Apc cy7 conjugated anti human cd3

CD3⁺T cells were isolated from PBMNC of HD by anti-CD3 microbeads (Miltenyi Biotec) and labeled with CellTrace^TM Violet Cell Proliferation kit (5 µM; Invitrogen, Waltham, USA). Isolated MDSC were co-cultured with allogeneic CD3⁺T cells for 72 h at ratios of 1:32, 1:16, 1:8, 1:4, 1:2 or 1:1 in the presence of anti-CD3/anti-CD28 Dynabeads^® (Gibco, Grand Island, USA). Cells were then stained with APC-Cy7-conjugated anti-human CD3, PerCP-Cy5.5-conjugated antihuman CD4 and APC-conjugated anti-human CD8 (Biolegend) antibodies. The proliferation of CD3⁺, CD3⁺CD4⁺ or CD3⁺CD8⁺ T cells was evaluated by flow cytometry.^{14 (link)} For the activation assay, cells were stained with APC-Cy7-con-jugated anti-human CD3, FITC-conjugated anti-human CD69 and PE-conjugated anti-human CD25 (Biolegend) antibodies.

Frequencies of peripheral CD4⁺ T cells expressing T cell activation markers CD25 and HLA-DR⁴⁷ were examined. In brief, PBMCs were stained with FITC-conjugated anti-human CD4, PerCP-conjugated anti-human CD8, APC-Cy7-conjugated anti-human CD3, Pacific Blue-conjugated anti-human CD25 (Biolegend), and PE-Cy7-conjugated anti-human HLA-DR (Biolegend) monoclonal antibodies. Stained cells were analyzed by BD FACS Canto II with FACS Diva v.8.0.1 and FlowJo v.9.2.