x56  (Bio-Rad)

Journal: PLoS ONE

Article Title: Identification of CD4 − CD8 − Double-Negative Natural Killer T Cell Precursors in the Thymus

doi: 10.1371/journal.pone.0003688

Figure Lengend Snippet: (A) FACS profiles of in vitro generated NKT cells. The expression of NK1.1, CD4, CD8 and CD44 antigens by NKT cells generated in vitro from B6 DN4 thymocytes on day 10 of culture are compared to those of ex vivo B6 thymic NKT cells. Viable αGC/CD1d dimer + NKT cells were gated for the analysis. Representative data from three independent experiments are shown. (B) Cytokine secretion profiles of in vitro generated NKT cells. αGC/CD1d dimer + NKT cells generated in vitro from B6 DN4 thymocytes cultured on OP9/Dll-1 were FACS sorted on day 10 of culture, as shown in , and co-cultured with αGalCer-pulsed GM-DCs from either B6 or CD1dKO mice. APC-conjugated αGC/CD1d dimer and anti-APC microbeads were used to enrich NKT cells by MACS. The supernatants were collected after 48 hours of culture and were assayed for IL-4 and IFN-γ levels using the CBA method. Freshly sorted B6 thymic NKT cells served as positive controls. Values are expressed as mean±SD from three independent experiments. ND, not detected.

Article Snippet: mAbs specific for B220 (RA3-6B2), CD3ε (145-2C11), CD4 (RM4-5) (GK1.5), CD8α (53-6.7), CD11b (M1/70), CD11c (HL3) CD19 (1D3), CD24 (M1/69), CD25 (PC61), CD44 (IM7), CD45 (30-F11), CD45.2 (104), CD62L (MEL-14), CD69 (H1.2F3), CD122 (TM-b1), Gr-1 (RB6-8C5), NK1.1 (PK136), TCRβ (H57-597), TCRγδ (GL3), TER-119 and isotype controls were purchased from BD Biosciences or eBioscience, and used as FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, or Pacific Blue conjugates. αGalCer- or vehicle-loaded soluble dimeric mouse CD1d:Ig fusion protein (BD Biosciences) was used together with APC-anti-mouse IgG 1 (X56; BD Biosciences) mAb to stain NKT cells.

Techniques: In Vitro, Generated, Expressing, Ex Vivo, Cell Culture

Journal: PLoS ONE

Article Title: Identification of CD4 − CD8 − Double-Negative Natural Killer T Cell Precursors in the Thymus

doi: 10.1371/journal.pone.0003688

Figure Lengend Snippet: (A) In vitro generation of αGC/CD1d dimer + NKT cells. Thymic DN4 fractions from B6 or CD1dKO mice were co-cultured with OP9/Dll-1 for 8 to 10 days as described in . The numbers are the percentages of αGC/CD1d dimer + NKT cells within the PI − CD45 + viable lymphocyte gate. Representative data from three independent experiments for each group of mice are shown. (B) Mixed co-culture of CD1dKO DN4 with WT DN4 fractions. Thymic DN4 fractions from B6.CD45.1 and CD1dKO (CD45.2 + ) mice were mixed at a ratio of 7∶3, and cultured for 8 days on OP9/Dll-1. Values indicate the percentage of αGC/CD1d dimer + NKT cells within CD45.2 + or CD45.2 − gated viable lymphocytes. APC-conjugated αGC/CD1d dimer and anti-APC microbeads were used to enrich NKT cells by MACS. Representative data from three independent experiments are shown.

Article Snippet: mAbs specific for B220 (RA3-6B2), CD3ε (145-2C11), CD4 (RM4-5) (GK1.5), CD8α (53-6.7), CD11b (M1/70), CD11c (HL3) CD19 (1D3), CD24 (M1/69), CD25 (PC61), CD44 (IM7), CD45 (30-F11), CD45.2 (104), CD62L (MEL-14), CD69 (H1.2F3), CD122 (TM-b1), Gr-1 (RB6-8C5), NK1.1 (PK136), TCRβ (H57-597), TCRγδ (GL3), TER-119 and isotype controls were purchased from BD Biosciences or eBioscience, and used as FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, or Pacific Blue conjugates. αGalCer- or vehicle-loaded soluble dimeric mouse CD1d:Ig fusion protein (BD Biosciences) was used together with APC-anti-mouse IgG 1 (X56; BD Biosciences) mAb to stain NKT cells.

Techniques: In Vitro, Cell Culture, Co-Culture Assay

Journal: PLoS ONE

Article Title: Identification of CD4 − CD8 − Double-Negative Natural Killer T Cell Precursors in the Thymus

doi: 10.1371/journal.pone.0003688

Figure Lengend Snippet: (A) Detection of αGC/CD1d dimer + NKT cells in the thymus. FACS sorted DN4 thymocytes (6×10 5 ) from CD1dKO mice were injected intrathymically into B6.CD45.1 congenic mice, and recipient mice were analyzed 3 weeks later by FACS. Numbers on FACS plots indicate the percentage of cells in the indicated gates. Anti-CD45.2 staining was used to distinguish between cells of donor and recipient origin. APC-conjugated αGC/CD1d dimer and anti-APC microbeads were used to enrich NKT cells by MACS. (B) FACS profiles of αGC/CD1d dimer + NKT cells generated by intrathymic injection of CD1dKO DN4 thymocytes. Histogram plots show the expression of NK1.1, CD4 and CD44 by in vivo generated NKT cells from CD45.2 + CD1dKO DN4 thymocytes compared with resident NKT cells of CD45.2 − B6.CD45.1 host mice. Staining controls are shown in gray. The data is representative of two independent experiments.

