ABSTRACT: Plasma cells from spleen were FACS sorted using Blmp1 and CD138 expression and gene expression compared to naïve B cells, FACS sorted using B220 and IgD
Project description:To understand the role of LSD1 in B cell differentiation, mice with B cell conditional deletion of LSD1 were intravenously inoculated with LPS. After 3 days, B220+GL7-CD138- naïve B cells and CD138+ plasmablasts were FACS-sorted from the spleens and RNA-seq was performed to identify LSD1-target regulated genes.
Project description:To understand the role of LSD1 in B cell differentiation, mice with B cell conditional deletion of LSD1 were intravenously inoculated with LPS. After 3 days, B220+GL7-CD138- naïve B cells and CD138+ plasmablasts were FACS-sorted from the spleens and RNA-seq was performed to identify LSD1-target regulated chromatin.
Project description:CD138+ B220- plasma cells were sorted from bone marrow and B220+ CD23+ mature follicular B cells were sorted from the spleens. Plasma cells were sorted from C57BL/6 mice 7 days after boosting with antigen, with mice first primed with an i.p. injection of KLH/IFA followed by boost at day 21 with KLH/PBS i.p. Mature B cells were sorted from antigen-naïve C57BL/6 mice. We compared expression profiles of plasma cells and mature B cells to identify differentially expressed transcripts.
Project description:CD138+ B220- plasma cells were sorted from bone marrow and B220+ CD23+ mature follicular B cells were sorted from the spleens. Plasma cells were sorted from C57BL/6 mice 7 days after boosting with antigen, with mice first primed with an i.p. injection of KLH/IFA followed by boost at day 21 with KLH/PBS i.p. Mature B cells were sorted from antigen-naïve C57BL/6 mice. We compared expression profiles of plasma cells and mature B cells to identify differentially expressed transcripts. Seven days after boosting with KLH, plasma cells were enriched by CD138-PE/anti-PE magnetic bead enrichment and then sorted to purity. Affymetrix microarrays were performed on 50,000-70,000 plasma cells and 2,000,000-3,000,000 naïve B cells.
Project description:in order to document molllecular signature of iMycCα B-cell lymphomas, RNA sequecing of CD138+ and CD138-B-cell lymphomas was performed and normalized expression data was obtained for all protein-coding genes Methods: B220+IgM+IgD+CD138+ and B220+IgM+IgD+CD138- B-cell lymphomas were investigated in the iMycCα mice. Total RNA from lymphoma cells (purified with B220-coupled beads from Miltenyi Biotech) was extracted and analysed by microarray (Génome et Transcriptome, GenoToul, Toulouse, France; get.genotoul.fr). RNA-seq paired-end libraries were prepared according to the Illumina protocol with some adjustments, using the TruSeq Stranded Total RNA Gold library prep Kit (Illumina, San Diego, USA). Libraries were quantified by qPCR using the KAPA Library Quantification Kit (Roche, Basel, Switzerland). Libraries quality was assessed by the HS NGS kit on the Fragment Analyzer (Agilent Technologies, Santa Clara, USA). Libraries were equimolarly pooled and RNA sequencing was then performed on one S4 lane of the Illumina NovaSeq 6000 instrument (Illumina, San Diego, USA), using the NovaSeq 6000 S4 v1.5 Reagent Kit (300 cycles), and a paired-end 2 x 150 pb strategy Results: Bioinformatic analysis defined CD138+ and CD138- B-cell lymphomas as two groups with a different transcriptome signature ( 2522 genes differentially expressed) driving various arrays of the immune responses and expressing different signaling/metabolic pathways. Enrichment of previously established human Burkitt lymphoma (BL) and multiple myeloma signatures was tested in our CD138+ B-cell lymphomas. In contrast to BL, myeloma up and down signatures were significantly enriched, emphasizing the molecular similarity of mouse CD138+ B-cell lymphomas to human multiple myeloma cells and the relevance of our mice as pertinent model of plasma B-cell lymphomas. Conclusions: Our mouse model carrying a homozygous insertion of c-myc into the IgH locus develops B-cell lymphoma with a large part of CD138+ plasma cell neoplasms. Its interesting and large transcriptome similarities with human myelomas make this mouse model an accurate, reliable experimental myeloma model.
Project description:The transcriptomic impact of Hells loss-of-function was quantified by RNA sequencing of sample from conditional knockout (Mb1-Cre+/WT HellsF/F) and littermate control mice (Mb1-CreWT/WT HellsF/F). Three time points were chosen to follow the kinetic: day 0 for naïve B cells collection, day 7 and day 14 post NP-CGG intraperitoneal immunization for germinal center B cells collection. At each time point, 4 mice of each genotype were used. Live cells were FACS sorted (AriaIII) using surface markers as follows: B220+CD21+CD23+ for naïve B cells, and B220+CD95+GL7+ for germinal center B cells. Due to their low frequency, germinal center B cells were FACS-sorted twice using the same gating strategy. Collected samples were lysed and processed for RNA extraction.
Project description:Long-lived plasma cells (LLPCs) develop under the help of follicular helper T (Tfh) cells and reside mainly in the bone marrow. However, these cells are unusually abundant in the spleen of several autoimmune models including K/BxNsf mice, yet their pathogenic impact remains unknown. To investigate a previously unappreciated role of splenic LLPCs, we sorted splenic plasma cells (PCs) from K/BxNsf and K/BxN mice, corresponding to LLPCs and conventional short-lived PCs, respectively, and compared their transcriptomes. The B220+CD138+ fraction from K/BxNsf and their control K/BxN mice were sorted by FACSariaIII.
Project description:Wild type or Aristolochic acid (AAI) nephropathy (AAN) mice were immunized with NP-KLH in Alum (100ug/mouse) On day 12 post-immunization, live NP-GcB cells were FACS-sorted from spleen according to B220+ GL7+ CD95+ NP+ expression directly into SmartSeq low-input RNA kit lysis buffer
Project description:The transcriptomes of FACS-sorted B220+GL7+CD95+ germinal center B cells and B220+GL7-CD95- non-germinal center B cells from lupus-prone B6.Sle1b were compared to the same cell types in B6.Sle1b mice without the type 1 interferon receptor (B6.Sle1b.IFNaR1-/-).
Project description:CD138-high plasma cell subsets were analyzed by single cell RNA-sequencing to identify gene expression differences between long- and short-lived plasma cells. Five different CD138-high subsets were analyzed based upon differential B220 expression and uptake of the fluorescent glucose analog 2NBDG: splenic B220+2NBDG-, B220+2NBDG+, B220-2NBDG-, B220-2NBDG+, and bone marrow B220-2NBDG+ cells.
