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1.
Figure 3

Figure 3. CD19- B220+ CD11c+ cells inhibit E47 protein in B cell precursors through a soluble factor. From: NK Cells in the CD19- B220+ Bone Marrow Fraction are Increased in Senescence and Reduce E2A and Surrogate Light Chain Proteins in B Cell Precursors.

A, Young CD19+ cells were sorted by magnetic beads and CD19-CD11c+ B220+ cells sorted from bone marrow by fluorescence flow cytometry and mixed at a 1:10 effector:target ratio of CD19- B220+ CD11c- to CD19+ cells, and cultured with IL-7 for 7 days. E47 and Ubc9 (loading control) proteins were detected by Western blot. B, CD19+ cells were co-cultured at varying ratios with CD19- B220+ CD11c+ cells, together with IL-7, for 7 days, either as cell mixtures or separated via transwells. Relative E47 levels of CD19+ cells as E47 to Ubc9 ratios were calculated.* p≤0.05 by Student's t-test for CD19+ only vs. CD19+ cells + inhibitory cells (n=5).

Anne M. King, et al. Mech Ageing Dev. ;130(6):384-392.
2.
Figure 1

Figure 1. CD19- B220+ cells are increased in aged mice and subsets include plamacytoid dendritic cells and NK lineage cells. From: NK Cells in the CD19- B220+ Bone Marrow Fraction are Increased in Senescence and Reduce E2A and Surrogate Light Chain Proteins in B Cell Precursors.

A, Bone marrow from young and aged BALB/c mice were harvested and surface stained for CD19, B220, analyzed by flow cytometry and numbers of CD19- B220+ cells were calculated based on total bone marrow cell numbers. Additionally, bone marrow from young and aged Balb/c mice was surface stained for B220, CD43 and IgM and analyzed by flow cytometry. Numbers of Fraction D early pre-B cells (B220+ CD43- IgM-) were calculated based on total bone marrow cell numbers. (n=10) *p≤0.05 by Student's t-test. B, Bone marrow from young and aged BALB/c mice were harvested and surface stained for CD19, B220, CD11c, Ly6C and DX5 and cell numbers of CD19- B220+ CD11c+ DX5+ (NK cells, n=4) and CD19- B220+ CD11c+ Ly6C+ (pDCs, n=10) in the bone marrow were calculated based on total bone marrow cell number *p≤0.05 by Student's t-test for young vs. aged groups. C, Freshly isolated bone marrow cells from young and aged BALB/c were surface stained for CD19, B220, CD11c, and DX5 and analyzed by flow cytometry (a: NK cells, b: pDCs). Data are representative of 4 individual pairs of young and aged mice.

Anne M. King, et al. Mech Ageing Dev. ;130(6):384-392.
3.
Figure 2

Figure 2. CD19- B220+ cells from young and aged mice inhibit E47 protein in CD19+ B cell precursors from young and aged mice. From: NK Cells in the CD19- B220+ Bone Marrow Fraction are Increased in Senescence and Reduce E2A and Surrogate Light Chain Proteins in B Cell Precursors.

CD19+ cells were magnetically bead separated from bone marrow of young BALB/c mice and co-cultured with magnetically sorted CD19- B220+ cells from young and aged mice at a 1:10 ratio (CD19- B220+:CD19+ cells) with IL-7 for 7 days. Non-adherent cells were harvested and E47 and actin proteins detected by Western blot. Shown in the graph below are cumulative data from 3 experiments where young and aged CD19+ cells were co-cultured with CD19- B220+ cells from young and aged mice. Aged CD19+ cells alone used in these experiments exhibited a 57% loss in E47 protein levels relative to young control CD19+ cells (data not shown). E47 values were normalized to their respective loading controls and then to young or aged CD19+ values in each experiment with young or aged CD19+ E47 values each set to 1. (*p≤0.05 by Student's t-test for all groups vs. young CD19+ cells alone based on loading control normalized values).

Anne M. King, et al. Mech Ageing Dev. ;130(6):384-392.
4.
Figure 6

Figure 6. TNFα is required for Fr. A/NK inhibition of E47 protein levels in B cell precursors. From: NK Cells in the CD19- B220+ Bone Marrow Fraction are Increased in Senescence and Reduce E2A and Surrogate Light Chain Proteins in B Cell Precursors.

CD19+ cells were sorted from young BALB/c bone marrow and plated with CD19- B220+ (Fr. A) bone marrow cells (1:5 effector:target ratio) or cell-sorted CD3-CD19- B220+ DX5+ bone marrow cells (1:10 effector:target ratio) in transwells. Control antibody (goat IgG), anti-IFNγ goat polyclonal antibody, or anti-TNFα goat polyclonal antibody were added at the inception of culture at 5μg/ml. Post 7 days of culture with IL-7, the CD19+ cells were harvested and E47 and Ubc9 and/or actin (loading control) proteins detected by Western blot. E47 values were normalized to both loading controls and to values for CD19+ cells cultured alone. Cumulative results are shown for 4 experiments. E47 values were normalized to their respective loading controls and then to young CD19+ values in each experiment with CD19+ E47 values each set to 1. (*p≤0.05 by Student's t-test for the anti-TNFα group vs. control Ab group; NS, not significant for experimental anti-IFNγ group vs. control Ab group).

