Nal FITC-conjugated Abs (BD Pharmingen or BioLegend/ Nordic Biosite) against human CD9 (M-LI3), CD19 (4G7), CD21 (B-ly4), CD23 (M-L233),J Immunol. Author manuscript; readily available in PMC 2014 September 24.Gutzeit et al.PageCD40 (5C3), CD63 (MEM-259), CD80 (2D10), CD81 (JS-81), CD86 (2331), TXA2/TP Antagonist site HLA-DR (L243), HLA-ABC (W6/32), IgG1 (MOPC-21), and IgG2a (MOPC-173). A total of 5 ?103 exosome-coated beads was acquired using a FACSCalibur (Becton Dickinson), and information were analyzed employing FlowJo software program (TreeStar). Nanoparticle tracking analysis The size distributions of B cell erived exosome preparations have been analyzed by measuring the rate of Brownian motion employing a NanoSight LM10 program, equipped using a rapid video capture and particle-tracking software program NTA two.2. Exosome preparations have been measured in triplicates at a concentration of five ?108 particles/ml. Immunoblot analysis DG75 cells (2 ?106) or exosomes (ten ) were separated by SDS-PAGE (ten ) and transferred to polyvinylidene difluoride membranes (Millipore). A total of 1 ?106 negatively selected B cells was incubated (37 , five CO2) for 15 h with 100 BJABex or LCL1ex in 500 complete medium (48-well plate; Becton Dickinson). B cells were washed 3 times with PBS to remove unbound exosomes and incubated for the remaining 24 or 48 h in total medium (37 , 5 CO2). Cell lysates have been separated by SDS-PAGE (NuPAGE four?two Bis-Tris Gel; Life Technologies) and transferred to polyvinylidene difluoride membranes (Millipore). Membranes have been stained as outlined by the manufacturer’s directions with Abs against LMP1 (CS. 1.four; Dako), EBNA2 (PE2; Novocastra), HLA-DR (TAL.1B5; Dako), CD81 (H-121; Santa Cruz Biotechnology), and -actin (I-19; Santa Cruz Biotechnology). Membranes have been visualized with ECL Prime Western Blotting Detection Reagent (GE Healthcare) and exposed on CL-XPosure films (Nordic Biolabs). Binding pattern of exosomes to B cells and monocytes in PBMCs PBMCs were isolated from buffy coat preparations of healthful blood donors (Blood Transfusion Center Solna, Stockholm, Sweden) by means of Ficoll-Paque Plus separation (GE Healthcare), as previously described (25). Exosomes (ten ) have been stained using a PKH67 Green Fluorescent Cell Linker Kit (Sigma-Aldrich), as previously described (25). Prefiltered (0.22- filter) PKH67-stained exosomes have been added to PBMCs (2.five ?105) for 1, 2, or 4 h at 37 , five CO2. A PKH67 dye pellet centrifuged in parallel with labeled exosomes served as adverse background control. PBMCs have been stained together 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 E-Cy5 (TU36; BD Biosciences), CD14-PE (HCD14; BioLegend), and CD3 Mcl-1 Inhibitor Storage & Stability 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 working with FlowJo application. Human major B cell isolation B cells had been isolated from PBMCs of healthier blood donors (Blood Transfusion Center Solna or Banc de Sang i Teixits, Barcelona, Spain). B cells were isolated either via adverse selection (B Cell Isolation Kit II; Miltenyi Biotec) or by constructive selection working with biotinylatedNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptJ Immunol. Author manuscript; accessible in PMC 2014 September 24.Gutzeit et al.Pageanti-IgD Ab (Southern Biotech) and Anti-Biotin MicroBeads (Mil.