igg quantitation enzyme linked immunosorbent assay Search Results


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  • 94
    Thermo Fisher igg2b
    Spontaneous hyper–Th1 phenotype in Foxp3 Cre xT-bet fl/fl mice. (A) Representative FACS plots of splenic T-bet + Foxp3 + Treg1 cells from naïve mice of the indicated genotypes. (B) Quantification of T-bet + Foxp3 + Treg1 cells in spleens and blood of naïve mice. (C and D) Quantification of (C) splenic and (D) blood T-bet + or IFN γ + Foxp3 − Th1 cells as indicated. (E) Quantification of chemokine receptor CXCR3 and CCR6 expression on Foxp3 − T helper cells in the blood of naïve mice. (F and G) Levels of total <t>IgG,</t> <t>IgG1,</t> and <t>IgG3</t> subclasses in the serum of naïve mice. ELISA data are shown as OD at 450 nm in serial dilutions as indicated. Numbers in FACS plots represent percentages of Foxp3 + cells; n =6 versus six animals for all analyses. Circles in B–E represent individual animals, and horizontal lines represent mean values. Error bars represent SEM. * P
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    Millipore igg
    1B9B9 suppressed the bioactivity of <t>FGF-2.</t> ( A ) Recombinant FGF-2 was incubated with 1B9B9 or <t>IgG</t> for 1 h and then used to treat FBHEs for 30 min. Phosphorylation of FGFR was analyzed by immunoblotting. Data are representative of three independent experiments that yielded similar results. ( B ) FBHE (2×10 4 ) cells were cultured in 2% FBS-DMEM/RPMI 1640 (1:1) for 24 h. The cells were then cultured with IgG (10 μg/mL) as a negative control or 1B9B9 (10 μg/mL, 5 μg/mL, 1 μg/mL) in the presence of FGF-2 (2 ng/mL). After 3 days, the cell number was assessed by Trypan blue staining. * p
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    Bethyl igg quantitation enzyme linked immunosorbent assay
    1B9B9 suppressed the bioactivity of <t>FGF-2.</t> ( A ) Recombinant FGF-2 was incubated with 1B9B9 or <t>IgG</t> for 1 h and then used to treat FBHEs for 30 min. Phosphorylation of FGFR was analyzed by immunoblotting. Data are representative of three independent experiments that yielded similar results. ( B ) FBHE (2×10 4 ) cells were cultured in 2% FBS-DMEM/RPMI 1640 (1:1) for 24 h. The cells were then cultured with IgG (10 μg/mL) as a negative control or 1B9B9 (10 μg/mL, 5 μg/mL, 1 μg/mL) in the presence of FGF-2 (2 ng/mL). After 3 days, the cell number was assessed by Trypan blue staining. * p
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    Bethyl mouse igg2b elisa quantitation kit
    VSV-IFN therapeutic efficacy in mice bearing orthotopic myeloma 5TGM1 cells were implanted IV in syngeneic C57Bl/KalWrij mice. ( A ) Mean serum <t>IgG2b</t> concentration was quantified at 7 day intervals (n=26 mice). Mean serum IgG2b was measured by <t>ELISA</t> and compared using a t-test indicating a significant change in serum IgG2b from day 14 to day 21 post 5TGM1 implantation (*P=0.0019) ( B ) Survival response in myeloma bearing mice following a single IV dose of PBS, or 1×10 8 VSV-mIFNβ or VSV-hIFNβ. Survival was compared by log-rank analysis. VSV-mIFNβ and VSV-hIFNβ treatment significantly prolonged survival compared to PBS (P=0.0008*** and P=0.017* respectively), and survival of mice treated with VSV-mIFNβ is significantly prolonged compared to VSV-hIFNβ treated mice (P=0.021*) ( C ) Myeloma burden in response to IV treatment with PBS, VSV-mIFNβ or VSV-hIFNβ measured by serum IgG2b ELISA.
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    Bethyl igg2a enzyme linked immunosorbent assay elisa quantitation kits
    Fraxinellone inhibits B cell maturation. ( A , B ) CD19 + B cells were activated by lipopolysaccharide. After treatment with or without fraxinellone (40 μM), the relative expression levels of AID and Blimp-1 in CD19 + B cells were determined with RT-PCR. ( C ) The level of immunoglobulin G in the culture supernatant was measured using <t>ELISA</t> in the presence of fraxinellone at doses of 0–40 μM. In all culture conditions, CD19 + B cells were stimulated by lipopolysaccharide. Data represent the mean of three independent experiments ± SEM. Nil, no stimulation; LPS, stimulation with lipopolysaccharide; Frx, fraxinellone; AID, activation-induced cytidine deaminase; Blimp-1, B lymphocyte-induced maturation protein-1; <t>IgG,</t> immunoglobulin G; * p
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    Bethyl human igg enzyme linked immunosorbent assay elisa quantitation set
    Isotyping and <t>IgG</t> subclass examination. ( A ) Hybridoma culture supernatants were tested by <t>ELISA</t> to determine the isotypes of the HuMAbs. Each ELISA included three positive controls (Ctrl; Ctrl IgA, Ctrl IgG, and Ctrl IgM). ( B ) IgG subclasses were determined
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    Roche mouse igg enzyme linked immunosorbent assay elisa quantitation kits
    Isotyping and <t>IgG</t> subclass examination. ( A ) Hybridoma culture supernatants were tested by <t>ELISA</t> to determine the isotypes of the HuMAbs. Each ELISA included three positive controls (Ctrl; Ctrl IgA, Ctrl IgG, and Ctrl IgM). ( B ) IgG subclasses were determined
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    Bethyl human igg enzyme linked immunosorbent assay elisa quantitation kit
    Isotyping and <t>IgG</t> subclass examination. ( A ) Hybridoma culture supernatants were tested by <t>ELISA</t> to determine the isotypes of the HuMAbs. Each ELISA included three positive controls (Ctrl; Ctrl IgA, Ctrl IgG, and Ctrl IgM). ( B ) IgG subclasses were determined
    Human Igg Enzyme Linked Immunosorbent Assay Elisa Quantitation Kit, supplied by Bethyl, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam igg enzyme linked immunsorbent assay elisa quantitation kit
    Effects of AMPCol and AMP on the levels of the relative weight of spleen ( A ), IgM ( B ), IgA ( C ) and <t>IgG</t> (D) from the serum were measured using <t>ELISA.</t> The data represent mean ± SD, *p
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    ZeptoMetrix immuno tek quantitative human igg enzyme linked immunosorbent assay elisa kit
    Effects of AMPCol and AMP on the levels of the relative weight of spleen ( A ), IgM ( B ), IgA ( C ) and <t>IgG</t> (D) from the serum were measured using <t>ELISA.</t> The data represent mean ± SD, *p
    Immuno Tek Quantitative Human Igg Enzyme Linked Immunosorbent Assay Elisa Kit, supplied by ZeptoMetrix, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bethyl isotype specific igg enzyme linked immunosorbent assay elisa quantitation
    Effects of AMPCol and AMP on the levels of the relative weight of spleen ( A ), IgM ( B ), IgA ( C ) and <t>IgG</t> (D) from the serum were measured using <t>ELISA.</t> The data represent mean ± SD, *p
    Isotype Specific Igg Enzyme Linked Immunosorbent Assay Elisa Quantitation, supplied by Bethyl, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bethyl mouse igg enzyme linked immunosorbent assay elisa quantification kit
    Effects of AMPCol and AMP on the levels of the relative weight of spleen ( A ), IgM ( B ), IgA ( C ) and <t>IgG</t> (D) from the serum were measured using <t>ELISA.</t> The data represent mean ± SD, *p
    Mouse Igg Enzyme Linked Immunosorbent Assay Elisa Quantification Kit, supplied by Bethyl, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    R&D Systems igg2b
    CD154-induced B cell class switching and differentiation into PNA hi B cells and plasma cells are inhibited by CpG in the absence of BCR crosslinking (flow cytometry analysis). ( a ) CSR to <t>IgG1</t> (top panels) and expression of PNA-binding lectins (bottom panels) in B cells stimulated with CD154 (3 unit/ml) plus IL-4 in the absence or presence of CpG (0.3 μM) for 4 d. ( b ) CSR to IgG1 (top panels) and expression of PNA-binding lectins (bottom panels) in B cells stimulated with CD154 (3 unit/ml) plus IFN-γ in the absence or presence of CpG for 4 d. ( c ) CSR to IgG1, IgE or <t>IgG2a</t> in B cells stimulated with CD154 plus IL-4 or IFN-γ in the absence or presence of CpG for 4 d. Data are mean and s.d. from three independent experiments ( p values calculated by paired student t test). ( d ) Formation of B220 lo CD138 hi plasma cells in cultures of B cells stimulated with LPS (1 μg/ml) plus IL-4 or IFN-γ in the absence or presence of CpG (0.3 μM) for 4 d. Data are mean and s.d. from three independent experiments ( p values calculated by paired student t test).
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    Sanquin igg3
    IL-21 drives partial plasma cell differentiation in BCL6-transduced B cells. A , PB CD19 + B cells expressing BCL6-IRES-YFP were cultured in the presence of IL-2 and IL-4 or with rmIL-21 alone. At the indicated time points expression of BLIMP1 and ACTB were measured by qRT-PCR. BLIMP1 expression was normalized to ACTB expression within each sample and the means ± SD of triplicate measurements in two different donors is plotted. B , BCL6-YFP expressing B cells were cultured with CD40L in the presence of IL-2 and IL-4 or with IL-21 and BLIMP1, BCL6, and tubulin protein expression were determined by immunoblotting 3 or 7 days after IL-21 treatment. C , CD19 + B cells were transduced with LZRS-BCL6-IRES-ΔNGFR and cultured for 10 days on CD40L in the presence of IL-2 and IL-4 or with IL-21 alone. Cells were stained with anti-CD19, anti-NGFR, anti-CD38, and anti-CD20 and analyzed by flow cytometry. The rightmost four plots show CD38/CD20 expression on CD19 + BCL6–ΔNGFR– and CD19 + BCL6–ΔNGFR + cells. D , CD38/CD20 expression on BCL6-IRES-YFP-transduced B cells after 21 days of culture with IL-2 and IL-4 or with IL-21 alone. E , Surface Igλ and Igκ BCR expression on cells cultured as in C . F , Expression of CD138, HLA-DR, CD86, CD27, CD25, and CD3 on BCL6 + B cells cultured as in C . Dotted histogram is iso-type control. G , CD19 + BCL6-overexpressing B cells maintained on CD40L with either IL-2 and IL-4 (○)or with IL-21 (•). Cumulative expansion was calculated based on absolute cell numbers over a 17-day period. Data shown are means ± SD of three independent experiments involving different donors and constructs (BCL6-IRES-ΔNGFR or BCL6-IRES-GFP). H , BCL6-IRES-ΔNGFR + cells were cultured for 6 days on CD40L-L cells in the presence of IL-2 and IL-4 or with IL-21. Cell numbers were counted and IgM and <t>IgG</t> in culture supernatants were measured by ELISA to obtain per cell values of IgM and IgG. Data are representative of three independent experiments. I , Total PB CD19 + BCL6-GFP + cells were cultured with CD40L in the presence of IL-2 and IL-4 or with IL-21. After 7 days, equal numbers of cells were plated in serial dilutions and in triplicate and IgG and IgM production were determined by ELISPOT.
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    SouthernBiotech igg3
    Defective Ig production in Atm −/− B cells activated in vitro. CD19 + B cells were isolated from the spleens of Atm −/− and WT mice and cultured with CD40L and IL-4 <t>(IgG1,</t> <t>IgG2a,</t> and IgM), CD40L, IL-4, IL-5, TGF-β, and anti-IgD dextran (IgA) or LPS <t>(IgG2b,</t> <t>IgG3).</t> Results are displayed as the mean of three to four experiments ± SEM Atm −/− titer as a percentage of the Atm +/+ controls.
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    Jackson Immuno igg3
    ICOS-signalling promotes humoral immune responses during blood-stage infection. (A) Flow cytometric gating strategy employed to analyze splenic CD4 + T-cell responses throughout the manuscript. (B) Representative FACS plots and time-course analysis of cell-surface ICOS expression on splenic CD4 + T-cells from WT mice (n = 3/time point) during Py 17XNL infection. (C-F) WT mice (n = 6/group) were treated with anti-ICOSL blocking monoclonal antibody (α-ICOSL) or its isotype control (rat-IgG2a) prior to and during infection with Py 17XNL. (C) Representative FACS plots (gated on B220 + CD19 + live singlets), proportions and numbers of splenic GC B-cells (GL-7 + Fas + ), (D) numbers of splenic Ig-class switched (IgD lo IgM lo ) B-cells, and (E) representative FACS plots (gated on CD4 + TCRβ + live singlets), proportions and numbers of splenic Tfh cells (PD1 + CXCR5 + ), in naïve mice and infected, α-ICOSL and control IgG-treated mice, 16 days p . i . (F) Py 17XNL-specific IgM, total IgG, IgG2b and <t>IgG3</t> levels in serum of naïve and infected, α-ICOSL and control IgG-treated mice, 16 days p . i . (G) Parasitemias in WT mice (n = 6/group) infected with Py 17XNL, and treated every three days with α-ICOSL or control IgG until day 21 p . i . (depicted by arrows, with estimated period of cover highlighted with shaded grey box—an α-ICOSL-treated mouse succumbed to infection on each of days 15, 17 and 30 p . i ., and one control-IgG treated mouse succumbed on day 20 p . i . Data representative of two independent experiments in (B-E), and two pooled independent experiments in (F), with experiment in (G) being conducted once. Statistics: Mann-Whitney U test, *P
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    Becton Dickinson igg1
    M2 expressing cells have an activated, pre-plasma memory phenotype. (A) M2-transduced cells were CD19 + , CD25 high , GL7 high , B220 low , I-A b low , sIgD − , sIgG + , and CD138 low when compared to untransduced cells within the culture. Representative flow cytometry histograms at day 4 post-transduction. The black line open histograms reflect staining of the transduced (Thy1.1 + ) B cell population, while the filled gray histograms reflect staining of the untransduced (Thy1.1 − ) population. The top panels depict data from M2-transduced cultures; bottom panels depict data from M2.Stop-transduced cells. Data is representative of three samples per time-point with at least three independent experiments per stain. (B and C) ELISA quantitation of the levels of IgM and <t>IgG</t> in supernatants of M2 and M2.Stop transduced B cell cultures. Three samples were analyzed per time point, and the data shown is representative of three independent experiments. Significance of differences in IgG secretion was determined by two-tailed, unpaired Student's T test with a confidence level of 95%. * p = 0.0182, ** p = 0.0352, *** p = 0.0118.
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    Bethyl igg2a
    In vivo effect of Fn14-Fc on Ig production in SLE mice. Sanroque mice were injected intraperitoneally with Fn14-Fc (100 μg/mouse) or control-Fc (100 μg/mouse) (n = 5/group) for 3 weeks. Mice were sacrificed on day 21 after the first injection. The serum total IgG, IgG1 and <t>IgG2a</t> levels were determined by ELISA. Data are expressed as means ± SDs. * P
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    Thermo Fisher igg4 quantification
    Correlation between specific IgA or <t>IgG</t> to Dp (A) or Bt (B) and Der p 1 (A) or Blo t 5 (B) allergens. Correlation coefficients were determined using Spearman’s tests; ** p
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    R&D Systems mouse igg1 isotype control
    Altered spatiotemporal level of CCL20 in the spinal cord after SCI. a The temporal profile (from 0 h to 28 days post-injury) of CCL20 mRNA expression in the spinal cord, as determined by qRT-PCR, shows that SCI leads to increased CCL20 mRNA level in the spinal cord, especially during the early period of SCI. b Mouse <t>IgG</t> levels of rat serum (from 0 h to 28 days post-injury), as determined by ELISA, are significantly increased in CCL20 mAb group and isotype control group from 6 h to 28 days post-SCI. CCL20 immunostaining at 1 day post-injury in the sham group (c) , SCI group (d) , negative control of the sham group (e) , and negative control of the SCI group (f) indicates that CCL20 is mainly localized in the cytoplasm of gray matter neurons and glial cells. The brown staining represents positive CCL20 expression. Black arrow indicates the CCL20 positive neuron and glial cell. Scale bar = 100 μm. + P
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    BioLegend igg3
    Immunoglobulin isotypes in serum from SAMR1 and SAMP8 mice determined by ELISA. ( a ) Serum IgM and IgG1 from 2- and 10-month-old SAMR1 and SAMP8 mice. ( b ) IgG2a, IgG2b and <t>IgG3</t> isotypes were quantified on sera from 10-month-old SAMP8 and SAMR1 mice. Data are measurements of individual mice. Means are indicated by the horizontal lines. Comparisons were made with the unpaired two-tailed Student’s t -test: ** P
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    Image Search Results


