Nevertheless, the clustering, in combination with the observation that these cells release CD24-bearing EMV, suggest that engagement of CD24 could preferentially affect neighbouring cells via the directed exchange of CD24 protein. Previous studies on EMV have shown they can participate in a wide range of cell processes including the regulation of lymphocyte activation, cellular proliferation, the transfer of signalling molecules, the epigenetic modification of cells and the delivery of active second messengers or microRNAs into cells.43,55,56,68,70,74,75 The transfer of EMV in blood transfusions can deliver CD55 and CD59, inhibitors of complement-mediated lysis, to host red blood cells, preventing their complement-mediated destruction.76,77 Although defining the precise function of CD24-bearing EMV in B-cell development requires further investigation, given their broad range of functions and their ubiquitous nature, the production of these CD24-bearing microvesicles from immature B cells may have the potential to affect differentiating B cells as well as the supporting stromal cell environment. Acknowledgments We thank Dr Nicholas Fairbridge for critical discussions and Dr David Schneider for statistical advice. a secondary antibody increased B-cell apoptosis, so demonstrating a dose-dependent regulation of CD24-mediated cell death.13 CD24 engagement also activates several caspase proteins, including Caspase-2, -3, -7 and -8, which are known to be involved in the induction of apoptosis.14 Engagement of CD24 causes the translocation of the Src-family tyrosine kinase, Lyn, into lipid rafts,5 which is presumed to activate downstream signalling. However, other plasma membrane-proximal events that occur in response to CD24 engagement have not been identified. We used transcriptomics data from the Immunological Genome project15 to identify additional potential functions of CD24. We found that genes with a similar expression profile to CD24 are significantly associated with cytoskeletal organization and vesicle trafficking. In support of the hypothesis that CD24 regulates vesicle trafficking, we found that antibody-mediated engagement of CD24 causes immediate and dramatic changes in its own cell surface expression in both mouse bone marrow-derived primary B cells and in the WEHI-231 immature B-cell line. This dynamic shift is not caused through classical endocytosis or exocytosis events, but is associated with the generation of CD24-bearing extracellular microvesicles (EMV) that can transport RBM45 CD24 between cells. Materials and methods Bioinformatics analysis Microarray-based expression data were retrieved from the Gene Expression Omnibus (GEO) using accession number “type”:”entrez-geo”,”attrs”:”text”:”GSE15907″,”term_id”:”15907″GSE15907. RMA normalization of gene expression and identification of differentially expressed genes was performed in R 2.15.016 via TinnR 2.3.7.1.17 using the Bioconductor,18 Biobase,18 Oligo,19 Limma20 and Affycoretools21 packages and the pd.mogene1.1 annotation file. False Discovery Rate was used for multiple testing correction. Unsupervised hierarchical clustering was performed in Genesis 1.7.6.22 The network interaction map 1,5-Anhydrosorbitol was created using the online GeneMANIA tool.23 The Caspase-7 gene was included with the co-expressed genes as it is known to be a target of CD24 signalling14 and was identified in the bioinformatics screen as being expressed during Hardy fractions A through C, when CD24 expression is highest. Genes lists were annotated automatically for gene ontology (GO) functions by GeneMANIA and AmiGO2,24 and manually annotated using the National Centre for Biotechnology Information Gene database and Vesiclepedia.25 Animal care The Institutional Animal Care committee at Memorial University of Newfoundland approved all animal procedures. Three-week-old C57BL/6N male mice were obtained from the Quebec facility of Charles River Laboratories (Wilmington, MA). Cell culture All materials for cell culture were obtained from Life Technologies (Carlsbad, CA) unless otherwise indicated. Isolated bone-marrow derived immature B cells and the BALB/c??NZB mouse WEHI-231 pre-B-cell 1,5-Anhydrosorbitol lymphoma cell line (ATCC, Manassas, VA) were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 1% antibiotic/antimycotic, 1% sodium pyruvate and 01% mercaptoethanol (complete media) at 37 and 5% CO2. Primary bone marrow B-cell isolation Femurs were removed from euthanized male C57BL/6N mice (3C6?weeks of age) and bone marrow was flushed out with Quin saline (QS; 25?mm NaHEPES, 125?mm NaCl, 5?mm KCl, 1?mm CaCl2, 1?mm Na2HPO4, 05?mm MgSO4, 1?g/l glucose, 2?mm glutamine, 1?mm sodium pyruvate, 50?m 2-mercaptoethanol, pH 72), using a 21-gauge needle. Single-cell suspensions were produced using a 100-m nylon mesh. The EasySep Mouse B Cell Isolation 1,5-Anhydrosorbitol Kit (cat. no. 19854; StemCell Technologies, Vancouver, BC, Canada) was used 1,5-Anhydrosorbitol to enrich bone marrow isolates following the manufacturers protocol. Fc-receptors were blocked on the B cells in this isolation using anti-mouse CD16/CD32 (Fcfor 5?min to remove unbound antibody and then resuspended in QS. Equal amounts of FITC-labelled and eFluor660-labelled cells were mixed either on ice (control) or at 37 for the indicated times. Cells were washed with FACS buffer and then analysed by flow cytometry. Inhibition of endocytosis and exocytosis WEHI-231 cells, resuspended at 50??105?cells/ml in QS, were pre-incubated in 200?m Pitstop 2 (Abcam, Cambridge, UK), 50?m Dynasore (Abcam), 40?m Exo1 (Abcam), 10?m Brefreldin A (Life Technologies) or vehicle control (DMSO) at 37 for 30?min and then treated with primary and secondary antibodies, as above, with inhibitor concentrations maintained at half the original concentration, for up to 1?hr. Transmission electron microscopy WEHI-231 cells were resuspended and stimulated as described above and then centrifuged and resuspended in Karnovsky fixative for 24?hr. Transmission electron microscopy (TEM) was performed by the Medical Education and Laboratory Support Services facility (Memorial University) according to standard protocols. Briefly, 85-nm resin-embedded sections were mounted on 300-mesh copper grids, and then stained with 3% uranyl acetate in a 30% ethanol. Grids were examined using a JEOL 1200 Ex lover electron microscope (JEOL, Peabody, MA) and images were captured using an SIA-L3C digital camera (SIA, Duluth, GA). Isolation of extracellular microvesicles WEHI-231 cells in QS were left untreated, or stimulated as explained above for 15?min or 60?min at 37 with either 10?g/ml of M1/69 anti-mouse CD24 antibody that had been.