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  • 96
    Bio X Cell rat igg2 b isotype control antibody
    Rat Igg2 B Isotype Control Antibody, supplied by Bio X Cell, used in various techniques. Bioz Stars score: 96/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher igg2b
    Total and virus-specific antibodies in class II-deficient (Ab°) and class I-deficient β 2 m [−/−]) mice. Total and TMEV-specific <t>IgG</t> and IgM in serum of chronically TMEV-infected Ab° (N = 6), β 2 m (−/−) (N = 4), and nonmutant C57BL/6 (N = 3) mice were determined by indirect ELISA. (A) Total IgG in Ab° was comparable to nonmutant C57BL/6 whereas (B) IgM antibodies were higher in Ab° mice than in C57BL/6 and β 2 m (−/−) mice. (C) TMEV-specific IgG was not present in Ab° mice but was detected in C57BL/6 and β 2 m (−/−) mice. (D) Ab° mice showed TMEV-specific IgM which was not observed in nonmutant C57BL/6 and β 2 m (−/−) mice. Because sera from Ab° mice did not neutralize TMEV in vitro (Insert in D), whereas sera from C57BL/6 and β 2 m (−/−) did, this indicated that the TMEV-neutralizing antibodies are predominantly IgG. Pooled serum from noninfected C57BL/6 mice was used as a negative control.
    Igg2b, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1653 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Bio-Rad igg 2b
    Total and virus-specific antibodies in class II-deficient (Ab°) and class I-deficient β 2 m [−/−]) mice. Total and TMEV-specific <t>IgG</t> and IgM in serum of chronically TMEV-infected Ab° (N = 6), β 2 m (−/−) (N = 4), and nonmutant C57BL/6 (N = 3) mice were determined by indirect ELISA. (A) Total IgG in Ab° was comparable to nonmutant C57BL/6 whereas (B) IgM antibodies were higher in Ab° mice than in C57BL/6 and β 2 m (−/−) mice. (C) TMEV-specific IgG was not present in Ab° mice but was detected in C57BL/6 and β 2 m (−/−) mice. (D) Ab° mice showed TMEV-specific IgM which was not observed in nonmutant C57BL/6 and β 2 m (−/−) mice. Because sera from Ab° mice did not neutralize TMEV in vitro (Insert in D), whereas sera from C57BL/6 and β 2 m (−/−) did, this indicated that the TMEV-neutralizing antibodies are predominantly IgG. Pooled serum from noninfected C57BL/6 mice was used as a negative control.
    Igg 2b, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Miltenyi Biotec isotype control mouse igg 2b pe
    Total and virus-specific antibodies in class II-deficient (Ab°) and class I-deficient β 2 m [−/−]) mice. Total and TMEV-specific <t>IgG</t> and IgM in serum of chronically TMEV-infected Ab° (N = 6), β 2 m (−/−) (N = 4), and nonmutant C57BL/6 (N = 3) mice were determined by indirect ELISA. (A) Total IgG in Ab° was comparable to nonmutant C57BL/6 whereas (B) IgM antibodies were higher in Ab° mice than in C57BL/6 and β 2 m (−/−) mice. (C) TMEV-specific IgG was not present in Ab° mice but was detected in C57BL/6 and β 2 m (−/−) mice. (D) Ab° mice showed TMEV-specific IgM which was not observed in nonmutant C57BL/6 and β 2 m (−/−) mice. Because sera from Ab° mice did not neutralize TMEV in vitro (Insert in D), whereas sera from C57BL/6 and β 2 m (−/−) did, this indicated that the TMEV-neutralizing antibodies are predominantly IgG. Pooled serum from noninfected C57BL/6 mice was used as a negative control.