    Spontaneous hyper–Th1 phenotype in Foxp3 Cre xT-bet fl/fl mice. (A) Representative FACS plots of splenic T-bet + Foxp3 + Treg1 cells from naïve mice of the indicated genotypes. (B) Quantification of T-bet + Foxp3 + Treg1 cells in spleens and blood of naïve mice. (C and D) Quantification of (C) splenic and (D) blood T-bet + or IFN γ + Foxp3 − Th1 cells as indicated. (E) Quantification of chemokine receptor CXCR3 and CCR6 expression on Foxp3 − T helper cells in the blood of naïve mice. (F and G) Levels of total IgG, IgG1, and IgG3 subclasses in the serum of naïve mice. ELISA data are shown as OD at 450 nm in serial dilutions as indicated. Numbers in FACS plots represent percentages of Foxp3 + cells; n =6 versus six animals for all analyses. Circles in B–E represent individual animals, and horizontal lines represent mean values. Error bars represent SEM. * P

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: T-Bet Enhances Regulatory T Cell Fitness and Directs Control of Th1 Responses in Crescentic GN

    doi: 10.1681/ASN.2015070820

    Figure Lengend Snippet: Spontaneous hyper–Th1 phenotype in Foxp3 Cre xT-bet fl/fl mice. (A) Representative FACS plots of splenic T-bet + Foxp3 + Treg1 cells from naïve mice of the indicated genotypes. (B) Quantification of T-bet + Foxp3 + Treg1 cells in spleens and blood of naïve mice. (C and D) Quantification of (C) splenic and (D) blood T-bet + or IFN γ + Foxp3 − Th1 cells as indicated. (E) Quantification of chemokine receptor CXCR3 and CCR6 expression on Foxp3 − T helper cells in the blood of naïve mice. (F and G) Levels of total IgG, IgG1, and IgG3 subclasses in the serum of naïve mice. ELISA data are shown as OD at 450 nm in serial dilutions as indicated. Numbers in FACS plots represent percentages of Foxp3 + cells; n =6 versus six animals for all analyses. Circles in B–E represent individual animals, and horizontal lines represent mean values. Error bars represent SEM. * P

    Article Snippet: ELISA plates were incubated with mouse serum, and the following secondary antibodies were used for detection: total IgG (Southern Biotech, Birmingham, AL), IgG1 (Southern Biotech), IgG2b (Invitrogen, Carlsbad, CA), IgG2c (Bethyl, Montgomery, TX), and IgG3 (Jackson ImmunoResearch Laboratories).

    Techniques: Mouse Assay, FACS, Expressing, Enzyme-linked Immunosorbent Assay

    Intact Treg cell–suppressive function in the absence of T-bet activation. (A–C) In vitro suppression assays were performed by coculturing wild–type CD4 + Teffs with Treg cells from Foxp3 Cre xT-bet fl/fl mice or Foxp3 Cre controls at the indicated ratios ( n =3 per group). Cytokine levels of (A) IL-2, (B) IL-10, and (C) IFN γ were analyzed in coculture supernatants as indicated. Dotted lines represent Teffs alone without Treg cells ( n =3). (D) Quantification of IL-10, IL35/EBI-3, and TGF- β 1 mRNA by quantitative RT-PCR from the indicated spleen cell populations FACS sorted at day 12 after immunization with sheep IgG. (E) Representative FACS plots and (F) quantification of KI67 + proliferating CD4 + Foxp3 − Teffs from spleens of immunized Rag1 −/− recipients harboring Treg cells from the indicated mouse strains. (G) FACS analysis of splenic CD4 + Teff activation from immunized Rag1 −/− recipients harboring Treg cells of the indicated genotype; n =6 versus six mice were analyzed in E–G. Circles represent individual animals, and horizontal lines represent mean values. Error bars represent SEM. KO, Foxp3 Cre xT-bet fl/fl mice; WT, Foxp3 Cre controls.

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: T-Bet Enhances Regulatory T Cell Fitness and Directs Control of Th1 Responses in Crescentic GN

    doi: 10.1681/ASN.2015070820

    Figure Lengend Snippet: Intact Treg cell–suppressive function in the absence of T-bet activation. (A–C) In vitro suppression assays were performed by coculturing wild–type CD4 + Teffs with Treg cells from Foxp3 Cre xT-bet fl/fl mice or Foxp3 Cre controls at the indicated ratios ( n =3 per group). Cytokine levels of (A) IL-2, (B) IL-10, and (C) IFN γ were analyzed in coculture supernatants as indicated. Dotted lines represent Teffs alone without Treg cells ( n =3). (D) Quantification of IL-10, IL35/EBI-3, and TGF- β 1 mRNA by quantitative RT-PCR from the indicated spleen cell populations FACS sorted at day 12 after immunization with sheep IgG. (E) Representative FACS plots and (F) quantification of KI67 + proliferating CD4 + Foxp3 − Teffs from spleens of immunized Rag1 −/− recipients harboring Treg cells from the indicated mouse strains. (G) FACS analysis of splenic CD4 + Teff activation from immunized Rag1 −/− recipients harboring Treg cells of the indicated genotype; n =6 versus six mice were analyzed in E–G. Circles represent individual animals, and horizontal lines represent mean values. Error bars represent SEM. KO, Foxp3 Cre xT-bet fl/fl mice; WT, Foxp3 Cre controls.

    Article Snippet: ELISA plates were incubated with mouse serum, and the following secondary antibodies were used for detection: total IgG (Southern Biotech, Birmingham, AL), IgG1 (Southern Biotech), IgG2b (Invitrogen, Carlsbad, CA), IgG2c (Bethyl, Montgomery, TX), and IgG3 (Jackson ImmunoResearch Laboratories).

    Techniques: Activation Assay, In Vitro, Mouse Assay, Quantitative RT-PCR, FACS

    Skewing of renal and systemic immunity toward Th1 in Treg1 cell–deficient mice. (A) Quantification of spleen cell numbers. (B and C) Quantification of (B) splenic and (C) renal Foxp3 − T helper cells expressing IFN γ and IL-17. (D) Representative FACS plots of renal T helper cells expressing the indicated cytokines. (E) Expression of the indicated chemokine receptors on renal Foxp3 − T helper cells. Analyses in A–E were performed at day 15 after NTN induction. (F) Serum levels of IgG1 and IgG3 anti–sheep globulin antibodies at day 12 after sheep IgG immunization. ELISA data are shown as OD at 450 nm in serial dilutions as indicated. Numbers in FACS plots represent percentages of CD4 + cells. Nine Foxp3 Cre versus 11 Foxp3 Cre xT-bet fl/fl mice were analyzed in A–E, and five Foxp3 Cre versus five Foxp3 Cre xT-bet fl/fl mice were analyzed in F. Circles in B, C, and E represent individual animals, and horizontal lines represent mean values. Error bars represent SEM. * P

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: T-Bet Enhances Regulatory T Cell Fitness and Directs Control of Th1 Responses in Crescentic GN

    doi: 10.1681/ASN.2015070820

    Figure Lengend Snippet: Skewing of renal and systemic immunity toward Th1 in Treg1 cell–deficient mice. (A) Quantification of spleen cell numbers. (B and C) Quantification of (B) splenic and (C) renal Foxp3 − T helper cells expressing IFN γ and IL-17. (D) Representative FACS plots of renal T helper cells expressing the indicated cytokines. (E) Expression of the indicated chemokine receptors on renal Foxp3 − T helper cells. Analyses in A–E were performed at day 15 after NTN induction. (F) Serum levels of IgG1 and IgG3 anti–sheep globulin antibodies at day 12 after sheep IgG immunization. ELISA data are shown as OD at 450 nm in serial dilutions as indicated. Numbers in FACS plots represent percentages of CD4 + cells. Nine Foxp3 Cre versus 11 Foxp3 Cre xT-bet fl/fl mice were analyzed in A–E, and five Foxp3 Cre versus five Foxp3 Cre xT-bet fl/fl mice were analyzed in F. Circles in B, C, and E represent individual animals, and horizontal lines represent mean values. Error bars represent SEM. * P

    Article Snippet: ELISA plates were incubated with mouse serum, and the following secondary antibodies were used for detection: total IgG (Southern Biotech, Birmingham, AL), IgG1 (Southern Biotech), IgG2b (Invitrogen, Carlsbad, CA), IgG2c (Bethyl, Montgomery, TX), and IgG3 (Jackson ImmunoResearch Laboratories).

    Techniques: Mouse Assay, Expressing, FACS, Enzyme-linked Immunosorbent Assay

    1B9B9 suppressed the bioactivity of FGF-2. ( A ) Recombinant FGF-2 was incubated with 1B9B9 or IgG for 1 h and then used to treat FBHEs for 30 min. Phosphorylation of FGFR was analyzed by immunoblotting. Data are representative of three independent experiments that yielded similar results. ( B ) FBHE (2×10 4 ) cells were cultured in 2% FBS-DMEM/RPMI 1640 (1:1) for 24 h. The cells were then cultured with IgG (10 μg/mL) as a negative control or 1B9B9 (10 μg/mL, 5 μg/mL, 1 μg/mL) in the presence of FGF-2 (2 ng/mL). After 3 days, the cell number was assessed by Trypan blue staining. * p

    Journal: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

    Article Title: Establishment of Neutralizing Rat Monoclonal Antibodies for Fibroblast Growth Factor-2

    doi: 10.1089/mab.2013.0085

    Figure Lengend Snippet: 1B9B9 suppressed the bioactivity of FGF-2. ( A ) Recombinant FGF-2 was incubated with 1B9B9 or IgG for 1 h and then used to treat FBHEs for 30 min. Phosphorylation of FGFR was analyzed by immunoblotting. Data are representative of three independent experiments that yielded similar results. ( B ) FBHE (2×10 4 ) cells were cultured in 2% FBS-DMEM/RPMI 1640 (1:1) for 24 h. The cells were then cultured with IgG (10 μg/mL) as a negative control or 1B9B9 (10 μg/mL, 5 μg/mL, 1 μg/mL) in the presence of FGF-2 (2 ng/mL). After 3 days, the cell number was assessed by Trypan blue staining. * p

    Article Snippet: IgG from rat serum and recombinant FGF-2 were obtained from Sigma-Aldrich and Invitrogen (Carlsbad, CA), respectively.

    Techniques: Recombinant, Incubation, Cell Culture, Negative Control, Staining

    1B9B9 recognized both recombinant and endogenous FGF-2. ( A ) GST-FGF-2 (0.5 μg) or GST (0.5 μg) was immunoprecipitated with 1B9B9 followed by immunoblotting with an anti-FGF2 polyclonal antibody (upper panel) and GST antibody (lower panel). ( B ) HUVECs were treated with GST-FGF-2 (10 ng/mL) for 1 h before lysis. Cell lysates were immunoprecipitated with rat IgG or 1B9B9 followed by immunoblotting with an anti-FGF-2 polyclonal antibody. ( C ) HUVECs were immunostained with 1B9B9 and phalloidin. A representative microscopic image of the cells is shown. Scale bar, 40 μm.

    Journal: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

    Article Title: Establishment of Neutralizing Rat Monoclonal Antibodies for Fibroblast Growth Factor-2

    doi: 10.1089/mab.2013.0085

    Figure Lengend Snippet: 1B9B9 recognized both recombinant and endogenous FGF-2. ( A ) GST-FGF-2 (0.5 μg) or GST (0.5 μg) was immunoprecipitated with 1B9B9 followed by immunoblotting with an anti-FGF2 polyclonal antibody (upper panel) and GST antibody (lower panel). ( B ) HUVECs were treated with GST-FGF-2 (10 ng/mL) for 1 h before lysis. Cell lysates were immunoprecipitated with rat IgG or 1B9B9 followed by immunoblotting with an anti-FGF-2 polyclonal antibody. ( C ) HUVECs were immunostained with 1B9B9 and phalloidin. A representative microscopic image of the cells is shown. Scale bar, 40 μm.

    Article Snippet: IgG from rat serum and recombinant FGF-2 were obtained from Sigma-Aldrich and Invitrogen (Carlsbad, CA), respectively.

    Techniques: Recombinant, Immunoprecipitation, Lysis

    1B9B9 suppressed the activity of ERK1,2 and Akt in HUVECs. ( A ) (upper) Serum-starved HUVECs were stimulated for 30 min with FGF-2, which had been incubated with 1B9B9 (1 μg/mL, 5 μg/mL, 10 μg/mL) or IgG (10 μg/mL) for 1 h. Thereafter lysates were immunoblotted with pAkt or pERK1,2 antibodies. (lower) Densitometric quantitation of each protein using β-actin for normalization. * p

    Journal: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

    Article Title: Establishment of Neutralizing Rat Monoclonal Antibodies for Fibroblast Growth Factor-2

    doi: 10.1089/mab.2013.0085

    Figure Lengend Snippet: 1B9B9 suppressed the activity of ERK1,2 and Akt in HUVECs. ( A ) (upper) Serum-starved HUVECs were stimulated for 30 min with FGF-2, which had been incubated with 1B9B9 (1 μg/mL, 5 μg/mL, 10 μg/mL) or IgG (10 μg/mL) for 1 h. Thereafter lysates were immunoblotted with pAkt or pERK1,2 antibodies. (lower) Densitometric quantitation of each protein using β-actin for normalization. * p

    Article Snippet: IgG from rat serum and recombinant FGF-2 were obtained from Sigma-Aldrich and Invitrogen (Carlsbad, CA), respectively.

