biotinylated antibodies to gr1  (SouthernBiotech)

Journal: Nature Communications

Article Title: The concerted change in the distribution of cell cycle phases and zone composition in germinal centers is regulated by IL-21

doi: 10.1038/s41467-021-27477-0

Figure Lengend Snippet: A – C WT and Il21r −/− mice were immunized i.p. with NP-KLH in alum and on day 7, spleen NP-binding GC B cells (CD138 − IgM − IgD − Gr1 − B220 + NP + IgG1 + CD38 − ) were sorted and RNA-seq performed. A Mean-difference plot of WT vs Il21r −/− GC B cells depicts genes that were significantly upregulated in WT compared to Il21r −/− GC B cells shown in red and significantly downregulated ones in blue (false discovery rate <0.05). B Gene expression fold changes in WT compared to Il21r −/− GC B cells. Significantly DE genes were ranked by FDR and the 50 lowest then ordered by FC, colored red if upregulated in WT compared to Il21r −/− , blue if downregulated. C , D limma barcode plots testing for GC zone signature or cMyc signature genes among DE genes from WT vs Il21r −/− GC B cells as in A . Genes were ranked left to right as most up in Il21r −/− to most up in WT GC. For zone signature ( C ), vertical red bars mark DZ signature genes while vertical blue bars mark LZ signature genes. Worms show relative enrichment of DZ and LZ signature genes and camera p values assess significance. The plot shows DZ genes enriched among those up in WT while LZ genes were enriched among those up in Il21r −/− GC. For the Myc signature ( D ), vertical red bars mark upregulated genes in Myc + cells while vertical blue bars mark genes upregulated in Myc − cells. Worms show relative enrichment of Myc signature genes and camera p values assess significance. The plot shows Myc +- associated genes were significantly up in WT GC B cells.

Article Snippet: GC B cell was enriched by magnetic sorting by staining with biotinylated antibodies to GR1 (clone 8C5, WEHI Antibody Facility; 1 in 200 dilution), IgD (clone 11-26, Southern Biotech, Cat. 1120-08; 1 in 200 dilution), CD4 (clone GK1.5, WEHI Antibody Facility; 1 in 200 dilution), CD8 (clone YTS.169, WEHI Antibody Facility; 1 in 200 dilution), and CD138 (clone 281-2, BD Biosciences, Cat. 553713; 1 in 200 dilution) followed by staining with anti-biotin microbeads (Miltenyi Biotec, Cat. 130-090-485; 1 in 200 dilution) and depletion using LS Columns (Miltenyi Biotec, Cat. 130-042-401) supported by a MACS separator magnetic stand (Miltenyi Biotec).

Techniques: Binding Assay, RNA Sequencing, Gene Expression

Journal: bioRxiv

Article Title: An Erg driven transcriptional program controls B-lymphopoiesis

doi: 10.1101/861542

Figure Lengend Snippet: Expression and targeted disruption of Erg in lymphopoiesis. A. Wild-type ( Erg) , Erg tm1a(KOMP)wtsi , lacZ reporter ( Erg KI ), conditional ( Erg fl ), and Cre recombinase-deleted ( Erg Δ ) alleles with exons, Cre (loxP) and Flp (frt) recombinase recognition sites. IRES, internal ribosome entry site; Neo, neomycin-resistance cassette. B. Erg transcriptional activity by lacZ expression in Erg KI bone marrow (BM) and thymus cell populations (see Materials and Methods and , ). Mean fluorescent intensity (MFI) ratio ± S.D of Erg KI (n=4) to C57BL/6 (n=4). P adj < 0.027 corrected using Holm’s modification for multiple testing for each population except BM Ter119 + and NK1.1 + , and thymic DP, CD4 + CD8 - and CD8 + CD4 - populations ( P adj > 0.05). C. Representative flow cytometry plots: BM pre-proB (blue), proB (green) and preB (orange) and control B220 + IgM - IgD - (black) (left) with lacZ MFI (right). D. Erg expression by RNA-seq (mean±S.D, Fragments Per Kilobase of transcript per Million mapped reads, FPKM) in Erg fl/fl pre-proB, proB and preB cells (n = 2) E. Erg RNA-seq (FPKM) in Erg fl/fl and Rag1Cre T/+ ;Erg Δ/Δ pre-proB cells (n=2) (left; *, P = 1.41e-5, Table S4 ). Erg locus RNA-seq in Erg fl/fl (WT) and Rag1Cre T/+ ;Erg Δ/Δ (Erg KO, with pink highlighting absent expression) in pre-proB cells, H3K4me3 and H3k27ac ChIP-seq and chromatin accessibility (ATAC-seq, blue). F. Erg fl/fl (n=4) and Rag1Cre T/+; Erg Δ/Δ (n=7) B220 + B-cell, Gr1 + Mac1 + myeloid-cell, and CD3 + T-cell blood counts, mean±S.E.M; *, P = 6e-8 (top left). B-lymphoid populations in Erg fl/fl (n=9) and Rag1Cre T/+ ;Erg Δ/Δ (n=10) BM as ratio of cell number to Erg fl/fl (bottom left, see ). *, P adj < 0.04 corrected using Holm’s modification for multiple testing. Representative flow cytometry plots (right).

Article Snippet: B-cell progenitors were obtained from bone marrow that was lineage depleted using biotinylated Ter119, Mac1, Gr1, CD3, CD4, and CD8 antibodies, anti-biotin microbeads and LS columns (Miltenyi Biotec) and cultured on OP9 stromal cells in Iscove’s Modified Dulbecco’s Medium (Gibco, Invitrogen) supplemented with 10% (v/v) foetal calf serum (Gibco, Invitrogen), 50μM β-mercaptoethanol as well as murine interleukin-7 (10ng/mL) at 37°C in 10% CO 2 for 7 days.

Techniques: Expressing, Disruption, Activity Assay, Modification, Flow Cytometry, RNA Sequencing Assay, ChIP-sequencing

Journal: Cell

Article Title: IFNγ-Dependent Tissue-Immune Homeostasis Is Co-opted in the Tumor Microenvironment

doi: 10.1016/j.cell.2017.06.016

Figure Lengend Snippet: Key Resources Table

Article Snippet: T cell Reconstitution For T cell reconstitution experiments, CD3 + T cells were isolated by magnetic bead depletion using a biotinylated cocktail of antibodies against CD11b, Ter119, B220, Gr1, DX5, and IAIE, followed by negative selection with anti-biotin microbeads (Miltenyi).

Techniques: Western Blot, Immunofluorescence, Virus, Recombinant, Sequencing, Isolation, Software