GaCD40L (100 ng/ml; Enzo Life Sciences) and IL-21 (50 ng/ml; Invitrogen) or with 15 B cell erived exosomes/well. Subsequently, B cells had been incubated with purified human Ig (Sigma-Aldrich) and stained with anti-CD19allophycocyanin (HIB19; BD Pharmingen) for 30 min at four . Cells have been washed with PBS (350 g, 5 min) and stained with an FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen), in accordance with the manufacturer’s guidelines. A total of 2 104 cells was acquired by flow cytometry (LSR II or Fortessa; BD) and analyzed with FlowJo software. Cells were gated as follows: forward scatter (FSC) region (FSC-A) versus side scatter (SSC) area (SSC-A), FSC-A versus FSC height (FSC-H) for doublet exclusion, CD19+. The morphology of cells was documented applying a light microscope (Axiovert 25; Zeiss) and Leica application (Zeiss).NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptJ Immunol. Author manuscript; obtainable in PMC 2014 September 24.Gutzeit et al.PageProliferation of PBMCs PBMCs were isolated from healthful blood donors (Blood Transfusion Center Solna). A total of ten 106 PBMCs was labeled at room temperature with 1.5 CFSE (CellTrace CFSE Cell Proliferation Kit; Invitrogen) for 3 min. Labeled PBMCs have been washed 3 instances with comprehensive medium (400 g, 7 min) and cultured in full medium at a density of 2.five 105 cells/200 (96-well flat bottom plates; BD). PBMCs have been either left unstimulated (unstimulated handle [co]) or stimulated with 1.5 PHA (PHA-M; Life Technologies Life Technologies) or 25 DG75-COex, DG75-LMP1ex, or DG75-EBVex per effectively. Immediately after 5 d of culture (37 , 5 CO2), cells had been incubated with purified human Ig (Sigma-Aldrich) and stained for CD3-allophycocyanin (UCHT1L; BioLegend) and CD19 exas Red (B4 [LYTIC]-ECD, CYTO-STAT; Beckman Coulter). DAPI (5.7 ; Sigma-Aldrich) was utilised as live/dead marker. Cells were acquired applying an LSR II or Fortessa (BD), and data had been analyzed with FlowJo computer software (TreeStar). Cells had been gated as follows: FSC-A-SSC-A, FSC-A-FSC-H, DAPI- (alive). B cell proliferation and differentiation B cells were isolated (negative choice), and purity was 92 (n = 4; two donors from Barcelona and two donors from Stockholm). A total of 2 106 B cells was labeled for 5 min at room temperature with 1.25 CFSE (CellTrace CFSE Cell Proliferation Kit; Invitrogen). Labeled B cells were washed three occasions (350 g, 7 min) and cultured in total culture medium at a density of 1.five 105 cells/200 (96-well round-bottom plates; BD). B cells had been either left unstimulated or stimulated with IL-21 (one hundred ng/ml; Invitrogen), MegaCD40L (500 ng/ml; Enzo Life Sciences), or five or 25 exosomes/well. After five d of cultivation (37 , 5 CO2), cells have been incubated with purified human Ig (Sigma-Aldrich) and subsequently stained with CD19-allophycocyanin (HIB19), CD20PerCP-Cy5.Setipiprant Epigenetics five (2H7) and CD38-PeCy (HB7; all from BD).Eact custom synthesis DAPI (five.PMID:28322188 7 ; Sigma-Aldrich) was made use of as live/dead marker. Cells had been analyzed with flow cytometry and gated as follows: FSC-A-SSC-A, FSC-A-FSC-H, DAPI- (alive), CD19+. Quantitative real-time PCR IgD+ B cells had been applied with a purity 96 (two donors from Barcelona). B cells (1.2 105/200 in 96-well round-bottom plates; BD) had been cultivated for 3 d in comprehensive culture medium (37 , 5 CO2) and either left unstimulated or stimulated with soluble MegaCD40L (500 ng/ml; Enzo Life Sciences) and IL-21 (100 ng/ml) or with 12.5 DG75 exosomes. RNA from 5 105 B cells was extracted (High Pure RNA Isolation.