    Techniques: Activity Assay, Incubation, Quantitation Assay

    VSV-IFN therapeutic efficacy in mice bearing orthotopic myeloma 5TGM1 cells were implanted IV in syngeneic C57Bl/KalWrij mice. ( A ) Mean serum IgG2b concentration was quantified at 7 day intervals (n=26 mice). Mean serum IgG2b was measured by ELISA and compared using a t-test indicating a significant change in serum IgG2b from day 14 to day 21 post 5TGM1 implantation (*P=0.0019) ( B ) Survival response in myeloma bearing mice following a single IV dose of PBS, or 1×10 8 VSV-mIFNβ or VSV-hIFNβ. Survival was compared by log-rank analysis. VSV-mIFNβ and VSV-hIFNβ treatment significantly prolonged survival compared to PBS (P=0.0008*** and P=0.017* respectively), and survival of mice treated with VSV-mIFNβ is significantly prolonged compared to VSV-hIFNβ treated mice (P=0.021*) ( C ) Myeloma burden in response to IV treatment with PBS, VSV-mIFNβ or VSV-hIFNβ measured by serum IgG2b ELISA.

    Journal: Cancer Gene Therapy

    Article Title: Potent systemic therapy of Multiple Myeloma utilizing Oncolytic Vesicular stomatitis virus coding for Interferon-beta

    doi: 10.1038/cgt.2012.14

    Figure Lengend Snippet: VSV-IFN therapeutic efficacy in mice bearing orthotopic myeloma 5TGM1 cells were implanted IV in syngeneic C57Bl/KalWrij mice. ( A ) Mean serum IgG2b concentration was quantified at 7 day intervals (n=26 mice). Mean serum IgG2b was measured by ELISA and compared using a t-test indicating a significant change in serum IgG2b from day 14 to day 21 post 5TGM1 implantation (*P=0.0019) ( B ) Survival response in myeloma bearing mice following a single IV dose of PBS, or 1×10 8 VSV-mIFNβ or VSV-hIFNβ. Survival was compared by log-rank analysis. VSV-mIFNβ and VSV-hIFNβ treatment significantly prolonged survival compared to PBS (P=0.0008*** and P=0.017* respectively), and survival of mice treated with VSV-mIFNβ is significantly prolonged compared to VSV-hIFNβ treated mice (P=0.021*) ( C ) Myeloma burden in response to IV treatment with PBS, VSV-mIFNβ or VSV-hIFNβ measured by serum IgG2b ELISA.

    Article Snippet: Myeloma burden was monitored by measurement of serum IgG2b using Mouse IgG2b ELISA Quantitation kit (Bethyl laboratories, E90–109).

    Techniques: Mouse Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Fraxinellone inhibits B cell maturation. ( A , B ) CD19 + B cells were activated by lipopolysaccharide. After treatment with or without fraxinellone (40 μM), the relative expression levels of AID and Blimp-1 in CD19 + B cells were determined with RT-PCR. ( C ) The level of immunoglobulin G in the culture supernatant was measured using ELISA in the presence of fraxinellone at doses of 0–40 μM. In all culture conditions, CD19 + B cells were stimulated by lipopolysaccharide. Data represent the mean of three independent experiments ± SEM. Nil, no stimulation; LPS, stimulation with lipopolysaccharide; Frx, fraxinellone; AID, activation-induced cytidine deaminase; Blimp-1, B lymphocyte-induced maturation protein-1; IgG, immunoglobulin G; * p

    Journal: International Journal of Molecular Sciences

    Article Title: Fraxinellone Attenuates Rheumatoid Inflammation in Mice

    doi: 10.3390/ijms19030829

    Figure Lengend Snippet: Fraxinellone inhibits B cell maturation. ( A , B ) CD19 + B cells were activated by lipopolysaccharide. After treatment with or without fraxinellone (40 μM), the relative expression levels of AID and Blimp-1 in CD19 + B cells were determined with RT-PCR. ( C ) The level of immunoglobulin G in the culture supernatant was measured using ELISA in the presence of fraxinellone at doses of 0–40 μM. In all culture conditions, CD19 + B cells were stimulated by lipopolysaccharide. Data represent the mean of three independent experiments ± SEM. Nil, no stimulation; LPS, stimulation with lipopolysaccharide; Frx, fraxinellone; AID, activation-induced cytidine deaminase; Blimp-1, B lymphocyte-induced maturation protein-1; IgG, immunoglobulin G; * p

    Article Snippet: The concentrations of total IgG1 and IgG2a were measured using mouse IgG1 and IgG2a enzyme-linked immunosorbent assay (ELISA) Quantitation Kits (Bethyl Laboratories, Montgomery, TX, USA), respectively.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Activation Assay

    Fraxinellone attenuates inflammatory arthritis in mice. ( A ) Mice with collagen-induced arthritis (CIA) were treated with fraxinellone (7.5 mg/kg) or the vehicle control ( n = 10 in each group). The arthritic score was defined as the sum of the scores of three paws (excluding the boosted paw); scores ranged from 0 to 12. ( B ) Tarsal joint tissues of CIA mice treated with fraxinellone or the control vehicle were stained with haematoxylin eosin. The scale bars at the bottom right of the images indicate 200 μm. ( C ) Serum levels of IgG1 and IgG2a of CIA mice were determined using an enzyme-linked immunosorbent assay (ELISA). ( D ) Splenocytes of CIA mice were stimulated with or without anti-CD3 antibodies. The level of TNF-α and IFN-γ in culture media were measured by ELISA. Data are expressed as mean ± standard error of the mean (SEM). IgG1, immunoglobulin G1; IgG2a, immunoglobulin G2a; Frx, fraxinellone; TNF-α, tumor necrosis factor-α; IFN-γ, interferon-γ; Nil, no treatment; aCD3, stimulation with anti-CD3 antibodies; NS, not significant; ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Fraxinellone Attenuates Rheumatoid Inflammation in Mice

    doi: 10.3390/ijms19030829

    Figure Lengend Snippet: Fraxinellone attenuates inflammatory arthritis in mice. ( A ) Mice with collagen-induced arthritis (CIA) were treated with fraxinellone (7.5 mg/kg) or the vehicle control ( n = 10 in each group). The arthritic score was defined as the sum of the scores of three paws (excluding the boosted paw); scores ranged from 0 to 12. ( B ) Tarsal joint tissues of CIA mice treated with fraxinellone or the control vehicle were stained with haematoxylin eosin. The scale bars at the bottom right of the images indicate 200 μm. ( C ) Serum levels of IgG1 and IgG2a of CIA mice were determined using an enzyme-linked immunosorbent assay (ELISA). ( D ) Splenocytes of CIA mice were stimulated with or without anti-CD3 antibodies. The level of TNF-α and IFN-γ in culture media were measured by ELISA. Data are expressed as mean ± standard error of the mean (SEM). IgG1, immunoglobulin G1; IgG2a, immunoglobulin G2a; Frx, fraxinellone; TNF-α, tumor necrosis factor-α; IFN-γ, interferon-γ; Nil, no treatment; aCD3, stimulation with anti-CD3 antibodies; NS, not significant; ** p

    Article Snippet: The concentrations of total IgG1 and IgG2a were measured using mouse IgG1 and IgG2a enzyme-linked immunosorbent assay (ELISA) Quantitation Kits (Bethyl Laboratories, Montgomery, TX, USA), respectively.

    Techniques: Mouse Assay, Staining, Enzyme-linked Immunosorbent Assay

    In vivo effect of Fn14-Fc on Ig production in SLE mice. Sanroque mice were injected intraperitoneally with Fn14-Fc (100 μg/mouse) or control-Fc (100 μg/mouse) (n = 5/group) for 3 weeks. Mice were sacrificed on day 21 after the first injection. The serum total IgG, IgG1 and IgG2a levels were determined by ELISA. Data are expressed as means ± SDs. * P

    Journal: Journal of Translational Medicine

    Article Title: Fn14-Fc suppresses germinal center formation and pathogenic B cells in a lupus mouse model via inhibition of the TWEAK/Fn14 Pathway

    doi: 10.1186/s12967-016-0846-4

    Figure Lengend Snippet: In vivo effect of Fn14-Fc on Ig production in SLE mice. Sanroque mice were injected intraperitoneally with Fn14-Fc (100 μg/mouse) or control-Fc (100 μg/mouse) (n = 5/group) for 3 weeks. Mice were sacrificed on day 21 after the first injection. The serum total IgG, IgG1 and IgG2a levels were determined by ELISA. Data are expressed as means ± SDs. * P

    Article Snippet: Total IgG, IgG1, IgG2a, and anti-double-stranded (ds) DNA Abs were measured using a mouse total IgG, IgG1 and IgG2a enzyme-linked immunosorbent assay (ELISA) quantitation kit (Bethyl Laboratories, Montgomery, TX, USA).

    Techniques: In Vivo, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay

    Isotyping and IgG subclass examination. ( A ) Hybridoma culture supernatants were tested by ELISA to determine the isotypes of the HuMAbs. Each ELISA included three positive controls (Ctrl; Ctrl IgA, Ctrl IgG, and Ctrl IgM). ( B ) IgG subclasses were determined

    Journal: The American Journal of Tropical Medicine and Hygiene

    Article Title: A Highly Conserved Region Between Amino Acids 221 and 266 of Dengue Virus Non-Structural Protein 1 Is a Major Epitope Region in Infected Patients

    doi: 10.4269/ajtmh.13-0624

    Figure Lengend Snippet: Isotyping and IgG subclass examination. ( A ) Hybridoma culture supernatants were tested by ELISA to determine the isotypes of the HuMAbs. Each ELISA included three positive controls (Ctrl; Ctrl IgA, Ctrl IgG, and Ctrl IgM). ( B ) IgG subclasses were determined

    Article Snippet: To determine the subclasses of the MAbs, hybridomas culture supernatants were tested using a Human IgG Enzyme-Linked Immunosorbent Assay (ELISA) Quantitation Set (E80-104; Bethyl Laboratories, Inc., Montgomery, TX) in accordance with the manufacturer's instructions.

    Techniques: Enzyme-linked Immunosorbent Assay

    Effects of AMPCol and AMP on the levels of the relative weight of spleen ( A ), IgM ( B ), IgA ( C ) and IgG (D) from the serum were measured using ELISA. The data represent mean ± SD, *p

    Journal: Scientific Reports

    Article Title: Biological and immunotoxicity evaluation of antimicrobial peptide-loaded coatings using a layer-by-layer process on titanium

    doi: 10.1038/srep16336

    Figure Lengend Snippet: Effects of AMPCol and AMP on the levels of the relative weight of spleen ( A ), IgM ( B ), IgA ( C ) and IgG (D) from the serum were measured using ELISA. The data represent mean ± SD, *p

    Article Snippet: Immunoglobulin levels were analyzed using a mouse IgM, IgA and IgG enzyme-linked immunsorbent assay (ELISA) Quantitation Kit (IgM and IgG from Abcam, Cambridge, UK; IgA from Sigma, USA) following the manufacturer’s instructions.

    Techniques: Enzyme-linked Immunosorbent Assay

    CD154-induced B cell class switching and differentiation into PNA hi B cells and plasma cells are inhibited by CpG in the absence of BCR crosslinking (flow cytometry analysis). ( a ) CSR to IgG1 (top panels) and expression of PNA-binding lectins (bottom panels) in B cells stimulated with CD154 (3 unit/ml) plus IL-4 in the absence or presence of CpG (0.3 μM) for 4 d. ( b ) CSR to IgG1 (top panels) and expression of PNA-binding lectins (bottom panels) in B cells stimulated with CD154 (3 unit/ml) plus IFN-γ in the absence or presence of CpG for 4 d. ( c ) CSR to IgG1, IgE or IgG2a in B cells stimulated with CD154 plus IL-4 or IFN-γ in the absence or presence of CpG for 4 d. Data are mean and s.d. from three independent experiments ( p values calculated by paired student t test). ( d ) Formation of B220 lo CD138 hi plasma cells in cultures of B cells stimulated with LPS (1 μg/ml) plus IL-4 or IFN-γ in the absence or presence of CpG (0.3 μM) for 4 d. Data are mean and s.d. from three independent experiments ( p values calculated by paired student t test).

    Journal: Autoimmunity

    Article Title: B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: modulation by BCR and CD40, and relevance to T-independent antibody responses

    doi: 10.3109/08916934.2014.993027

    Figure Lengend Snippet: CD154-induced B cell class switching and differentiation into PNA hi B cells and plasma cells are inhibited by CpG in the absence of BCR crosslinking (flow cytometry analysis). ( a ) CSR to IgG1 (top panels) and expression of PNA-binding lectins (bottom panels) in B cells stimulated with CD154 (3 unit/ml) plus IL-4 in the absence or presence of CpG (0.3 μM) for 4 d. ( b ) CSR to IgG1 (top panels) and expression of PNA-binding lectins (bottom panels) in B cells stimulated with CD154 (3 unit/ml) plus IFN-γ in the absence or presence of CpG for 4 d. ( c ) CSR to IgG1, IgE or IgG2a in B cells stimulated with CD154 plus IL-4 or IFN-γ in the absence or presence of CpG for 4 d. Data are mean and s.d. from three independent experiments ( p values calculated by paired student t test). ( d ) Formation of B220 lo CD138 hi plasma cells in cultures of B cells stimulated with LPS (1 μg/ml) plus IL-4 or IFN-γ in the absence or presence of CpG (0.3 μM) for 4 d. Data are mean and s.d. from three independent experiments ( p values calculated by paired student t test).

    Article Snippet: Recombinant IL-4 (3 ng/ml) was added for CSR to IgG1 and IgE, IFN-γ (50 ng/ml) for CSR to IgG2a, and TGF-β (2 ng/ml) for CSR to IgG2b (all from R & D Systems).

    Techniques: Flow Cytometry, Cytometry, Expressing, Binding Assay

    LPS induces B cells to undergo CSR and plasma cell differentiation in vitro in a dose- and time-dependent manner (flow cytometry analysis). ( a, b ) CSR to IgG1 in B cells stimulated with LPS at the indicated concentrations plus IL-4 for 4 d ( a , top panels), and formation of B220 lo CD138 hi plasma cells in the same cell culture ( a , bottom panels). Mean and s.d. of data from four independent experiments were depicted in ( b ). p values calculated by paired student t test. ( c ) CSR to IgG1 in B cells stimulated with LPS plus IL-4, starting at d 0, and harvested at d 2, d 3, d 4, d 5 and d 7, as indicated. ( d ) Formation of B220 lo CD138 hi plasma cells (top panels) as well as the proportion of IgG1 + (middle panels) and IgM + (bottom panels) within those plasma cells in cultures of B cells stimulated with LPS plus IL-4, starting at d 0, and harvested at d 2, d 3, d 4, d 5 and d 7, as indicated. ( e ) CSR to IgG1 in B cells stimulated with LPS plus IL-4 for the period indicated, pelleted to remove LPS, and then cultured in RPMI-FBS only until being harvested at d 4. Data in ( a , c , d , e ) are representative of three independent experiments.