Article Snippet: mAbs specific for B220 (RA3-6B2), CD3ε (145-2C11), CD4 (RM4-5) (GK1.5), CD8α (53-6.7), CD11b (M1/70), CD11c (HL3) CD19 (1D3), CD24 (M1/69), CD25 (PC61), CD44 (IM7), CD45 (30-F11), CD45.2 (104), CD62L (MEL-14), CD69 (H1.2F3), CD122 (TM-b1), Gr-1 (RB6-8C5), NK1.1 (PK136), TCRβ (H57-597), TCRγδ (GL3), TER-119 and isotype controls were purchased from BD Biosciences or eBioscience, and used as FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, or Pacific Blue conjugates. αGalCer- or vehicle-loaded soluble dimeric mouse CD1d:Ig fusion protein (BD Biosciences) was used together with APC-anti-mouse IgG 1 (X56; BD Biosciences) mAb to stain NKT cells.

Techniques: Injection, Staining, Generated, Expressing, In Vivo

Journal: Immunology and Cell Biology

Article Title: CXCL11 expressing C57BL/6 mice have intact adaptive immune responses to viral infection

doi: 10.1111/imcb.12541

Figure Lengend Snippet: Lymphocytic choriomeningitis virus (LCMV) infected CXCL11 KI mice exhibit an intact germinal center response. Splenocytes were analyzed from CXCL11 B6 and CXCL11 KI mice D8 following LCMV infection. (a) Representative plots and (b) frequency and (c) total numbers of CD4 + Tfh (CD44 + CD162 − Ly6C ‐ CD279 + CXCR5 + ) in CXCL11 B6 and CXCL11 KI cells. (d) Representative plots and (e) total numbers of Tfh (CD44 + CD162 − Ly6C − CD279 + CXCR5 + ) GP66 tetramer + CD44 + cells. (f) Total numbers of B220 + B cells in CXCL11 B6 and CXCL11 KI mice. (g) Representative plots, (h) frequency and (i) total numbers of GC B cells (B220 + IgD − CD95 + ). (j) Representative plots, (k) frequency and (l) total numbers of IgG1 + and IgG2a/c + GC B cells in CXCL11 B6 and CXCL11 KI . Data are representative of three independent experiments of 3–5 mice per group. Data are mean ± s.e.m.

Article Snippet: For B cell analysis : anti‐B220 (clone RA3‐6B2), anti‐CD95 (clone JO2), anti‐IgG1 (clone X56), IgG2a/b (clone R2‐40) and anti‐CD138 (clone 281‐2) from BD Biosciences, anti‐CD38 (clone 90) from eBioscience (San Diego, CA, USA) and anti‐IgD (clone 1126c) from WEHI antibody facility.

Techniques: Infection

Journal: Immunology and Cell Biology

Article Title: CXCL11 expressing C57BL/6 mice have intact adaptive immune responses to viral infection

doi: 10.1111/imcb.12541

Figure Lengend Snippet: Polyclonal and antigen‐specific response to influenza A remains intact in CXCL11 KI mice. (a) CXCL11 protein in spleen tissue lysates from CXCL11 B6 and CXCL11 KI mice 4 days following influenza infection. Data are median, minimum and maximum of 5 mice per group. (b–h, j–l) CXCL11 B6 and CXCL11 KI mice were analyzed D8 following influenza infection. (b, c) Total numbers of splenic CD8 + effector (CD44 + KLRG1 + CD62L − ) and memory precursor (CD44 + KLRG1 − CD62L + ) (b) polyclonal and (c) NP366 tetramer + cells in CXCL11 B6 and CXCL11 KI mice. (d, e) Total numbers of splenic CD4 + Th1 (CD44 + CD162 + Ly6C + ) and Tfh (CD44 + CD162 ‐ Ly6C ‐ CD279 + CXCR5 + ) (d) polyclonal and (e) NP311 tetramer + cells in CXCL11 B6 and CXCL11 KI mice. (f) Total numbers of splenic GC B cells (B220 + IgD ‐ CD95 + ) and (g) total numbers of IgG1 + and IgG2a/c + GC B cells in CXCL11 B6 and CXCL11 KI . (h) Representative confocal micrographs of splenic GCs D8 of CXCL11 B6 and CXCL11 KI mice stained with IgD (magenta, follicle), GL7 (cyan, GC structure) and CD35 (yellow, follicular dendritic cell stain). The scale bar represents 200 μm. (i) CXCL11 protein expression in lung tissue lysates from CXCL11 B6 and CXCL11 KI mice 4 days following influenza infection. Data are median, minimum and maximum of 5 mice per group. (j) Representative plots and (k) frequency of B220 + B cells, CD4 + and CD8 + T cells, and NKp46 + NK cells in CD45 + cells infiltrating the lung parenchyma of CXCL11 B6 and CXCL11 KI . (l) Representative immunohistochemistry staining of lung tissue sections CXCL11 B6 and CXCL11 KI mice. The scale bar represents 500 μm. Data are representative of three independent experiments of 3–5 mice per group. Data are mean ± s.e.m.

Article Snippet: For B cell analysis : anti‐B220 (clone RA3‐6B2), anti‐CD95 (clone JO2), anti‐IgG1 (clone X56), IgG2a/b (clone R2‐40) and anti‐CD138 (clone 281‐2) from BD Biosciences, anti‐CD38 (clone 90) from eBioscience (San Diego, CA, USA) and anti‐IgD (clone 1126c) from WEHI antibody facility.

Techniques: Infection, Staining, Expressing, Immunohistochemistry