Anne M. King, et al. Mech Ageing Dev. ;130(6):384-392.
5.
Figure 5

Figure 5. Stromal cells and macrophages from young or aged bone marrow microenvironments do not inhibit E47 expression in young CD19+ cells. From: NK Cells in the CD19- B220+ Bone Marrow Fraction are Increased in Senescence and Reduce E2A and Surrogate Light Chain Proteins in B Cell Precursors.

Young and aged BALB/c bone marrow cells were cultured with IL-7 for 7 days. Following the 7 day culture, adherent cells were harvested after addition of trypsin and magnetically separated based on expression of CD45. Once separated, stromal cells (CD45-) and macrophages (CD45+) from young and aged mice were cultured separately for an additional 3 days. Magnetically sorted young CD19+ B cell precursors (from separate mice) were then co-cultured with either young or aged stromal cells (CD45-) or macrophages (CD45+) for 7 days with IL-7. E47 protein was determined by Western blot and shown as E47 to Ubc9 signal ratios (n=4). No groups differed from the CD19+ alone control at the p<0.05 level by Student's t test. Data for E47 protein was normalized to loading control values [E47/Ubc9 ratio] in Western blots, not relative to the CD19+ alone control group.

Anne M. King, et al. Mech Ageing Dev. ;130(6):384-392.
6.
Figure 4

Figure 4. DX5+ bone marrow cells, but not dendritic cells, inhibit E47 expression and increase pERK expression in B cell precursors. From: NK Cells in the CD19- B220+ Bone Marrow Fraction are Increased in Senescence and Reduce E2A and Surrogate Light Chain Proteins in B Cell Precursors.

A and B, B cell precursors (CD19+) from young BALB/c mice were sorted by flow cytometry and co-cultured with the following sorted bone marrow cell subsets from young mice and placed in transwells: total DC (CD19- CD11c+); pDCs (CD19- B220+ CD11c+); or NK cells (DX5+) at 1:5 ratios. Cultures also contained 5ng/ml IL-7 and were harvested at 7 days and Western blots performed for E47, pERK and the loading control Ubc9. Cumulative results for E47 protein levels for 3 experiments are shown in C and for phospho-ERK protein in D normalized to loading controls. * p≤0.05 by Student's t-test for CD19+ vs. CD19+ cells + NK, tDC, or pDC groups. Data for E47 protein was normalized to loading control values [E47/Ubc9 ratio or pErk/Ubc9 ratio] in Western blots.

Anne M. King, et al. Mech Ageing Dev. ;130(6):384-392.
7.
Figure 8

Figure 8. Depletion of NK cells in aged mice increases λ5 protein expression in pro-B cells in vivo. From: NK Cells in the CD19- B220+ Bone Marrow Fraction are Increased in Senescence and Reduce E2A and Surrogate Light Chain Proteins in B Cell Precursors.

BALB/c young adult (3 mo.) and aged (24 mo.) mice were administered 200μg rabbit anti-asialo GM1 antibody or control antibody i.p. daily for 5 days. On day 7, bone marrow pro-B cells were enumerated as IgM- CD2- CD19+ B220+ cells and cytoplasmic λ5 protein levels assessed by fluorescence flow cytometry.
A, Representative fluorescence histograms for λ5 staining of young pro-B cells from mice treated with anti-asialo GM1 antibody or controls are shown. Proportions of λ5 high cells as well as mean fluorescent intensities are indicated. B, Representative fluorescence histograms for λ5 staining of aged pro-B cells from mice treated with anti-asialo GM1 antibody or control are shown. Proportions indicated are pro-B cells having λ5 protein levels above isotype control staining, designated “λ5Hi”. C, Cumulative data for 5-14 mice (n) used per group; abbreviations are NK depleted (NK Dep) and control (Ctrl). P values calculated by Student's t-test for the paired groups as indicated.

Anne M. King, et al. Mech Ageing Dev. ;130(6):384-392.
8.
Figure 7

Figure 7. TNFα impairs E47 and λ5 protein expression in B cell precursors and preferentially results in loss of pro-B cells in vitro. From: NK Cells in the CD19- B220+ Bone Marrow Fraction are Increased in Senescence and Reduce E2A and Surrogate Light Chain Proteins in B Cell Precursors.

A, CD19+ B cell precursors were co-cultured with recombinant murine TNFα (100ng/ml) added daily. E47 and λ5 protein expression was determined by fluorescence flow cytometry at day 7 of culture. Proportions of cells with high λ5 levels, above that of the isotype control, are indicated. B, Pro-B cells (CD2-) and pre-B cells (CD2+) from young and aged BALB/c mice in IL-7 supplemented cultures were assessed at day 7 after TNFα (100ng/ml) treatment. Since the expansion of B cell precursors differed in control cultures in individual experiments, the data on pro-B and pre-B cell numbers has been normalized to that of the analogous cells in control cultures in each experiment (control=100% expansion). TNFα effects on both young and aged B cell precursors are shown for experiments using 12 young and 4 aged BALB/c individuals. C, CD2- pro-B cells from young and aged mice were cultured with TNFα (100ng/ml) and proportions of cells expressing high λ5 levels (above isotype control staining) are indicated as determined by cytoplasmic fluorescence flow cytometry. In all fluorescence flow cytometry analyses, only live cells were assayed as determined by lack of Annexin V staining. *, p<0.05 for −TNFα vs. +TNFα groups in each panel.

Anne M. King, et al. Mech Ageing Dev. ;130(6):384-392.

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