Human cd27 apc efluor780

Fresh negatively selected CD3 and CD14 fractions by Miltenyi according to manufacturer’s instruction. Initially the cells were blocked with nonspecific staining by incubating cells with 5% normal mouse serum (NMS; Jackson ImmunoResearch) in PBS for 10 min at RT. Cells were washed and stained with human CD3-PE-Cy5.5, human CD14-PE-Cy5.5 (LifeTech); human CD19-PE-Cy7, human IgD-FITC, human CD27-APC-eFluor780, human CD38-v450, and human CD138-APC (BD Biosciences). After washing, blood ASC (CD19⁺CD27^hiCD38^hi) were sorted on the FACSAria II sorter (BD Biosciences). The ASC populations were generally ~90–95% pure. Post-sort ASC were cultured immediately.

PBMC isolation and blood ASC purification were performed^{1 (link),36 (link),47 (link),48 (link)}. Briefly, PBMC were separated from freshly collected PBL samples by Ficoll-Hypaque (GE Healthcare) or Lymphocyte Separation Medium (LSM; Cellgro/Corning) density-gradient centrifugation^{49 (link)}. T cells and monocytes were removed by CD3 and CD14 beads (Miltenyi) and flow through stained with the following panel: human CD3-PE-Cy5.5 (or human CD3-BV711), human CD14-PE-Cy5.5 (or human CD14-BV711) (Life Tech); human CD19-PE-Cy7, human IgD-FITC, human CD27-APC-eFluor780 (or human CD27-PE), human CD38-v450, and human CD138-APC (BD Biosciences). After washing, PBMC were sorted on the FACSAria II sorter (BD Biosciences). The resultant blood ASC populations (CD19⁺CD27^hiCD38^hi) were generally ~85–99% pure, as assessed by flow cytometric re-analysis of post-sort cells. Post-sort ASC were subjected to cultures immediately.