D not proliferate through overnight culture, Raji cells slightly proliferated, and Jurkat and JINB8 proliferated by a issue of 3. PMNs remained alive throughout overnight culture but showed a trend to display an activated phenotype characterized by a reduce of SSC and an increase of FSC and according to the stimulation counts importantly decreased (shown in Figure S2B). This uncontrolled weak activation of PMNs in the course of their preparation was previously described (31) and resulted in spontaneous cytotoxicity or protection of T cells from spontaneous death ranging among 50 and 150 . Experiments had been excluded exactly where this effect was over 20 to concentrate around the effects of antibodies. Inhibition of respiratory burst was performed by adding 50 mg/ml of catalase to digest H2O2 and ten mM of Diphenyleneiodonium chloride (DPI) to inhibit NADPH oxidase (Sigma-Aldrich, Merck) for the duration of the overnight coculture. DPI couldn’t be made use of with cell lines as a target because it inhibited cell proliferation. Since cell linesCytotoxicity Assayproliferated for the duration of the overnight incubation cytotoxicity could no more be quantified since it was primarily based on the absolute counting of cells.Flow CytometryCell suspensions had been stained with Near-IR LIVE/DEADTM (Life Technologies), CD3-PC5 or CD19-PC5, and CD11b-PE when indicated (BD Biosciences).Solasodine Formula Samples have been acquired on a FORTESSA cytometer (BD Biosciences). Information have been exported and analyzed with FlowJo (RRID : SCR_008520; version 9-2, MacOS X). Counting beads and cells had been gated on forward scatter-area/side-scatter area- (FSC-A/SSC-A) (shown in Figure 1C). Doublets had been excluded on FSC-A/FSC-H. Dead cells have been excluded on the expression with the viability dye (shown in Figure S1A). PMNs have been gated as SSChi 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 cellline) had been gated on SSC lowFSC hi , cell-trace, and CD3 or CD19 expression.Lithocholic acid In Vitro TrogocytosisTarget cells were stained with all the membrane-dye PKH67 (Sigma-Aldrich) and cocultured with PMNs at ratios ranging from 1:1 to 3:1 for 3 h.PMID:23563799 Trogocytosis was also analyzed in PMNs recovered from cytotoxicity assays following an overnight incubation with targets stained with CellTrace. Percentages of trogocytosis have been determined by the expression on the T cell dye in PMNs just after setting gates on PMNs cultured alone.ABCFIGURE 1 | Anti-CD47 mAbs induce killing of main T cells by PMNs. (A) Cytotoxicity to primary T cells induced by anti-CD47 mAb clone CC2C6 alone or in the presence of PMNs (n = 95 distinct donors; ratio of PMN to T cells = 2). Iso, isotype; CD3, anti-CD3 mAbs; CD47, anti-CD47 mAbs. (B) Induction of PMNs’ cytotoxicity to Raji lymphoma B cells by combinations of Rituximab (RTX) plus anti-CD47 mAb clone CC2C6 (CD47) (n = three; ratio of PMN to target = 3). For (A, B), cytotoxicity is represented by the of live targets in indicated conditions compared to targets cultivated overnight alone. Median and IQR (interquartile range) are shown. P-values from Kruskall allis test is indicated on best of groups, P-values from Dunn’s multiple comparison post-test on top of pairs: P 0.05; P 0.01; P 0.0001. (C) Cytotoxicity assay. The left plot shows the coculture of PMN with Cell-Trace stained T cells on day 0. The following plots show cell-trace and viability staining of indicated cocultures right after overnight incubation. Gates and counts of beads, l.
