA-DR (LB3.1; gift of J. Strominger, Harvard University, Cambridge, MA) antibodies, followed by incubation with magnetic pan-mouse IgG beads (Invitrogen). Memory T cells were isolated by further incubating T cells with mouse anti-CD45RA (eBioscience) and pan-mouse IgG beads. Isolates were routinely >99% CD4⁺ HLA-DR⁻CD45RA⁻ by flow cytometry.
Human umbilical vein ECs were isolated from umbilical cords by collagenase digestion. ECs were serially cultured on 0.1% gelatin–coated tissue culture plates in M199 (Invitrogen) supplemented with 20% FBS, 2 mM l-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.1% EC growth supplement (Collaborative Biomedical Research), and 100 µg/ml porcine heparin (Sigma-Aldrich). EC cultures were used at passage levels 2–5, at which time the cultured cells are uniformly positive for the EC marker CD31 and are devoid of CD45⁺ contaminating leukocytes.