    Journal: Autoimmunity

    Article Title: B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: modulation by BCR and CD40, and relevance to T-independent antibody responses

    doi: 10.3109/08916934.2014.993027

    Figure Lengend Snippet: LPS induces B cells to undergo CSR and plasma cell differentiation in vitro in a dose- and time-dependent manner (flow cytometry analysis). ( a, b ) CSR to IgG1 in B cells stimulated with LPS at the indicated concentrations plus IL-4 for 4 d ( a , top panels), and formation of B220 lo CD138 hi plasma cells in the same cell culture ( a , bottom panels). Mean and s.d. of data from four independent experiments were depicted in ( b ). p values calculated by paired student t test. ( c ) CSR to IgG1 in B cells stimulated with LPS plus IL-4, starting at d 0, and harvested at d 2, d 3, d 4, d 5 and d 7, as indicated. ( d ) Formation of B220 lo CD138 hi plasma cells (top panels) as well as the proportion of IgG1 + (middle panels) and IgM + (bottom panels) within those plasma cells in cultures of B cells stimulated with LPS plus IL-4, starting at d 0, and harvested at d 2, d 3, d 4, d 5 and d 7, as indicated. ( e ) CSR to IgG1 in B cells stimulated with LPS plus IL-4 for the period indicated, pelleted to remove LPS, and then cultured in RPMI-FBS only until being harvested at d 4. Data in ( a , c , d , e ) are representative of three independent experiments.

    Article Snippet: Recombinant IL-4 (3 ng/ml) was added for CSR to IgG1 and IgE, IFN-γ (50 ng/ml) for CSR to IgG2a, and TGF-β (2 ng/ml) for CSR to IgG2b (all from R & D Systems).

    Techniques: Cell Differentiation, In Vitro, Flow Cytometry, Cytometry, Cell Culture

    LPS-induced plasma cell differentiation and secretion of class-switched Ig are inhibited by CpG in the absence of BCR crosslinking. ( a ) Formation of B220 lo CD138 hi plasma cells in cultures of B cells stimulated with LPS (1 μg/ml) plus IL-4 in the absence or presence of CpG (0.3 μM) for 4 d (flow cytometry analysis). ( b ) Formation of B220 lo CD138 hi plasma cells in cultures of B cells stimulated with LPS plus IL-4, nil or IL-4, IL-5 and TGF-β, as indicated, in the absence or presence of CpG for 4 d (flow cytometry analysis). ( c ) ELISA of IgG1, IgG3 or IgA secreted into the supernatant of B cell cultures after stimulation with LPS plus IL-4, nil or IL-4, IL-5 and TGF-β, as indicated, in the absence or presence of CpG for 4 d. Data are mean and s.d. from three independent experiments ( p values calculated by paired student t test).

    Journal: Autoimmunity

    Article Title: B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: modulation by BCR and CD40, and relevance to T-independent antibody responses

    doi: 10.3109/08916934.2014.993027

    Figure Lengend Snippet: LPS-induced plasma cell differentiation and secretion of class-switched Ig are inhibited by CpG in the absence of BCR crosslinking. ( a ) Formation of B220 lo CD138 hi plasma cells in cultures of B cells stimulated with LPS (1 μg/ml) plus IL-4 in the absence or presence of CpG (0.3 μM) for 4 d (flow cytometry analysis). ( b ) Formation of B220 lo CD138 hi plasma cells in cultures of B cells stimulated with LPS plus IL-4, nil or IL-4, IL-5 and TGF-β, as indicated, in the absence or presence of CpG for 4 d (flow cytometry analysis). ( c ) ELISA of IgG1, IgG3 or IgA secreted into the supernatant of B cell cultures after stimulation with LPS plus IL-4, nil or IL-4, IL-5 and TGF-β, as indicated, in the absence or presence of CpG for 4 d. Data are mean and s.d. from three independent experiments ( p values calculated by paired student t test).

    Article Snippet: Recombinant IL-4 (3 ng/ml) was added for CSR to IgG1 and IgE, IFN-γ (50 ng/ml) for CSR to IgG2a, and TGF-β (2 ng/ml) for CSR to IgG2b (all from R & D Systems).

    Techniques: Cell Differentiation, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    CpG-mediated inhibition of LPS- or CD154-induced CSR requires co-stimulation of B cells by CpG with LPS or CD154. ( a ) Levels of transcripts, as indicated, in B cells stimulated with CpG plus IL-4 for 0, 4 or 24 h (real-time qRT-PCR analysis). ( b, c ) CSR to IgG1 in CFSE-labeled B cells stimulated with LPS (3 μg/ml, b ) or CD154 (3 unit/ml, c ) plus CpG (0.3 mM) for the period as indicated plus IL4 (flow cytometry analysis). Data are representative of three independent experiments.

    Journal: Autoimmunity

    Article Title: B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: modulation by BCR and CD40, and relevance to T-independent antibody responses

    doi: 10.3109/08916934.2014.993027

    Figure Lengend Snippet: CpG-mediated inhibition of LPS- or CD154-induced CSR requires co-stimulation of B cells by CpG with LPS or CD154. ( a ) Levels of transcripts, as indicated, in B cells stimulated with CpG plus IL-4 for 0, 4 or 24 h (real-time qRT-PCR analysis). ( b, c ) CSR to IgG1 in CFSE-labeled B cells stimulated with LPS (3 μg/ml, b ) or CD154 (3 unit/ml, c ) plus CpG (0.3 mM) for the period as indicated plus IL4 (flow cytometry analysis). Data are representative of three independent experiments.

    Article Snippet: Recombinant IL-4 (3 ng/ml) was added for CSR to IgG1 and IgE, IFN-γ (50 ng/ml) for CSR to IgG2a, and TGF-β (2 ng/ml) for CSR to IgG2b (all from R & D Systems).

    Techniques: Inhibition, Quantitative RT-PCR, Labeling, Flow Cytometry, Cytometry

    LPS-induced CSR is inhibited by CpG and R-848 in the absence of BCR crosslinking (flow cytometry analysis). ( a ) The proportion of switched IgG1 + IgM − B cells and IgM + IgG1 − unswitched B cells in B cells after stimulation with LPS (3 μg/ml) plus IL-4 in the presence of nil, Pam 3 CSK 4 , R-848, CpG or GpC for 4 d. ( b ) CSR to IgG1 in CFSE-labeled B cells stimulated with LPS (3 μg/ml) plus IL-4 in the absence or presence of GpC (0.3 μM) or CpG (0.3 μM; left panels) for 4 d and depiction of the proportion of switched IgG1 + cells in B cells that had completed each cell division (right panels). ( c ) CSR to different IgG isotypes or IgA in B cells stimulated with LPS (3 μg/ml) plus appropriate cytokines (as indicated) in the absence or presence of CpG (0.3 μM) (mean and s.d. of data from three independent experiments; p values calculated by paired student t test). Data in ( a ) and ( b ) are representative of three independent experiments.

    Journal: Autoimmunity

    Article Title: B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: modulation by BCR and CD40, and relevance to T-independent antibody responses

    doi: 10.3109/08916934.2014.993027

    Figure Lengend Snippet: LPS-induced CSR is inhibited by CpG and R-848 in the absence of BCR crosslinking (flow cytometry analysis). ( a ) The proportion of switched IgG1 + IgM − B cells and IgM + IgG1 − unswitched B cells in B cells after stimulation with LPS (3 μg/ml) plus IL-4 in the presence of nil, Pam 3 CSK 4 , R-848, CpG or GpC for 4 d. ( b ) CSR to IgG1 in CFSE-labeled B cells stimulated with LPS (3 μg/ml) plus IL-4 in the absence or presence of GpC (0.3 μM) or CpG (0.3 μM; left panels) for 4 d and depiction of the proportion of switched IgG1 + cells in B cells that had completed each cell division (right panels). ( c ) CSR to different IgG isotypes or IgA in B cells stimulated with LPS (3 μg/ml) plus appropriate cytokines (as indicated) in the absence or presence of CpG (0.3 μM) (mean and s.d. of data from three independent experiments; p values calculated by paired student t test). Data in ( a ) and ( b ) are representative of three independent experiments.

    Article Snippet: Recombinant IL-4 (3 ng/ml) was added for CSR to IgG1 and IgE, IFN-γ (50 ng/ml) for CSR to IgG2a, and TGF-β (2 ng/ml) for CSR to IgG2b (all from R & D Systems).

    Techniques: Flow Cytometry, Cytometry, Gel Permeation Chromatography, Labeling

    LPS-induced CSR to IgG1 is enhanced by Pam 3 CSK 4 and CpG in the presence of BCR signaling (flow cytometry analysis). ( a ) CSR to IgG1 in CFSE-labeled B cells stimulated with suboptimal doses of LPS, Pam 3 CSK 4 , R-848 and CpG (as indicated), alone or in pair-wise combination, all in the presence of anti–δ mAb/dex (100 ng/ml) and IL-4 for 4 d. ( b ) CSR to IgG1 in B cells stimulated with different concentrations of Pam 3 CSK 4 and LPS in the presence of anti–δ mAb/dex (100 ng/ml) and IL-4 for 4 d.

    Journal: Autoimmunity

    Article Title: B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: modulation by BCR and CD40, and relevance to T-independent antibody responses

    doi: 10.3109/08916934.2014.993027

    Figure Lengend Snippet: LPS-induced CSR to IgG1 is enhanced by Pam 3 CSK 4 and CpG in the presence of BCR signaling (flow cytometry analysis). ( a ) CSR to IgG1 in CFSE-labeled B cells stimulated with suboptimal doses of LPS, Pam 3 CSK 4 , R-848 and CpG (as indicated), alone or in pair-wise combination, all in the presence of anti–δ mAb/dex (100 ng/ml) and IL-4 for 4 d. ( b ) CSR to IgG1 in B cells stimulated with different concentrations of Pam 3 CSK 4 and LPS in the presence of anti–δ mAb/dex (100 ng/ml) and IL-4 for 4 d.

    Article Snippet: Recombinant IL-4 (3 ng/ml) was added for CSR to IgG1 and IgE, IFN-γ (50 ng/ml) for CSR to IgG2a, and TGF-β (2 ng/ml) for CSR to IgG2b (all from R & D Systems).

    Techniques: Flow Cytometry, Cytometry, Labeling

    NP-LPS induces long-term and secondary antibody response in vivo. Three C57BL/6 mice (8-12 week old) were injected intraperitoneally with NP-LPS (25 μg) in PBS at d 0 and re-injected with NP-LPS (10 μg) in PBS at d 90 (second arrow). Sera were collected prior to the first injection and at different days, and analyzed for titers of NP 3 -binding IgG2b and IgG3 (depicted as relative units). Titers at d 0 were beyond the detection limit and set as 1 for the graph purpose.

    Journal: Autoimmunity

    Article Title: B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: modulation by BCR and CD40, and relevance to T-independent antibody responses

    doi: 10.3109/08916934.2014.993027

    Figure Lengend Snippet: NP-LPS induces long-term and secondary antibody response in vivo. Three C57BL/6 mice (8-12 week old) were injected intraperitoneally with NP-LPS (25 μg) in PBS at d 0 and re-injected with NP-LPS (10 μg) in PBS at d 90 (second arrow). Sera were collected prior to the first injection and at different days, and analyzed for titers of NP 3 -binding IgG2b and IgG3 (depicted as relative units). Titers at d 0 were beyond the detection limit and set as 1 for the graph purpose.

    Article Snippet: Recombinant IL-4 (3 ng/ml) was added for CSR to IgG1 and IgE, IFN-γ (50 ng/ml) for CSR to IgG2a, and TGF-β (2 ng/ml) for CSR to IgG2b (all from R & D Systems).

    Techniques: In Vivo, Mouse Assay, Injection, Binding Assay

    Co-stimulation of B cells by LPS and CD154 hampers CSR and plasma cell differentiation (flow cytometry analysis). ( a, b ) CSR to IgG1 ( a ) and formation of B220 lo CD138 hi plasma cells ( b ) in B cell cultures after stimulation with LPS (3 μg/ml), CD154 (3 unit/ml) or both plus IL-4 for 4 d. Data are representative of three independent experiments.

    Journal: Autoimmunity

    Article Title: B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: modulation by BCR and CD40, and relevance to T-independent antibody responses

    doi: 10.3109/08916934.2014.993027

    Figure Lengend Snippet: Co-stimulation of B cells by LPS and CD154 hampers CSR and plasma cell differentiation (flow cytometry analysis). ( a, b ) CSR to IgG1 ( a ) and formation of B220 lo CD138 hi plasma cells ( b ) in B cell cultures after stimulation with LPS (3 μg/ml), CD154 (3 unit/ml) or both plus IL-4 for 4 d. Data are representative of three independent experiments.

    Article Snippet: Recombinant IL-4 (3 ng/ml) was added for CSR to IgG1 and IgE, IFN-γ (50 ng/ml) for CSR to IgG2a, and TGF-β (2 ng/ml) for CSR to IgG2b (all from R & D Systems).

    Techniques: Cell Differentiation, Flow Cytometry, Cytometry

    IL-21 drives partial plasma cell differentiation in BCL6-transduced B cells. A , PB CD19 + B cells expressing BCL6-IRES-YFP were cultured in the presence of IL-2 and IL-4 or with rmIL-21 alone. At the indicated time points expression of BLIMP1 and ACTB were measured by qRT-PCR. BLIMP1 expression was normalized to ACTB expression within each sample and the means ± SD of triplicate measurements in two different donors is plotted. B , BCL6-YFP expressing B cells were cultured with CD40L in the presence of IL-2 and IL-4 or with IL-21 and BLIMP1, BCL6, and tubulin protein expression were determined by immunoblotting 3 or 7 days after IL-21 treatment. C , CD19 + B cells were transduced with LZRS-BCL6-IRES-ΔNGFR and cultured for 10 days on CD40L in the presence of IL-2 and IL-4 or with IL-21 alone. Cells were stained with anti-CD19, anti-NGFR, anti-CD38, and anti-CD20 and analyzed by flow cytometry. The rightmost four plots show CD38/CD20 expression on CD19 + BCL6–ΔNGFR– and CD19 + BCL6–ΔNGFR + cells. D , CD38/CD20 expression on BCL6-IRES-YFP-transduced B cells after 21 days of culture with IL-2 and IL-4 or with IL-21 alone. E , Surface Igλ and Igκ BCR expression on cells cultured as in C . F , Expression of CD138, HLA-DR, CD86, CD27, CD25, and CD3 on BCL6 + B cells cultured as in C . Dotted histogram is iso-type control. G , CD19 + BCL6-overexpressing B cells maintained on CD40L with either IL-2 and IL-4 (○)or with IL-21 (•). Cumulative expansion was calculated based on absolute cell numbers over a 17-day period. Data shown are means ± SD of three independent experiments involving different donors and constructs (BCL6-IRES-ΔNGFR or BCL6-IRES-GFP). H , BCL6-IRES-ΔNGFR + cells were cultured for 6 days on CD40L-L cells in the presence of IL-2 and IL-4 or with IL-21. Cell numbers were counted and IgM and IgG in culture supernatants were measured by ELISA to obtain per cell values of IgM and IgG. Data are representative of three independent experiments. I , Total PB CD19 + BCL6-GFP + cells were cultured with CD40L in the presence of IL-2 and IL-4 or with IL-21. After 7 days, equal numbers of cells were plated in serial dilutions and in triplicate and IgG and IgM production were determined by ELISPOT.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: STAT3-Mediated Up-Regulation of BLIMP1 Is Coordinated with BCL6 Down-Regulation to Control Human Plasma Cell Differentiation 1

    doi:

