Cd19 pc5

Cell suspensions were stained with Near-IR LIVE/DEAD™ (Life Technologies), CD3-PC5 or CD19-PC5, and CD11b-PE when indicated (BD Biosciences). Samples were acquired on a FORTESSA cytometer (BD Biosciences). Data were exported and analyzed with FlowJo (RRID : SCR_008520; version 9-2, MacOS X). Counting beads and cells were gated on forward scatter-area/side-scatter area- (FSC-A/SSC-A) (shown in Figure 1C). Doublets were excluded on FSC-A/FSC-H. Dead cells were excluded on the expression of the viability dye (shown in Figure S1A). PMNs were gated as SSC^hi cells and CD11b expression. For analysis of trogocytosis of PMNs, target T cells were excluded on the expression of CD3. For cytotoxicity assays, targets (T cells, Jurkat T cell-lines, and Raji B lymphoma cell-line) were gated on SSC^lowFSC^hi, cell-trace, and CD3 or CD19 expression.

Briefly, MSC immunophenotype was established by flow cytometry using the following monoclonal antibodies: anti-CD73-PE (Miltenyi Biotec, Bergisch Gladbach, Germany), anti-CD90-PE (Miltenyi Biotec), anti-CD105-FITC (Ancell Corporation, Bayport, USA), anti-CD45-PC7 (BD Biosciences), anti-CD34-PE (Miltenyi), anti-CD14-PC7 (BD Biosciences), CD11b-APC (Miltenyi Biotec) and CD19-PC5 (BD Biosciences). After labeling, acquired results were analyzed by using a MACSQuant analyzer (Miltenyi Biotec). Cells were incubated for 30 min with these antibodies and after washing with PBS, the cells were fixed with 8% formaldehyde. BM-MSC were cultured in appropriate induction medium to assess their adipogenic, osteogenic and chondrogenic lineage differentiation capacities (NH media, Miltenyi Biotec). Lipid vacuole formation, mineralization (calcium deposits) and presence of proteoglycans corresponding to each lineage commitment were demonstrated by Oil Red O (Sigma-Aldrich), Alizarin Red S (Sigma-Aldrich) and Alcian Blue (Sigma-Aldrich) staining respectively.