ABSTRACT: Goals/objectives: to identify various gene expression in B cell subsets derived from human PBMC and cord blood Overall design: Sample: human PBMC and human cord blood Cell subsets: memory B cell (CD20+CD10-CD27+), naive B cell (CD20+CD10-CD27-), CD21lo transitional B cell (CD20+CD10+CD27-CD21lo), CD21hi transitional B cell (CD20+CD10+CD27-CD21hi) Number of sample: PBMC (pooled from 2 donors), cord blood (pooled from 2-3 donors) Number of repeats: 2 Microarray: Affymetrix HU133Plus2 RNA extraction: Trizol (Invitrogen) RNA preparation/hybridization: All according to affymetrix protocol
INSTRUMENT(S): [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array
Project description:Goals/objectives: to identify various gene expression in B cell subsets derived from human PBMC and cord blood Sample: human PBMC and human cord blood Cell subsets: memory B cell (CD20+CD10-CD27+), naive B cell (CD20+CD10-CD27-), CD21lo transitional B cell (CD20+CD10+CD27-CD21lo), CD21hi transitional B cell (CD20+CD10+CD27-CD21hi) Number of sample: PBMC (pooled from 2 donors), cord blood (pooled from 2-3 donors) Number of repeats: 2 Microarray: Affymetrix HU133Plus2 RNA extraction: Trizol (Invitrogen) RNA preparation/hybridization: All according to affymetrix protocol
Project description:B lymphocytes in the neonates are poorly define and some of which express the marker CD5. The experiment is based on the flow cytometry cell sorting of 2 populations of B lymphocytes defined according to their phenotype : 1/ CD5 positive sorted as CD20+CD19+CD3-CD5+CD10- ; 2/ CD5 negative B lymphocytes sorted as CD20+CD19+CD3-CD5-CD10-. Cells were isolated from fresh heparinised cord blood used within 24 hours after sample collect, without any additional treatment Overall design: Comparison of CD5-positive and CD5-negative B lymphocytes isolated from cord blood obtained from 4 donors
Project description:Cord blood (CB) samples from normal donors were obtained with informed consent. Fresh CB samples were processed within 18-34h after collection. Mononuclear cells were isolated and CD34+ fraction was separated. CB CD34+ enriched fraction was lineage depleted by staining with purified anti-human CD2, CD3, CD4, CD7, CD8a, CD11b, CD14, CD19, CD20, CD56, CD235a followed by Qdot 605 conjugated goat F(ab')2 anti-mouse IgG (H+L). Cells were also stained with anti-human CD38-FITC, CD45RA-PE or -BV650, CD123-PE Cy7, CD90-biotin, CD34- PerCP and CD10-APC. Finally, cells were incubated with streptavidin-conjugated APC-eF780 and Hoechst 33258 (Invitrogen, final concentration: 1 g/ml). Populations were defined, as follows: HSC - Lin-CD34+CD38-CD90+CD45RA-CD10-, MPP - Lin-CD34+CD38-CD90-CD45RA-CD10-, LMPP - Lin-CD34+CD38-CD90-/loCD45RA+CD10-, MLP - Lin-CD34+CD38-CD90-/loCD45RA+CD10+, GMP - Lin-CD34+CD38+CD123+CD45RA+CD10-, CMP - Lin-CD34+CD38+CD123+CD45RA-CD10-, MEP - Lin-CD34+CD38+CD123-CD45RA-CD10-.
Project description:Non-switched memory (ME-M) B cells are an enigmatic population of IgM+ memory lymphocytes that are thought to emerge from germinal centers during systemic antibody responses against T cell-dependent antigens. To gain new insights into the properties of ME-M B cells generated during intestinal antibody responses, we performed global gene transcriptome expression analysis on naïve, ME-M and canonical memory class-switched (ME-SW) B cells purified from human gut samples. Marginal zone (MZ) and ME-SW B cells isolated from human spleen samples were used for comparison. Overall design: Naive (N) B cells (CD45+CD19+CD38-CD10-IgD++IgM+CD27-), MZ B cells (CD45+CD19+CD38-CD10-IgD+IgM++CD27+), ME-M B cells (CD45+CD19+CD38-CD10-IgD-IgM+CD27+) as well as ME-SW B cells (CD45+CD19+CD38-CD10-IgD-IgM-) were FACSorted from histologically normal tissue samples obtained from human terminal ileum (4 adult donors undergoing right hemicolectomy) and/or human spleen (4 adults donors).