    Figure Lengend Snippet: IL-21 drives partial plasma cell differentiation in BCL6-transduced B cells. A , PB CD19 + B cells expressing BCL6-IRES-YFP were cultured in the presence of IL-2 and IL-4 or with rmIL-21 alone. At the indicated time points expression of BLIMP1 and ACTB were measured by qRT-PCR. BLIMP1 expression was normalized to ACTB expression within each sample and the means ± SD of triplicate measurements in two different donors is plotted. B , BCL6-YFP expressing B cells were cultured with CD40L in the presence of IL-2 and IL-4 or with IL-21 and BLIMP1, BCL6, and tubulin protein expression were determined by immunoblotting 3 or 7 days after IL-21 treatment. C , CD19 + B cells were transduced with LZRS-BCL6-IRES-ΔNGFR and cultured for 10 days on CD40L in the presence of IL-2 and IL-4 or with IL-21 alone. Cells were stained with anti-CD19, anti-NGFR, anti-CD38, and anti-CD20 and analyzed by flow cytometry. The rightmost four plots show CD38/CD20 expression on CD19 + BCL6–ΔNGFR– and CD19 + BCL6–ΔNGFR + cells. D , CD38/CD20 expression on BCL6-IRES-YFP-transduced B cells after 21 days of culture with IL-2 and IL-4 or with IL-21 alone. E , Surface Igλ and Igκ BCR expression on cells cultured as in C . F , Expression of CD138, HLA-DR, CD86, CD27, CD25, and CD3 on BCL6 + B cells cultured as in C . Dotted histogram is iso-type control. G , CD19 + BCL6-overexpressing B cells maintained on CD40L with either IL-2 and IL-4 (○)or with IL-21 (•). Cumulative expansion was calculated based on absolute cell numbers over a 17-day period. Data shown are means ± SD of three independent experiments involving different donors and constructs (BCL6-IRES-ΔNGFR or BCL6-IRES-GFP). H , BCL6-IRES-ΔNGFR + cells were cultured for 6 days on CD40L-L cells in the presence of IL-2 and IL-4 or with IL-21. Cell numbers were counted and IgM and IgG in culture supernatants were measured by ELISA to obtain per cell values of IgM and IgG. Data are representative of three independent experiments. I , Total PB CD19 + BCL6-GFP + cells were cultured with CD40L in the presence of IL-2 and IL-4 or with IL-21. After 7 days, equal numbers of cells were plated in serial dilutions and in triplicate and IgG and IgM production were determined by ELISPOT.

    Article Snippet: Bound IgG was detected with a mix of HRP-labeled IgG1 (1/1000), IgG2 (1/2000), IgG3 (1/2000) and IgG4 (1/1000) (Sanquin Reagents).

    Techniques: Cell Differentiation, Expressing, Cell Culture, Quantitative RT-PCR, Transduction, Staining, Flow Cytometry, Cytometry, Construct, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

    Specific activation of STAT3 in BCL6-transduced B cells. 100% pure PB CD19 + BCL6-IRES-YFP + B cells were transduced with LZRS-STAT3ER-IRES-GFP and expanded on CD40L-L cells with IL-2 and IL-4. Double positive GFP + YFP + cells were sorted by FACS. A , Cumulative expansion of BCL6/ STAT3ER cells cultured with medium only, with IL-2 and IL-4, or with only 1 μ M 4-HT for 22 days. B , IgG and IgA production in BCL6-YFP + /STAT3ER-GFP + cells treated for 4 days in the presence or absence of 4-HT. IgM was not detected in these cultures. C , Semi-quantitative RT-PCR for BLIMP1 and HPRT1 mRNA expression in BCL6-YFP + /STAT3ER-GFP + cells cultured for 4 days in the presence or absence of 4-HT. Triangles represent 5-fold dilutions of cDNA in the PCR reaction. Data are representative of two independent experiments. D , PB CD19 + B cells coexpressing BCL6 and STAT3ER were cultured in the presence or absence of 4-HT and at different time points after 4-HT addition, BLIMP1, BCL6, and tubulin protein levels were determined by immunoblot analysis. E and F , Raji cells expressing control-IRES-GFP or STAT3ER-IRES-GFP were cultured for 4 days in the presence of 1 μ M 4-HT. E , BLIMP1 and BCL6 ( F ) mRNA levels were determined by qRT-PCR and normalized to ACTB expression.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: STAT3-Mediated Up-Regulation of BLIMP1 Is Coordinated with BCL6 Down-Regulation to Control Human Plasma Cell Differentiation 1

    doi:

    Figure Lengend Snippet: Specific activation of STAT3 in BCL6-transduced B cells. 100% pure PB CD19 + BCL6-IRES-YFP + B cells were transduced with LZRS-STAT3ER-IRES-GFP and expanded on CD40L-L cells with IL-2 and IL-4. Double positive GFP + YFP + cells were sorted by FACS. A , Cumulative expansion of BCL6/ STAT3ER cells cultured with medium only, with IL-2 and IL-4, or with only 1 μ M 4-HT for 22 days. B , IgG and IgA production in BCL6-YFP + /STAT3ER-GFP + cells treated for 4 days in the presence or absence of 4-HT. IgM was not detected in these cultures. C , Semi-quantitative RT-PCR for BLIMP1 and HPRT1 mRNA expression in BCL6-YFP + /STAT3ER-GFP + cells cultured for 4 days in the presence or absence of 4-HT. Triangles represent 5-fold dilutions of cDNA in the PCR reaction. Data are representative of two independent experiments. D , PB CD19 + B cells coexpressing BCL6 and STAT3ER were cultured in the presence or absence of 4-HT and at different time points after 4-HT addition, BLIMP1, BCL6, and tubulin protein levels were determined by immunoblot analysis. E and F , Raji cells expressing control-IRES-GFP or STAT3ER-IRES-GFP were cultured for 4 days in the presence of 1 μ M 4-HT. E , BLIMP1 and BCL6 ( F ) mRNA levels were determined by qRT-PCR and normalized to ACTB expression.

    Article Snippet: Bound IgG was detected with a mix of HRP-labeled IgG1 (1/1000), IgG2 (1/2000), IgG3 (1/2000) and IgG4 (1/1000) (Sanquin Reagents).

    Techniques: Activation Assay, Transduction, FACS, Cell Culture, Quantitative RT-PCR, Expressing, Polymerase Chain Reaction

    Defective Ig production in Atm −/− B cells activated in vitro. CD19 + B cells were isolated from the spleens of Atm −/− and WT mice and cultured with CD40L and IL-4 (IgG1, IgG2a, and IgM), CD40L, IL-4, IL-5, TGF-β, and anti-IgD dextran (IgA) or LPS (IgG2b, IgG3). Results are displayed as the mean of three to four experiments ± SEM Atm −/− titer as a percentage of the Atm +/+ controls.

    Journal: The Journal of Experimental Medicine

    Article Title: Immunoglobulin Class Switch Recombination Is Impaired in Atm-deficient Mice

    doi: 10.1084/jem.20041074

    Figure Lengend Snippet: Defective Ig production in Atm −/− B cells activated in vitro. CD19 + B cells were isolated from the spleens of Atm −/− and WT mice and cultured with CD40L and IL-4 (IgG1, IgG2a, and IgM), CD40L, IL-4, IL-5, TGF-β, and anti-IgD dextran (IgA) or LPS (IgG2b, IgG3). Results are displayed as the mean of three to four experiments ± SEM Atm −/− titer as a percentage of the Atm +/+ controls.

    Article Snippet: Total IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, or IgE were captured with purified goat anti–mouse IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, or IgE (Southern Biotechnology Associates, Inc.) and detected with horseradish peroxidase (HRP)–conjugated goat anti–mouse α, γ1, γ2a, γ2b, γ3, or μ (Southern Biotechnology Associates, Inc.) or biotinylated rat anti–mouse IgE, and HRP-conjugated streptavidin (Southern Biotechnology Associates, Inc.).

    Techniques: In Vitro, Isolation, Mouse Assay, Cell Culture

    Real-time RT-PCR analysis of germline and productive IgG1 and IgE transcripts in Atm −/− B cells activated in vitro. Total RNA was isolated from B cells activated in vitro, and quantitative real-time RT-PCR was performed as described in Materials and Methods. Germline transcripts were measured at day 3 of culture and productive transcripts were measured at day 6 of culture. The data were normalized to GAPDH and presented as the increase in transcript level over unstimulated wild-type B cells. The data shown are representative of two independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Immunoglobulin Class Switch Recombination Is Impaired in Atm-deficient Mice

    doi: 10.1084/jem.20041074

    Figure Lengend Snippet: Real-time RT-PCR analysis of germline and productive IgG1 and IgE transcripts in Atm −/− B cells activated in vitro. Total RNA was isolated from B cells activated in vitro, and quantitative real-time RT-PCR was performed as described in Materials and Methods. Germline transcripts were measured at day 3 of culture and productive transcripts were measured at day 6 of culture. The data were normalized to GAPDH and presented as the increase in transcript level over unstimulated wild-type B cells. The data shown are representative of two independent experiments.

    Article Snippet: Total IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, or IgE were captured with purified goat anti–mouse IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, or IgE (Southern Biotechnology Associates, Inc.) and detected with horseradish peroxidase (HRP)–conjugated goat anti–mouse α, γ1, γ2a, γ2b, γ3, or μ (Southern Biotechnology Associates, Inc.) or biotinylated rat anti–mouse IgE, and HRP-conjugated streptavidin (Southern Biotechnology Associates, Inc.).

    Techniques: Quantitative RT-PCR, In Vitro, Isolation

    Surface Ig expression of IgG1 and IgE isotypes on in vitro–stimulated wild type and Atm −/− B cells. Cells were harvested after 4 d of in vitro stimulation with CD40L and IL-4, and flow cytometric analysis was used to determine surface IgG1 and IgE expression. Numbers on the dot plots show the percentage of switched cells as a proportion of B220 + B cells. Results are representative of four independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Immunoglobulin Class Switch Recombination Is Impaired in Atm-deficient Mice

    doi: 10.1084/jem.20041074

    Figure Lengend Snippet: Surface Ig expression of IgG1 and IgE isotypes on in vitro–stimulated wild type and Atm −/− B cells. Cells were harvested after 4 d of in vitro stimulation with CD40L and IL-4, and flow cytometric analysis was used to determine surface IgG1 and IgE expression. Numbers on the dot plots show the percentage of switched cells as a proportion of B220 + B cells. Results are representative of four independent experiments.

    Article Snippet: Total IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, or IgE were captured with purified goat anti–mouse IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, or IgE (Southern Biotechnology Associates, Inc.) and detected with horseradish peroxidase (HRP)–conjugated goat anti–mouse α, γ1, γ2a, γ2b, γ3, or μ (Southern Biotechnology Associates, Inc.) or biotinylated rat anti–mouse IgE, and HRP-conjugated streptavidin (Southern Biotechnology Associates, Inc.).

    Techniques: Expressing, In Vitro, Flow Cytometry

    Clonal expansion of IgG1-switched B cells. CFSE-stained resting B cells were stimulated in vitro with CD40L and IL-4 for 3–6 d and counterstained with goat anti–mouse IgG1 as described in Materials and Methods. (A) CFSE staining profiles of Atm +/+ and Atm −/− B cells are presented in the first two columns. The third column shows the percentage of recovered Atm −/− and Atm +/+ cells that have undergone the indicated number of cell divisions. (B) Two-color flow cytometric profiles indicate IgG1 expression on CFSE stained cells. The percentage of B cells expressing IgG1 is indicated for Atm +/+ B cells and Atm −/− B cells that have undergone the indicated numbers of cell divisions. Results are representative of three independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Immunoglobulin Class Switch Recombination Is Impaired in Atm-deficient Mice

    doi: 10.1084/jem.20041074

    Figure Lengend Snippet: Clonal expansion of IgG1-switched B cells. CFSE-stained resting B cells were stimulated in vitro with CD40L and IL-4 for 3–6 d and counterstained with goat anti–mouse IgG1 as described in Materials and Methods. (A) CFSE staining profiles of Atm +/+ and Atm −/− B cells are presented in the first two columns. The third column shows the percentage of recovered Atm −/− and Atm +/+ cells that have undergone the indicated number of cell divisions. (B) Two-color flow cytometric profiles indicate IgG1 expression on CFSE stained cells. The percentage of B cells expressing IgG1 is indicated for Atm +/+ B cells and Atm −/− B cells that have undergone the indicated numbers of cell divisions. Results are representative of three independent experiments.

    Article Snippet: Total IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, or IgE were captured with purified goat anti–mouse IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, or IgE (Southern Biotechnology Associates, Inc.) and detected with horseradish peroxidase (HRP)–conjugated goat anti–mouse α, γ1, γ2a, γ2b, γ3, or μ (Southern Biotechnology Associates, Inc.) or biotinylated rat anti–mouse IgE, and HRP-conjugated streptavidin (Southern Biotechnology Associates, Inc.).

    Techniques: Staining, In Vitro, Flow Cytometry, Expressing

    Impaired Ig production in Atm −/− mice. (A) Sera from eight Atm -deficient mice and littermate controls were assayed for total Ig levels by ELISA. IgE was undetectable by ELISA in sera from unimmunized mice. Results are displayed as the mean ± SEM Atm −/− titer as a percentage of the Atm +/+ controls. (B–D) Three Atm −/− mice and their WT littermates were immunized with TNP-KLH/Alum i.p., and serum was collected at the indicated time points. Serum levels of Ag-specific IgM (B) and IgG1 (C) were assayed by ELISA; data points represent the geometric mean ± geometric SEM of the ELISA titers. (D) Mice received a second immunization of TNP-KLH/Alum 40 d after the first immunization, and serum was collected after 8 d. Serum levels of total IgE and Ag-specific IgG2b and IgG3 were assayed by ELISA, and data points are displayed as the mean ± SD Atm −/− titer as a percentage of the Atm +/+ controls. The dotted line indicates the limit of detection. IgE, IgG2b, and IgG3 were undetectable in Atm −/− mice. Results are representative of two independent experiments. (E) Serum levels of Ag-specific IgA were measured in mice (five per group) immunized with TNP-KLH p.o., and serum was collected 4 d after the second immunization.

    Journal: The Journal of Experimental Medicine

    Article Title: Immunoglobulin Class Switch Recombination Is Impaired in Atm-deficient Mice

    doi: 10.1084/jem.20041074

    Figure Lengend Snippet: Impaired Ig production in Atm −/− mice. (A) Sera from eight Atm -deficient mice and littermate controls were assayed for total Ig levels by ELISA. IgE was undetectable by ELISA in sera from unimmunized mice. Results are displayed as the mean ± SEM Atm −/− titer as a percentage of the Atm +/+ controls. (B–D) Three Atm −/− mice and their WT littermates were immunized with TNP-KLH/Alum i.p., and serum was collected at the indicated time points. Serum levels of Ag-specific IgM (B) and IgG1 (C) were assayed by ELISA; data points represent the geometric mean ± geometric SEM of the ELISA titers. (D) Mice received a second immunization of TNP-KLH/Alum 40 d after the first immunization, and serum was collected after 8 d. Serum levels of total IgE and Ag-specific IgG2b and IgG3 were assayed by ELISA, and data points are displayed as the mean ± SD Atm −/− titer as a percentage of the Atm +/+ controls. The dotted line indicates the limit of detection. IgE, IgG2b, and IgG3 were undetectable in Atm −/− mice. Results are representative of two independent experiments. (E) Serum levels of Ag-specific IgA were measured in mice (five per group) immunized with TNP-KLH p.o., and serum was collected 4 d after the second immunization.

