Cd3 pacific blue sp34 2

PBMCs were isolated from buffy coat preparations of healthy blood donors (Blood Transfusion Center Solna, Stockholm, Sweden) through Ficoll-Paque Plus separation (GE Healthcare), as previously described (25 (link)). Exosomes (10 µg) were stained with a PKH67 Green Fluorescent Cell Linker Kit (Sigma-Aldrich), as previously described (25 (link)). Prefiltered (0.22-µm filter) PKH67-stained exosomes were added to PBMCs (2.5 × 10⁵) for 1, 2, or 4 h at 37°C, 5% CO₂. A PKH67 dye pellet centrifuged in parallel with labeled exosomes served as negative background control. PBMCs were stained with the following Abs to distinguish B cells (CD3⁻CD19⁺HLA-DR⁺), monocytes (CD3⁻CD14⁺HLA-DR⁺), and T cells (CD3⁺CD19⁻): CD19-ECD (HD237; B4 lytic; Beckman Coulter); HLA-DR–PE-Cy5 (TU36; BD Biosciences), CD14-PE (HCD14; BioLegend), and CD3 Pacific Blue (SP34-2; BD Biosciences). Exosome binding to live PBMCs (LIVE/DEAD Fixable Aqua Dead Cell Stain Kit; Invitrogen) was measured (~150,000 events) using an LSR Fortessa (BD) or FACSAria (BD) and analyzed using FlowJo software.