Project description:This SuperSeries is composed of the following subset Series: GSE10512: Gene Expression Profiles of PBMC Subsets in Acute GVHD Following Cord Blood Transplantation (IMS v.1) GSE10513: Gene Expression Profiles of PBMC Subsets in Acute GVHD Following Cord Blood Transplantation (IMS v.2) Keywords: SuperSeries Refer to individual Series
Project description:Human B-1 cells (CD20+CD27+CD43+CD38lo/int) and pre-plasmablast like cells (CD20+CD27hiCD38hi) are new antibody secreting cells identified in circulation. We used microarray to compare and contrast expressed genes between these two cell population Cell subsets of interested were sort purified using BD Influx cell sorter and were subject for RNA extraction and hybridization on Human Gene 2.1 ST arrays.
Project description:We used microarrays to analyze the gene expression profile of CD34+CD45RA+CD7+, CD34+CD45RA+CD10+CD19- and CD34+CD45+CD7-CD10-CD19- HPCs isolated from umbilical cord blood CD34+CD45RA+CD7+(CD10-) and CD34+CD45RA+CD10+(CD7-CD19-) HPCs correspond respectively to prothymocytes and early pre-proB precursors. CD34+CD45RA+CD7-CD10-CD19- HPCs correspond to lympho-granulo-macrophagic precursors The corresponding populations were sorted from total CD34+ HPCs isolated from 2 or 3 individual donors
Project description:The role of B cells after transplant regarding allograft rejection or tolerance has become a topic of major interest. Recently, in renal transplant recipients, a B cell signature characterized by the overexpression of CD19+CD38hiCD24hi transitional B cells has been observed in operationally tolerant patients and in Belatacept treated patients with significant lower incidence of donor specific antibodies. The phenotypic and functional characterization of these transitional B cells is far to be exhaustive. We present the first transcriptomic and phenotypic analysis associated with this phenotype. Three populations were studied and compared: (i) transitional CD24hiCD38hi (ii) CD24+CD38- and (iii) CD24intCD38int populations. Peripheral blood mononuclear cells were isolated from 5 healthy donors and CD20+ B cells were isolated by magnetic beads. Three B cells populations were then sorted by flow cytometry: CD19+CD24hiCD38hi (test), CD19+CD24+CD38- (control 1) and CD19+CD24intCD38int (control 2)
Project description:The recent discovery of the human B1 cells, identified by the expression of CD20, CD27 and CD43 in absence of expression of CD70 and CD69 has been subject of debate. Some studies have raised the possibility that these cells are B cells differentiating towards the plasmablast and plasma cell stage rather than being the human counterpart of murine B1 cells. No further in depth studies have been performed. Therefore, a functional comparison was made between, the proposed B1 cells and plasmablasts. We observed that for several functional characteristics (distribution of isotypes of spontaneously producted antibodies, production of antigen-specific antibodies after vaccination with both T-cell dependent as well as T-cell independent antigen, the proposed B1 cells behaved similar to plasmablasts. In addition, we were able to differentiate the proposed B1 cells in vitro, indicating that they are not from a distinct lineage as the murine B1 cells. Gene expression analysis revealed that these cells cluster between memory B cells and plasmablasts, contradicting them being the genuine human counterpart of murine B1 cells, rather revealing a preplasmablast phenotype. Different B cells subsets were isolated from PBMC from healthy donors by a combination of magnetic and fluorescence activated cell sorting
Project description:Microarray deconvolution is a technique for quantifying the relative abundance of constituent cells in a mixture based on that mixture's microarray signature and the signatures of the purified constituents. It has been applied to yeast and other systems but not to blood samples. Here we test the ability of this technique to determine the fractions of subsets of memory T cells in peripheral blood mononuclear cell (PBMC) samples. Keywords: cell type comparison Overall design: PBMC samples from several donors were split. One portion of each PBMC sample was set aside, while the remainder of each was sorted into naive, central memory, and effector memory T cell subsets.