    Article Snippet: Total IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, or IgE were captured with purified goat anti–mouse IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, or IgE (Southern Biotechnology Associates, Inc.) and detected with horseradish peroxidase (HRP)–conjugated goat anti–mouse α, γ1, γ2a, γ2b, γ3, or μ (Southern Biotechnology Associates, Inc.) or biotinylated rat anti–mouse IgE, and HRP-conjugated streptavidin (Southern Biotechnology Associates, Inc.).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    ICOS-signalling promotes humoral immune responses during blood-stage infection. (A) Flow cytometric gating strategy employed to analyze splenic CD4 + T-cell responses throughout the manuscript. (B) Representative FACS plots and time-course analysis of cell-surface ICOS expression on splenic CD4 + T-cells from WT mice (n = 3/time point) during Py 17XNL infection. (C-F) WT mice (n = 6/group) were treated with anti-ICOSL blocking monoclonal antibody (α-ICOSL) or its isotype control (rat-IgG2a) prior to and during infection with Py 17XNL. (C) Representative FACS plots (gated on B220 + CD19 + live singlets), proportions and numbers of splenic GC B-cells (GL-7 + Fas + ), (D) numbers of splenic Ig-class switched (IgD lo IgM lo ) B-cells, and (E) representative FACS plots (gated on CD4 + TCRβ + live singlets), proportions and numbers of splenic Tfh cells (PD1 + CXCR5 + ), in naïve mice and infected, α-ICOSL and control IgG-treated mice, 16 days p . i . (F) Py 17XNL-specific IgM, total IgG, IgG2b and IgG3 levels in serum of naïve and infected, α-ICOSL and control IgG-treated mice, 16 days p . i . (G) Parasitemias in WT mice (n = 6/group) infected with Py 17XNL, and treated every three days with α-ICOSL or control IgG until day 21 p . i . (depicted by arrows, with estimated period of cover highlighted with shaded grey box—an α-ICOSL-treated mouse succumbed to infection on each of days 15, 17 and 30 p . i ., and one control-IgG treated mouse succumbed on day 20 p . i . Data representative of two independent experiments in (B-E), and two pooled independent experiments in (F), with experiment in (G) being conducted once. Statistics: Mann-Whitney U test, *P

    Journal: PLoS Pathogens

    Article Title: IFNAR1-Signalling Obstructs ICOS-mediated Humoral Immunity during Non-lethal Blood-Stage Plasmodium Infection

    doi: 10.1371/journal.ppat.1005999

    Figure Lengend Snippet: ICOS-signalling promotes humoral immune responses during blood-stage infection. (A) Flow cytometric gating strategy employed to analyze splenic CD4 + T-cell responses throughout the manuscript. (B) Representative FACS plots and time-course analysis of cell-surface ICOS expression on splenic CD4 + T-cells from WT mice (n = 3/time point) during Py 17XNL infection. (C-F) WT mice (n = 6/group) were treated with anti-ICOSL blocking monoclonal antibody (α-ICOSL) or its isotype control (rat-IgG2a) prior to and during infection with Py 17XNL. (C) Representative FACS plots (gated on B220 + CD19 + live singlets), proportions and numbers of splenic GC B-cells (GL-7 + Fas + ), (D) numbers of splenic Ig-class switched (IgD lo IgM lo ) B-cells, and (E) representative FACS plots (gated on CD4 + TCRβ + live singlets), proportions and numbers of splenic Tfh cells (PD1 + CXCR5 + ), in naïve mice and infected, α-ICOSL and control IgG-treated mice, 16 days p . i . (F) Py 17XNL-specific IgM, total IgG, IgG2b and IgG3 levels in serum of naïve and infected, α-ICOSL and control IgG-treated mice, 16 days p . i . (G) Parasitemias in WT mice (n = 6/group) infected with Py 17XNL, and treated every three days with α-ICOSL or control IgG until day 21 p . i . (depicted by arrows, with estimated period of cover highlighted with shaded grey box—an α-ICOSL-treated mouse succumbed to infection on each of days 15, 17 and 30 p . i ., and one control-IgG treated mouse succumbed on day 20 p . i . Data representative of two independent experiments in (B-E), and two pooled independent experiments in (F), with experiment in (G) being conducted once. Statistics: Mann-Whitney U test, *P

    Article Snippet: Following six washes, wells were incubated in the dark with biotinylated anti-IgM, total IgG, IgG1, IgG2b and IgG3 (Jackson ImmunoResearch) for 1hr at room temperature.

    Techniques: Infection, Flow Cytometry, FACS, Expressing, Mouse Assay, Blocking Assay, MANN-WHITNEY

    IFNAR1-signalling simultaneously limits splenic Th1 and Tfh cell responses. (A B) Representative FACS plots (gated on CD4 + TCRβ + live singlets), proportions and absolute numbers of splenic (A) Th1 (IFNγ + Tbet + ) and (B) emerging Tfh (PD1 + CXCR5 + ) cells in WT and Ifnar1 -/- mice (n = 6/group), 6 days p . i with Pc AS. Data representative of two independent experiments. (C) Numbers of splenic ICOS + Th1 cells (Tbet + IFNγ + CD4 + T cells) and Tfh cells (PD1 + CXCR5 + CD4 + T cells) 6 days p . i . with Pc AS. (D) Numbers of ICOS + Tfh cells (PD1 + CXCR5 + CD4 + T cells), 16 days p . i . with Py 17XNL infection. (E) Proportions and absolute numbers of splenic CD4 + T-cells expressing Ki-67 in naïve, WT and Ifnar1 -/- mice 6 days p . i . with Pc AS. (F) Proportions and absolute numbers of splenic CD4 + T-cells expressing Ki-67 in naïve mice, and WT mice, 6 days p . i . with Py 17XNL and treatment with α-IFNAR1 or Control IgG. (G) Absolute numbers of splenocytes, CD4 + T-cells and B-cells, in WT naïve, infected WT and Ifnar1 -/- mice 6 days (n = 17–18, pooled from three independent experiments (n = 5–6 per expt)) and 8 days (n = 29, pooled from five experiments (n = 5–6 per expt)) p . i . with Pc AS. (H) Absolute numbers of splenocytes, CD4 + T-cells and B-cells in WT naïve, infected WT and Ifnar1 -/- mice, 16 days p . i . with Py 17XNL (n = 17–18, pooled from three experiments (n = 5–6 per expt)) Mann-Whitney U-test **P

    Journal: PLoS Pathogens

    Article Title: IFNAR1-Signalling Obstructs ICOS-mediated Humoral Immunity during Non-lethal Blood-Stage Plasmodium Infection

    doi: 10.1371/journal.ppat.1005999

    Figure Lengend Snippet: IFNAR1-signalling simultaneously limits splenic Th1 and Tfh cell responses. (A B) Representative FACS plots (gated on CD4 + TCRβ + live singlets), proportions and absolute numbers of splenic (A) Th1 (IFNγ + Tbet + ) and (B) emerging Tfh (PD1 + CXCR5 + ) cells in WT and Ifnar1 -/- mice (n = 6/group), 6 days p . i with Pc AS. Data representative of two independent experiments. (C) Numbers of splenic ICOS + Th1 cells (Tbet + IFNγ + CD4 + T cells) and Tfh cells (PD1 + CXCR5 + CD4 + T cells) 6 days p . i . with Pc AS. (D) Numbers of ICOS + Tfh cells (PD1 + CXCR5 + CD4 + T cells), 16 days p . i . with Py 17XNL infection. (E) Proportions and absolute numbers of splenic CD4 + T-cells expressing Ki-67 in naïve, WT and Ifnar1 -/- mice 6 days p . i . with Pc AS. (F) Proportions and absolute numbers of splenic CD4 + T-cells expressing Ki-67 in naïve mice, and WT mice, 6 days p . i . with Py 17XNL and treatment with α-IFNAR1 or Control IgG. (G) Absolute numbers of splenocytes, CD4 + T-cells and B-cells, in WT naïve, infected WT and Ifnar1 -/- mice 6 days (n = 17–18, pooled from three independent experiments (n = 5–6 per expt)) and 8 days (n = 29, pooled from five experiments (n = 5–6 per expt)) p . i . with Pc AS. (H) Absolute numbers of splenocytes, CD4 + T-cells and B-cells in WT naïve, infected WT and Ifnar1 -/- mice, 16 days p . i . with Py 17XNL (n = 17–18, pooled from three experiments (n = 5–6 per expt)) Mann-Whitney U-test **P

    Article Snippet: Following six washes, wells were incubated in the dark with biotinylated anti-IgM, total IgG, IgG1, IgG2b and IgG3 (Jackson ImmunoResearch) for 1hr at room temperature.

    Techniques: FACS, Mouse Assay, Infection, Expressing, MANN-WHITNEY

    Splenic Germinal Centre B-cell and plasmablast responses are strongly dependent on CD4 + cells during blood-stage Plasmodium infection. (A) Parasitemia and (B) survival of WT mice (n = 6) treated with CD4-depleting monoclonal antibody (αCD4) or control IgG 1 day prior to infection with Py 17XNL. (C) Flow cytometric gating strategy employed to analyze splenic B-cell responses throughout the manuscript. (D-F) WT mice (n = 5) were administered αCD4 or control-IgG prior to Py 17XNL infection. Presented are representative FACS plots (gated on B220 + CD19 + live singlets), proportions and absolute numbers in the spleen of (D) plasmablasts (IgD lo CD138 hi ), (E) GC B-cells (GL-7 + Fas + ), and (F) Ig-switched B-cells (IgD lo IgM lo cells) from naïve and infected, control IgG and αCD4-treated WT mice, 10 days p . i . Statistics: Mann-Whitney U test, **P

    Journal: PLoS Pathogens

    Article Title: IFNAR1-Signalling Obstructs ICOS-mediated Humoral Immunity during Non-lethal Blood-Stage Plasmodium Infection

    doi: 10.1371/journal.ppat.1005999

    Figure Lengend Snippet: Splenic Germinal Centre B-cell and plasmablast responses are strongly dependent on CD4 + cells during blood-stage Plasmodium infection. (A) Parasitemia and (B) survival of WT mice (n = 6) treated with CD4-depleting monoclonal antibody (αCD4) or control IgG 1 day prior to infection with Py 17XNL. (C) Flow cytometric gating strategy employed to analyze splenic B-cell responses throughout the manuscript. (D-F) WT mice (n = 5) were administered αCD4 or control-IgG prior to Py 17XNL infection. Presented are representative FACS plots (gated on B220 + CD19 + live singlets), proportions and absolute numbers in the spleen of (D) plasmablasts (IgD lo CD138 hi ), (E) GC B-cells (GL-7 + Fas + ), and (F) Ig-switched B-cells (IgD lo IgM lo cells) from naïve and infected, control IgG and αCD4-treated WT mice, 10 days p . i . Statistics: Mann-Whitney U test, **P

    Article Snippet: Following six washes, wells were incubated in the dark with biotinylated anti-IgM, total IgG, IgG1, IgG2b and IgG3 (Jackson ImmunoResearch) for 1hr at room temperature.

    Techniques: Infection, Mouse Assay, Flow Cytometry, FACS, MANN-WHITNEY

    Antibody-mediated IFNAR1 blockade boosts humoral immune responses during blood-stage infection. WT mice (n = 5/group) were treated with anti-IFNAR1 blocking antibody (α-Ifnar1) or control IgG prior to and during infection with Pc AS. (A) Representative FACS plots (gated on CD4 + TCRβ + live singlets), proportions and absolute numbers of splenic ICOS + CD4 + T cells in naïve and infected mice on days 6 and 7 p . i . (B) Representative FACS plots (gated on CD4 + TCRβ + live singlets), proportions and numbers of splenic Tfh cells (as PD1 + CXCR5 + CD4 + T cells) in naïve and infected and antibody-treated mice, 7 days p . i . Bcl-6 expression is also shown in histograms for Tfh (PD1 + CXCR5 + ; red gate) and non-Tfh cells (PD1 - CXCR5 - ; blue gate), alongside Geometric Mean Bcl-6 expression by these populations in individual mice. (C and D) Numbers of splenic (C) plasmablasts and (D) GC B cells in naïve, and infected and treated mice, 7 days p . i . Data representative of 2 independent experiments. Mann-Whitney U test *P

    Journal: PLoS Pathogens

    Article Title: IFNAR1-Signalling Obstructs ICOS-mediated Humoral Immunity during Non-lethal Blood-Stage Plasmodium Infection

    doi: 10.1371/journal.ppat.1005999

    Figure Lengend Snippet: Antibody-mediated IFNAR1 blockade boosts humoral immune responses during blood-stage infection. WT mice (n = 5/group) were treated with anti-IFNAR1 blocking antibody (α-Ifnar1) or control IgG prior to and during infection with Pc AS. (A) Representative FACS plots (gated on CD4 + TCRβ + live singlets), proportions and absolute numbers of splenic ICOS + CD4 + T cells in naïve and infected mice on days 6 and 7 p . i . (B) Representative FACS plots (gated on CD4 + TCRβ + live singlets), proportions and numbers of splenic Tfh cells (as PD1 + CXCR5 + CD4 + T cells) in naïve and infected and antibody-treated mice, 7 days p . i . Bcl-6 expression is also shown in histograms for Tfh (PD1 + CXCR5 + ; red gate) and non-Tfh cells (PD1 - CXCR5 - ; blue gate), alongside Geometric Mean Bcl-6 expression by these populations in individual mice. (C and D) Numbers of splenic (C) plasmablasts and (D) GC B cells in naïve, and infected and treated mice, 7 days p . i . Data representative of 2 independent experiments. Mann-Whitney U test *P

    Article Snippet: Following six washes, wells were incubated in the dark with biotinylated anti-IgM, total IgG, IgG1, IgG2b and IgG3 (Jackson ImmunoResearch) for 1hr at room temperature.

