ABSTRACT: Expression data from murine irf4-/- and irf4+/+ mature B cells purified by magnetic cell separation and stimulated with anti-CD40 antibodies and IL-4
Project description:Gene expression profiling of murine irf4-/- and irf4+/+ splenic B cells identifies genes regulated by the transcription factor IRF4 in CD40+IL-4 activated mature B cells. B cells from 12-week old irf4-/- and irf4+/+ littermate mice were isolated by magnetic depletion of non-B cells from splenic mononuclear cells and cultures with mitogens in vitro. RNA was isolated. Labeled cRNA was hybridized to microarrays and genes specifically expressed in irf4-/- vs. irf4+/+ samples.
Project description:RNAseq analysis of CD40 + IgM in vitro-stimulated (6 hours) murine relafl/flCD19-Cre (furtheron designated as RELA) and CD19-Cre (furtheron designated as WT) splenic B cells identifies genes regulated by the transcription factor RELA in activated B cells. Splenic B cells from 12-week old relafl/flCD19-Cre and CD19-Cre littermate mice were isolated by magnetic cell separation from splenic mononuclear cells and stimulated in vitro for 6 hours with anti-CD40 and anti-IgM. RNA was isolated and submitted for RNA-sequencing on an Illumina HiSeq2000 instrument for 30 million single-ended reads.
Project description:RNAseq analysis of CD40 + IgM in vitro-stimulated (6 hours) murine relfl/flCD19-Cre (furtheron designated as REL) and CD19-Cre (furtheron designated as WT) splenic B cells identifies genes regulated by the transcription factor c-REL in activated B cells. Splenic B cells from 12-week old relfl/flCD19-Cre and CD19-Cre littermate mice were isolated by magnetic cell separation from splenic mononuclear cells and stimulated in vitro for 6 hours with anti-CD40 and anti-IgM. RNA was isolated and submitted for RNA-sequencing on an Illumina HiSeq2000 instrument for 30 million single-ended reads.
Project description:RNAseq analysis of CD40 + IgM in vitro-stimulated (24 hours) murine relfl/flCD19-Cre (furtheron designated as REL) and CD19-Cre (furtheron designated as WT) splenic B cells identifies genes regulated by the transcription factor c-REL in activated B cells. Splenic B cells from 12-week old relfl/flCD19-Cre and CD19-Cre littermate mice were isolated by magnetic cell separation from splenic mononuclear cells and stimulated in vitro for 24 hours with anti-CD40 and anti-IgM. RNA was isolated and submitted for RNA-sequencing on an Illumina HiSeq2000 instrument for 30 million single-ended reads.
Project description:Gene expression profiling of murine irf4-/- and irf4+/+ splenic B cells identifies genes regulated by the transcription factor IRF4 in CD40+IL-4 activated mature B cells.
Project description:Nuocytes are a recently described cell that responds to both IL-25 and IL-33 and produce high levels of IL-13 and IL-5 Mice were administered with 3 daily doses of IL-25 (400 ng/dose). Nuocytes were purified by flow cytometry. Spleen cells prepared from IL-25-treated mice were depleted of lineage+ cells before cell sorting by incubation with biotin-conjugated anti-CD3, anti-CD19, anti-CD11b and anti-FcεRI antibodies before removal of antibody-bound cells by magnetic separation using Dynabeads (Invitrogen). Lineage-depleted cells were stained with PE-conjugated antibodies against CD4, CD8, B220, TER-119 and CD11b, a FITC-conjugated antibody against CD45, and an APC-conjugated antibody against ICOS. PE−, FITC+, APC+ cells were collected using a Mo-flo cell sorter, and purity was checked by staining with lineage antibodies and antibodies against IL-17BR and T1/ST2. Following purification nuocytes were prepared directly for microarray gene expression analysis or cultured with IL-33 and IL-7 (both at 10 nanograms/ml) for 9 days before being prepared for microarray gene expression analysis.
Project description:To induce leukemia in NOD/SCID/γc−/− (NSG) mice, we injected 10^5 human B-ALL cells, into the tail vein of non-irradiated mice. Two different PDX mice models were studied (PDX-1; #1 and PDX-2; #2). The InVivoMAb anti-human CD40 antibody (BE0189, clone G28.5, BioXCell) was i.v. administrated (CD40) at the dose of 1 mg/kg, the same was done with the recommended isotype (Ig) (BE0083, MOPC-21, BioXCell). We administrated antibodies, at days 15 and 25. At day 35, when mice developed leukemia, B-ALL cells were purified from the BM of PDX-mice, with magnetic beads-conjugated to human CD45 antibodies. Three mice per condition were tested (replicat 1 to 3; mice m1 to m3).
Project description:We sought to identify genes necessary to induce cytoskeletal change in B cells. Using gene expression microarray, we compared B cells stimulated with Interleukin-4 (IL-4) and anti-CD40 antibodies that induce B cell spreading, cell motility, tight aggregates, and extensive microvilli with B cells stimulated with lipopolysaccharide (LPS) that lack these cytoskeletal changes.
Project description:Antibodies are widely used to purify protein from complex mixtures like human plasma. Assessing the specificity of anntibodies, or any protein binder, is essential for the development of a protein biomarker assay. In addition to antibodies, novel protein binders like affimers are developed with a high specificity against the target protein and without batch-to-batch variability. In this study, we compared the specificity of affimers and commercially available antibodies against two non-related proteins (IL-37 and proinsulin). We conducted protein capture experiments with antibodies coupled to magnetic protein G beads and (biotinylated) affimers coupled to streptavidin magnetic beads. Binding capacity of anti-IL-37 and anti-proinsulin antibodies and affimers was investigated via these magnetic-bead captures of their recombinant protein targets in human plasma. The purified fractions were transferred to an SDS-PAGE, followed by in-gel digestion of the visualized protein bands and subsequently analyzed with western blot or shotgun proteomics using LC-MS/MS. From the proteomics experiments we evaluated the amount of background proteins that were co-purified during the protein purification procedure. In addition we determined the relative abundance of the target protein relative to other non-related proteins.