    Techniques: Infection, Mouse Assay, Blocking Assay, FACS, Expressing, MANN-WHITNEY

    IFNAR1-deficiency boosts humoral immunity via enhanced ICOS-signalling. WT and Ifnar1 -/- mice (n = 6/group), infected with Pc AS, and treated with αICOSL (100μg) or control IgG2a throughout infection (days 0, 2, 4 and 6 p . i .), were assessed on day 8 p . i . for (A) parasite-specific IgM and IgG, as well as (B) Parasitemia. (C D) WT and Ifnar1 -/- mice were infected with Pc AS, and treated as in (A B) with α-ICOSL or control IgG2a: (C) Representative FACS plots (gated on CD4 + TCRβ + live singlets), proportions and absolute numbers of splenic Tfh cells, and (D) proportions and absolute numbers of GC B-cells (gated on B220 + CD19 + live singlets) on day 6 p . i . Statistics: One-way ANOVA, Tukey’s test for multiple comparisons, *P

    Journal: PLoS Pathogens

    Article Title: IFNAR1-Signalling Obstructs ICOS-mediated Humoral Immunity during Non-lethal Blood-Stage Plasmodium Infection

    doi: 10.1371/journal.ppat.1005999

    Figure Lengend Snippet: IFNAR1-deficiency boosts humoral immunity via enhanced ICOS-signalling. WT and Ifnar1 -/- mice (n = 6/group), infected with Pc AS, and treated with αICOSL (100μg) or control IgG2a throughout infection (days 0, 2, 4 and 6 p . i .), were assessed on day 8 p . i . for (A) parasite-specific IgM and IgG, as well as (B) Parasitemia. (C D) WT and Ifnar1 -/- mice were infected with Pc AS, and treated as in (A B) with α-ICOSL or control IgG2a: (C) Representative FACS plots (gated on CD4 + TCRβ + live singlets), proportions and absolute numbers of splenic Tfh cells, and (D) proportions and absolute numbers of GC B-cells (gated on B220 + CD19 + live singlets) on day 6 p . i . Statistics: One-way ANOVA, Tukey’s test for multiple comparisons, *P

    Article Snippet: Following six washes, wells were incubated in the dark with biotinylated anti-IgM, total IgG, IgG1, IgG2b and IgG3 (Jackson ImmunoResearch) for 1hr at room temperature.

    Techniques: Mouse Assay, Infection, FACS

    IFNAR1-signalling obstructs B-cell and parasite-specific antibody responses during blood-stage infection. (A) Time-course analysis of parasitemia in WT and Ifnar1 -/- mice (n = 5/group) infected with Py 17XNL. (B) ELISA quantitation of Py 17XNL-specific IgM, total IgG, IgG1, IgG2b, IgG3 in serum diluted from 1 in 400 in two-fold sequential dilutions to 1 in 3200, and (C) IgG2c at 1 in 400 in the sera of naïve and infected WT and Ifnar1 -/- mice, 16 days p . i . (D E) Representative FACS plots (gated on B220 + CD19 + live singlets), proportions and absolute numbers of (D) splenic GC B-cells (GL7 + Fas + ) and (E) Ig-class-switched B-cells (IgD lo IgM lo ) in naïve and infected WT and Ifnar1 -/- mice, 16 days p . i . (F) Representative FACS plots (gated on B220 + CD19 + live singlets), proportions and absolute numbers of splenic plasmablasts (IgD lo CD138 hi ) in naïve and infected WT and Ifnar1 -/- mice (n = 6), 6 days p . i . (G) Time course analysis of IgM, total IgG, IgG2b and IgG3 levels in naïve and infected WT and Ifnar1 -/- mice (n = 6). Statistics: Mann-Whitney U test (A C-G), Two way ANOVA and Tukey’s test for multiple comparisons in (B), *P

    Journal: PLoS Pathogens

    Article Title: IFNAR1-Signalling Obstructs ICOS-mediated Humoral Immunity during Non-lethal Blood-Stage Plasmodium Infection

    doi: 10.1371/journal.ppat.1005999

    Figure Lengend Snippet: IFNAR1-signalling obstructs B-cell and parasite-specific antibody responses during blood-stage infection. (A) Time-course analysis of parasitemia in WT and Ifnar1 -/- mice (n = 5/group) infected with Py 17XNL. (B) ELISA quantitation of Py 17XNL-specific IgM, total IgG, IgG1, IgG2b, IgG3 in serum diluted from 1 in 400 in two-fold sequential dilutions to 1 in 3200, and (C) IgG2c at 1 in 400 in the sera of naïve and infected WT and Ifnar1 -/- mice, 16 days p . i . (D E) Representative FACS plots (gated on B220 + CD19 + live singlets), proportions and absolute numbers of (D) splenic GC B-cells (GL7 + Fas + ) and (E) Ig-class-switched B-cells (IgD lo IgM lo ) in naïve and infected WT and Ifnar1 -/- mice, 16 days p . i . (F) Representative FACS plots (gated on B220 + CD19 + live singlets), proportions and absolute numbers of splenic plasmablasts (IgD lo CD138 hi ) in naïve and infected WT and Ifnar1 -/- mice (n = 6), 6 days p . i . (G) Time course analysis of IgM, total IgG, IgG2b and IgG3 levels in naïve and infected WT and Ifnar1 -/- mice (n = 6). Statistics: Mann-Whitney U test (A C-G), Two way ANOVA and Tukey’s test for multiple comparisons in (B), *P

    Article Snippet: Following six washes, wells were incubated in the dark with biotinylated anti-IgM, total IgG, IgG1, IgG2b and IgG3 (Jackson ImmunoResearch) for 1hr at room temperature.

    Techniques: Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay, Quantitation Assay, FACS, MANN-WHITNEY

    M2 expressing cells have an activated, pre-plasma memory phenotype. (A) M2-transduced cells were CD19 + , CD25 high , GL7 high , B220 low , I-A b low , sIgD − , sIgG + , and CD138 low when compared to untransduced cells within the culture. Representative flow cytometry histograms at day 4 post-transduction. The black line open histograms reflect staining of the transduced (Thy1.1 + ) B cell population, while the filled gray histograms reflect staining of the untransduced (Thy1.1 − ) population. The top panels depict data from M2-transduced cultures; bottom panels depict data from M2.Stop-transduced cells. Data is representative of three samples per time-point with at least three independent experiments per stain. (B and C) ELISA quantitation of the levels of IgM and IgG in supernatants of M2 and M2.Stop transduced B cell cultures. Three samples were analyzed per time point, and the data shown is representative of three independent experiments. Significance of differences in IgG secretion was determined by two-tailed, unpaired Student's T test with a confidence level of 95%. * p = 0.0182, ** p = 0.0352, *** p = 0.0118.

    Journal: PLoS Pathogens

    Article Title: The MHV68 M2 Protein Drives IL-10 Dependent B Cell Proliferation and Differentiation

    doi: 10.1371/journal.ppat.1000039

    Figure Lengend Snippet: M2 expressing cells have an activated, pre-plasma memory phenotype. (A) M2-transduced cells were CD19 + , CD25 high , GL7 high , B220 low , I-A b low , sIgD − , sIgG + , and CD138 low when compared to untransduced cells within the culture. Representative flow cytometry histograms at day 4 post-transduction. The black line open histograms reflect staining of the transduced (Thy1.1 + ) B cell population, while the filled gray histograms reflect staining of the untransduced (Thy1.1 − ) population. The top panels depict data from M2-transduced cultures; bottom panels depict data from M2.Stop-transduced cells. Data is representative of three samples per time-point with at least three independent experiments per stain. (B and C) ELISA quantitation of the levels of IgM and IgG in supernatants of M2 and M2.Stop transduced B cell cultures. Three samples were analyzed per time point, and the data shown is representative of three independent experiments. Significance of differences in IgG secretion was determined by two-tailed, unpaired Student's T test with a confidence level of 95%. * p = 0.0182, ** p = 0.0352, *** p = 0.0118.

    Article Snippet: Cells were stained with the following antibodies: Thy1.1-FITC, -PE, or –APC (eBiosciences), CD44-FITC (Caltag), CD62L-PE (Caltag), GL7-FITC, IgG1, 2a, 2b, 3 -FITC, CD25-PE, CD138-PE, I-Ab -PE, CD4-PerCP, CD11a-PE-Cy7, CD19-APC, B220-APC, CD8-PacficBlue (BD Pharmigen except where noted).

    Techniques: Expressing, Flow Cytometry, Cytometry, Transduction, Staining, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Two Tailed Test

    In vivo effect of Fn14-Fc on Ig production in SLE mice. Sanroque mice were injected intraperitoneally with Fn14-Fc (100 μg/mouse) or control-Fc (100 μg/mouse) (n = 5/group) for 3 weeks. Mice were sacrificed on day 21 after the first injection. The serum total IgG, IgG1 and IgG2a levels were determined by ELISA. Data are expressed as means ± SDs. * P

    Journal: Journal of Translational Medicine

    Article Title: Fn14-Fc suppresses germinal center formation and pathogenic B cells in a lupus mouse model via inhibition of the TWEAK/Fn14 Pathway

    doi: 10.1186/s12967-016-0846-4

    Figure Lengend Snippet: In vivo effect of Fn14-Fc on Ig production in SLE mice. Sanroque mice were injected intraperitoneally with Fn14-Fc (100 μg/mouse) or control-Fc (100 μg/mouse) (n = 5/group) for 3 weeks. Mice were sacrificed on day 21 after the first injection. The serum total IgG, IgG1 and IgG2a levels were determined by ELISA. Data are expressed as means ± SDs. * P

    Article Snippet: Total IgG, IgG1, IgG2a, and anti-double-stranded (ds) DNA Abs were measured using a mouse total IgG, IgG1 and IgG2a enzyme-linked immunosorbent assay (ELISA) quantitation kit (Bethyl Laboratories, Montgomery, TX, USA).

    Techniques: In Vivo, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay

    Correlation between specific IgA or IgG to Dp (A) or Bt (B) and Der p 1 (A) or Blo t 5 (B) allergens. Correlation coefficients were determined using Spearman’s tests; ** p

    Journal: PLoS ONE

    Article Title: Early Exposure to Respiratory Allergens by Placental Transfer and Breastfeeding

    doi: 10.1371/journal.pone.0139064

    Figure Lengend Snippet: Correlation between specific IgA or IgG to Dp (A) or Bt (B) and Der p 1 (A) or Blo t 5 (B) allergens. Correlation coefficients were determined using Spearman’s tests; ** p

    Article Snippet: For Bt- specific IgG4 quantification in maternal and newborn serum, we performed an amplified ELISA using an ELISA amplification system (19.589–019, Invitrogen) according to the manufacturer’s instructions.

    Techniques:

    Levels of specific IgG subclasses to D . pteronyssinus (Dp) and B . tropicalis (Bt) in both cord blood and mother blood sera. Horizontal lines represent the median antibody levels; *** p

    Journal: PLoS ONE

    Article Title: Early Exposure to Respiratory Allergens by Placental Transfer and Breastfeeding

    doi: 10.1371/journal.pone.0139064

    Figure Lengend Snippet: Levels of specific IgG subclasses to D . pteronyssinus (Dp) and B . tropicalis (Bt) in both cord blood and mother blood sera. Horizontal lines represent the median antibody levels; *** p

    Article Snippet: For Bt- specific IgG4 quantification in maternal and newborn serum, we performed an amplified ELISA using an ELISA amplification system (19.589–019, Invitrogen) according to the manufacturer’s instructions.

    Techniques:

    TACI requires MyD88 to induce CSR in mice ( a ) MyD88-binding site (BS) in human or mouse TACI. Numbers, amino-acid positions; bold letters, identical amino acids; box, MyD88-binding site in the THC domain. ( b ) QRT-PCR of AICDA , I γ 1-C γ 1 and I γ 1-C μ transcripts from WT (open bars) or MyD88 KO (solid bars) mouse B cells cultured for 4 d in the presence or absence of BAFF, APRIL or CpG DNA plus IL-4. Results are normalized to ACTB (encoding β-actin) mRNA; RE, relative expression compared to control (ctrl) unstimulated B cells. ( c ) ELISA of IgG1 and IgM from WT or MyD88 KO B cells cultured as in c for 8 days. ( d,e ) QRT-PCR of I α 1-C μ and flow cytometric analysis of surface IgA from WT or MyD88 KO B cells cultured as in b for 48 h (I α 1-C μ ) or 5 days (IgA). ( e ) QRT-PCR of AICDA and I γ 1-C γ 1 from WT or MyD88 KO B cells cultured with a ctrl antibody or anti-TACI for 2 d. * P

    Journal: Nature immunology

    Article Title: TACI triggers immunoglobulin class switching by activating B cells through the adaptor protein MyD88

    doi: 10.1038/ni.1914

    Figure Lengend Snippet: TACI requires MyD88 to induce CSR in mice ( a ) MyD88-binding site (BS) in human or mouse TACI. Numbers, amino-acid positions; bold letters, identical amino acids; box, MyD88-binding site in the THC domain. ( b ) QRT-PCR of AICDA , I γ 1-C γ 1 and I γ 1-C μ transcripts from WT (open bars) or MyD88 KO (solid bars) mouse B cells cultured for 4 d in the presence or absence of BAFF, APRIL or CpG DNA plus IL-4. Results are normalized to ACTB (encoding β-actin) mRNA; RE, relative expression compared to control (ctrl) unstimulated B cells. ( c ) ELISA of IgG1 and IgM from WT or MyD88 KO B cells cultured as in c for 8 days. ( d,e ) QRT-PCR of I α 1-C μ and flow cytometric analysis of surface IgA from WT or MyD88 KO B cells cultured as in b for 48 h (I α 1-C μ ) or 5 days (IgA). ( e ) QRT-PCR of AICDA and I γ 1-C γ 1 from WT or MyD88 KO B cells cultured with a ctrl antibody or anti-TACI for 2 d. * P

    Article Snippet: For co-immunoprecipitation assays, total B cell lysates were first pre-cleared with 100 μl Streptavidin Sepharose beads (GE healthcare) for 30 min at 4°C, and then incubated with 2.5 μg control IgG2a mAb or human-reactive biotin-conjugated anti-TACI 11H3 mAb (eBioscience) or mouse-reactive biotin-conjugated BAF104 mAb (R & D Systems) overnight.

    Techniques: Mouse Assay, Binding Assay, Quantitative RT-PCR, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry

    TACI triggers CSR by cooperating with TLR ligands ( a ) Immunofluorescence staining of tonsil (top) and splenic (bottom) tissues for IgD (green), TACI (red), and nuclei (blue). Dashed line, follicle; EP, epithelium; FM, follicular mantle; FO, follicle; GC, germinal center; MZ; marginal zone; SE, sub-epithelium; RP, red pulp. Original magnification, ×10 (left panels) or ×63 (right panels). ( b–e ) QRT-PCR of I γ 1-C γ 1, I γ 1-C μ and AICDA in naïve ( b–d ) or lymphoblastoid ( e ) B cells from healthy donors (HD) or CVID patients with various heterozygous TACI substitutions cultured for 2 or 4 days with or without anti-TACI, IL-10 and/or IL-4. Results are normalized to ACTB (encoding β-actin) mRNA; RE, relative expression compared to B cells incubated with a control antibody (ctrl). ( f ) Flow cytometry of TACI on primary CD19 + CD27 + B cells from a HD or CVID patient with homozygous S144X/S144X TACI substitution. Red histograms, ctrl; blue histograms, anti-TACI. ( g ) AICDA and I γ 1-C μ in primary naive B cells from CVID case shown in f incubated with BAFF or APRIL plus IL-4 for 6 d. ( h ) Flow cytometry of IgG, IgA and CD27 on primary naive B cells incubated for 7 days with ctr, anti-TACI, IL-10 and/or CpG DNA. Numbers indicate percentages. ( i ) Flow cytometry of IgG and IgA (upper panels) and ELISA of secreted IgG and IgA (bottom panels) from B cells stimulated as in h . * P

    Journal: Nature immunology

    Article Title: TACI triggers immunoglobulin class switching by activating B cells through the adaptor protein MyD88

    doi: 10.1038/ni.1914

    Figure Lengend Snippet: TACI triggers CSR by cooperating with TLR ligands ( a ) Immunofluorescence staining of tonsil (top) and splenic (bottom) tissues for IgD (green), TACI (red), and nuclei (blue). Dashed line, follicle; EP, epithelium; FM, follicular mantle; FO, follicle; GC, germinal center; MZ; marginal zone; SE, sub-epithelium; RP, red pulp. Original magnification, ×10 (left panels) or ×63 (right panels). ( b–e ) QRT-PCR of I γ 1-C γ 1, I γ 1-C μ and AICDA in naïve ( b–d ) or lymphoblastoid ( e ) B cells from healthy donors (HD) or CVID patients with various heterozygous TACI substitutions cultured for 2 or 4 days with or without anti-TACI, IL-10 and/or IL-4. Results are normalized to ACTB (encoding β-actin) mRNA; RE, relative expression compared to B cells incubated with a control antibody (ctrl). ( f ) Flow cytometry of TACI on primary CD19 + CD27 + B cells from a HD or CVID patient with homozygous S144X/S144X TACI substitution. Red histograms, ctrl; blue histograms, anti-TACI. ( g ) AICDA and I γ 1-C μ in primary naive B cells from CVID case shown in f incubated with BAFF or APRIL plus IL-4 for 6 d. ( h ) Flow cytometry of IgG, IgA and CD27 on primary naive B cells incubated for 7 days with ctr, anti-TACI, IL-10 and/or CpG DNA. Numbers indicate percentages. ( i ) Flow cytometry of IgG and IgA (upper panels) and ELISA of secreted IgG and IgA (bottom panels) from B cells stimulated as in h . * P

    Article Snippet: For co-immunoprecipitation assays, total B cell lysates were first pre-cleared with 100 μl Streptavidin Sepharose beads (GE healthcare) for 30 min at 4°C, and then incubated with 2.5 μg control IgG2a mAb or human-reactive biotin-conjugated anti-TACI 11H3 mAb (eBioscience) or mouse-reactive biotin-conjugated BAF104 mAb (R & D Systems) overnight.

    Techniques: Immunofluorescence, Staining, Quantitative RT-PCR, Cell Culture, Expressing, Incubation, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    Altered spatiotemporal level of CCL20 in the spinal cord after SCI. a The temporal profile (from 0 h to 28 days post-injury) of CCL20 mRNA expression in the spinal cord, as determined by qRT-PCR, shows that SCI leads to increased CCL20 mRNA level in the spinal cord, especially during the early period of SCI. b Mouse IgG levels of rat serum (from 0 h to 28 days post-injury), as determined by ELISA, are significantly increased in CCL20 mAb group and isotype control group from 6 h to 28 days post-SCI. CCL20 immunostaining at 1 day post-injury in the sham group (c) , SCI group (d) , negative control of the sham group (e) , and negative control of the SCI group (f) indicates that CCL20 is mainly localized in the cytoplasm of gray matter neurons and glial cells. The brown staining represents positive CCL20 expression. Black arrow indicates the CCL20 positive neuron and glial cell. Scale bar = 100 μm. + P

    Journal: Journal of Neuroinflammation

    Article Title: C-C motif chemokine ligand 20 regulates neuroinflammation following spinal cord injury via Th17 cell recruitment

    doi: 10.1186/s12974-016-0630-7

    Figure Lengend Snippet: Altered spatiotemporal level of CCL20 in the spinal cord after SCI. a The temporal profile (from 0 h to 28 days post-injury) of CCL20 mRNA expression in the spinal cord, as determined by qRT-PCR, shows that SCI leads to increased CCL20 mRNA level in the spinal cord, especially during the early period of SCI. b Mouse IgG levels of rat serum (from 0 h to 28 days post-injury), as determined by ELISA, are significantly increased in CCL20 mAb group and isotype control group from 6 h to 28 days post-SCI. CCL20 immunostaining at 1 day post-injury in the sham group (c) , SCI group (d) , negative control of the sham group (e) , and negative control of the SCI group (f) indicates that CCL20 is mainly localized in the cytoplasm of gray matter neurons and glial cells. The brown staining represents positive CCL20 expression. Black arrow indicates the CCL20 positive neuron and glial cell. Scale bar = 100 μm. + P

    Article Snippet: As previously described, the anti-rat CCL20 antibody (MAB540; R & D Systems Inc., Minneapolis, MN, USA) and mouse IgG1 isotype control antibody (MAB002; R & D Systems) were dissolved in phosphate-buffered saline (PBS) and diluted to 100 μg/ml [ ].

    Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Immunostaining, Negative Control, Staining

    Immunoglobulin isotypes in serum from SAMR1 and SAMP8 mice determined by ELISA. ( a ) Serum IgM and IgG1 from 2- and 10-month-old SAMR1 and SAMP8 mice. ( b ) IgG2a, IgG2b and IgG3 isotypes were quantified on sera from 10-month-old SAMP8 and SAMR1 mice. Data are measurements of individual mice. Means are indicated by the horizontal lines. Comparisons were made with the unpaired two-tailed Student’s t -test: ** P

    Journal: Cell Death & Disease

    Article Title: Altered marginal zone and innate-like B cells in aged senescence-accelerated SAMP8 mice with defective IgG1 responses

    doi: 10.1038/cddis.2017.351

    Figure Lengend Snippet: Immunoglobulin isotypes in serum from SAMR1 and SAMP8 mice determined by ELISA. ( a ) Serum IgM and IgG1 from 2- and 10-month-old SAMR1 and SAMP8 mice. ( b ) IgG2a, IgG2b and IgG3 isotypes were quantified on sera from 10-month-old SAMP8 and SAMR1 mice. Data are measurements of individual mice. Means are indicated by the horizontal lines. Comparisons were made with the unpaired two-tailed Student’s t -test: ** P

    Article Snippet: Standard curves for each Ig isotype were generated using purified myeloma proteins or antibodies: IgM (clone G155-228, BD Biosciences), IgG1 (clone 4.19, in-house), IgG2a (clone PK136, in-house), IgG2b (clone Y.3, in-house), IgG3 (clone MG3-3S, Biolegend, San Diego, CA, USA).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    Plasma cell B cell maturation and distribution of the memory B-cell compartments in aged SAMP8 mice. ( a ) Cells were stained with anti-CD19-Violet-421, anti-CD45R-PE and anti-CD138-APC. Absolute numbers of CD138 + plasmablasts were calculated from flow cytometry as in Figure 1c . Data are mean±S.E.M., ( n =4). ( b ) IgG1-specific ELISPOT analysis (see ‘Materials and Methods’) of purified samples of B1REL and B2 cells obtained from aged SAMR1 and SAMP8 mice. The numbers of IgG1- antibody secreting cells (ASCs) are shown. Data are mean±S.E.M., ( n =4). ( c ) RT-qPCR analyses were performed for Pax5, Xbp-1, Blimp1 and AID transcript expression in purified B1REL cells from 10-month-old SAMR1 and SAMP8 mice (see ‘Materials and Methods’). The Bio-Rad CFX Manager software (Bio-Rad, Hercules, CA, USA) was used to calculate the C T of each reaction. The amount of specific transcripts in each cDNA sample was determined as the 2 −ΔΔ C T , relative to that of the HPRT transcripts and normalized to those obtained for 2-month-old BALB/c samples used as reference. Data are mean±S.E.M. ( n =6–10 performed in duplicates). ( d–f ) Flow cytometry analyses 10-month-old SAMR1 and SAMP8 mice spleen samples. Representative dot-plots are shown for each mouse. Numbers inside the plots are frequencies of each population (mean±S.E.M.: n =4 for T FH cells and for GL7 + and IgD+ cells, and n =3 for memBC). Fluorescence scales are logarithmic. ( d ) Staining was performed with anti-CD4-APC, anti-CXCR5-Violet-421 and anti-PD1-biotynilated and revealed with streptavidin-PE-Cy7. T FH cells were gated as CD4 + CXCR5 + PD1 + cells (boxes). ( e ) MemBC were determined as CD11b − Gr1 − CD138 − IgM − IgD − CD19 + CD38 + IgG1 + by using anti-CD11b- and anti-Gr1-PE-Cy7, anti-CD138-PE, anti-IgM- and anti-IgD-FITC, anti-CD19-Violet-421, anti-CD38-APC and rat anti-mouse IgG1-biotynilated (clone A85-1, rat IgG1/k). The FMO control was a biotinylated rat IgG1/ k (clone R3-34). Biotynilated Abs were revealed with streptavidin-APC-Cy7. The histograms display the IgG1 + cells among the CD138 − CD38 + cells (empty, FMO isotype control; filled gray, IgG1 + cells). ( f ) Staining was performed with anti-CD19-Violet-421, anti-GL7-PE and anti-IgD-FITC, to determine GL7 + cells and naive IgD + cells on gated CD19 cells. ( g ) Bar graph shows the absolute number/spleen of T FH , GL7 + , memBC and naive B cells (right Y scale). These absolute numbers were calculated from the frequencies of each population. Shown are the mean±S.E.M. ( n =4 and 3 for memBC). The group comparisons were made using the unpaired two-tailed Student’s t -test: * P

    Journal: Cell Death & Disease

    Article Title: Altered marginal zone and innate-like B cells in aged senescence-accelerated SAMP8 mice with defective IgG1 responses

    doi: 10.1038/cddis.2017.351

    Figure Lengend Snippet: Plasma cell B cell maturation and distribution of the memory B-cell compartments in aged SAMP8 mice. ( a ) Cells were stained with anti-CD19-Violet-421, anti-CD45R-PE and anti-CD138-APC. Absolute numbers of CD138 + plasmablasts were calculated from flow cytometry as in Figure 1c . Data are mean±S.E.M., ( n =4). ( b ) IgG1-specific ELISPOT analysis (see ‘Materials and Methods’) of purified samples of B1REL and B2 cells obtained from aged SAMR1 and SAMP8 mice. The numbers of IgG1- antibody secreting cells (ASCs) are shown. Data are mean±S.E.M., ( n =4). ( c ) RT-qPCR analyses were performed for Pax5, Xbp-1, Blimp1 and AID transcript expression in purified B1REL cells from 10-month-old SAMR1 and SAMP8 mice (see ‘Materials and Methods’). The Bio-Rad CFX Manager software (Bio-Rad, Hercules, CA, USA) was used to calculate the C T of each reaction. The amount of specific transcripts in each cDNA sample was determined as the 2 −ΔΔ C T , relative to that of the HPRT transcripts and normalized to those obtained for 2-month-old BALB/c samples used as reference. Data are mean±S.E.M. ( n =6–10 performed in duplicates). ( d–f ) Flow cytometry analyses 10-month-old SAMR1 and SAMP8 mice spleen samples. Representative dot-plots are shown for each mouse. Numbers inside the plots are frequencies of each population (mean±S.E.M.: n =4 for T FH cells and for GL7 + and IgD+ cells, and n =3 for memBC). Fluorescence scales are logarithmic. ( d ) Staining was performed with anti-CD4-APC, anti-CXCR5-Violet-421 and anti-PD1-biotynilated and revealed with streptavidin-PE-Cy7. T FH cells were gated as CD4 + CXCR5 + PD1 + cells (boxes). ( e ) MemBC were determined as CD11b − Gr1 − CD138 − IgM − IgD − CD19 + CD38 + IgG1 + by using anti-CD11b- and anti-Gr1-PE-Cy7, anti-CD138-PE, anti-IgM- and anti-IgD-FITC, anti-CD19-Violet-421, anti-CD38-APC and rat anti-mouse IgG1-biotynilated (clone A85-1, rat IgG1/k). The FMO control was a biotinylated rat IgG1/ k (clone R3-34). Biotynilated Abs were revealed with streptavidin-APC-Cy7. The histograms display the IgG1 + cells among the CD138 − CD38 + cells (empty, FMO isotype control; filled gray, IgG1 + cells). ( f ) Staining was performed with anti-CD19-Violet-421, anti-GL7-PE and anti-IgD-FITC, to determine GL7 + cells and naive IgD + cells on gated CD19 cells. ( g ) Bar graph shows the absolute number/spleen of T FH , GL7 + , memBC and naive B cells (right Y scale). These absolute numbers were calculated from the frequencies of each population. Shown are the mean±S.E.M. ( n =4 and 3 for memBC). The group comparisons were made using the unpaired two-tailed Student’s t -test: * P

    Article Snippet: Standard curves for each Ig isotype were generated using purified myeloma proteins or antibodies: IgM (clone G155-228, BD Biosciences), IgG1 (clone 4.19, in-house), IgG2a (clone PK136, in-house), IgG2b (clone Y.3, in-house), IgG3 (clone MG3-3S, Biolegend, San Diego, CA, USA).

    Techniques: Mouse Assay, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunospot, Purification, Quantitative RT-PCR, Expressing, Software, Fluorescence, Two Tailed Test

    In vitro and in vivo responses of B-cell subsets from SAMP8 mice stimulated with LPS ( a ) and ( b ). The indicated B-cell subsets identified as in Figure 1b from spleens of 10-month-old SAMP8 and SAMR1 mice were FACS-purified, and then labeled with the CellTrace Violet kit before culturing for 72 h in the presence or absence of LPS (see Materials and Methods). After culture, the cells were washed and stained to detect CD138 and the incorporated violet dye by flow cytometry. ( a ) Representative results from B1REL, ABC and B2 cells after LPS stimulation are shown. Fluorescence scales are logarithmic. The numbers in the plots are the frequency in each quadrant. Data are means±S.E.M. ( n =3). ( b ) The IgM and IgG1 secreted into the culture medium after 72 h were determined by ELISA. Data are means±S.E.M. ( n =3). Comparisons were made using a two-tailed Student’s t -test: * P

    Journal: Cell Death & Disease

    Article Title: Altered marginal zone and innate-like B cells in aged senescence-accelerated SAMP8 mice with defective IgG1 responses

    doi: 10.1038/cddis.2017.351

    Figure Lengend Snippet: In vitro and in vivo responses of B-cell subsets from SAMP8 mice stimulated with LPS ( a ) and ( b ). The indicated B-cell subsets identified as in Figure 1b from spleens of 10-month-old SAMP8 and SAMR1 mice were FACS-purified, and then labeled with the CellTrace Violet kit before culturing for 72 h in the presence or absence of LPS (see Materials and Methods). After culture, the cells were washed and stained to detect CD138 and the incorporated violet dye by flow cytometry. ( a ) Representative results from B1REL, ABC and B2 cells after LPS stimulation are shown. Fluorescence scales are logarithmic. The numbers in the plots are the frequency in each quadrant. Data are means±S.E.M. ( n =3). ( b ) The IgM and IgG1 secreted into the culture medium after 72 h were determined by ELISA. Data are means±S.E.M. ( n =3). Comparisons were made using a two-tailed Student’s t -test: * P

    Article Snippet: Standard curves for each Ig isotype were generated using purified myeloma proteins or antibodies: IgM (clone G155-228, BD Biosciences), IgG1 (clone 4.19, in-house), IgG2a (clone PK136, in-house), IgG2b (clone Y.3, in-house), IgG3 (clone MG3-3S, Biolegend, San Diego, CA, USA).

    Techniques: In Vitro, In Vivo, Mouse Assay, FACS, Purification, Labeling, Staining, Flow Cytometry, Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Two Tailed Test