    Isotype Control Mouse Igg 2b Pe, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 89/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Nordic-Mubio igg
    Vacc-4x Ig antibody levels. Changes (Δ) from baseline to end of study in rectal <t>IgA,</t> nasal IgA and serum <t>IgG</t> antibody levels in the four dose groups. Adj = adjuvant, LD = low, MD = median and HD = high dose. IgA antibody levels are adjusted to total IgA in rectal and nasal samples (please note different scales). Data are given as medians, interquartile and overall ranges. Differences between dose groups and the adjuvant group with p-values less than 0.10 are indicated (Mann-Whitney U test). Kruskal-Wallis ANOVA analysis for all four groups yielded p = 0.044 (rectal IgA), p = 0.093 (nasal IgA) and p = 0.067 (serum IgG).
    Igg, supplied by Nordic-Mubio, used in various techniques. Bioz Stars score: 86/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Bio-Rad isotype matched control igg 2b
    Vacc-4x Ig antibody levels. Changes (Δ) from baseline to end of study in rectal <t>IgA,</t> nasal IgA and serum <t>IgG</t> antibody levels in the four dose groups. Adj = adjuvant, LD = low, MD = median and HD = high dose. IgA antibody levels are adjusted to total IgA in rectal and nasal samples (please note different scales). Data are given as medians, interquartile and overall ranges. Differences between dose groups and the adjuvant group with p-values less than 0.10 are indicated (Mann-Whitney U test). Kruskal-Wallis ANOVA analysis for all four groups yielded p = 0.044 (rectal IgA), p = 0.093 (nasal IgA) and p = 0.067 (serum IgG).
    Isotype Matched Control Igg 2b, supplied by Bio-Rad, 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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    85
    Becton Dickinson irrelevant antibody igg 2b
    Vacc-4x Ig antibody levels. Changes (Δ) from baseline to end of study in rectal <t>IgA,</t> nasal IgA and serum <t>IgG</t> antibody levels in the four dose groups. Adj = adjuvant, LD = low, MD = median and HD = high dose. IgA antibody levels are adjusted to total IgA in rectal and nasal samples (please note different scales). Data are given as medians, interquartile and overall ranges. Differences between dose groups and the adjuvant group with p-values less than 0.10 are indicated (Mann-Whitney U test). Kruskal-Wallis ANOVA analysis for all four groups yielded p = 0.044 (rectal IgA), p = 0.093 (nasal IgA) and p = 0.067 (serum IgG).
    Irrelevant Antibody Igg 2b, supplied by Becton Dickinson, 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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    91
    Becton Dickinson rat immunoglobulin g igg 2b
    Allo-Ab assay after intravenous injection of allogeneic B cells. B cells of SPL or PerC, or non-B cells of PerC isolated from Balb/c mice were injected into B6 mice trough the tail vein. To evaluate allo-Ab production, sera of recipient B6 mice were obtained every week after the adoptive transfer, and were incubated together with splenocytes from Balb/c mice as target cells. Mice in the control group received no adoptive transfer of cells. (A) Allo antibody response after the intravenous injection. Anti-Balb/c IgM or <t>IgG</t> was evaluated by FCM assay, and MFI was calculated (error bars, SEM). CD19 + B cells of the Balb/c splenocytes were excluded from the analyses, because they included surface IgM or IgG expressing cells. (B) Subclasses of anti-Balb/c IgGs produced after the intravenous injection. <t>Anti-IgG1,</t> <t>anti-IgG2a/2b,</t> or <t>anti-IgG3</t> was used to detect the production of allo-IgG subclasses and evaluated by FCM assay. Data are presented as MFI ± SEM; * p
    Rat Immunoglobulin G Igg 2b, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Agilent technologies mn normal mouse igg2 b
    Allo-Ab assay after intravenous injection of allogeneic B cells. B cells of SPL or PerC, or non-B cells of PerC isolated from Balb/c mice were injected into B6 mice trough the tail vein. To evaluate allo-Ab production, sera of recipient B6 mice were obtained every week after the adoptive transfer, and were incubated together with splenocytes from Balb/c mice as target cells. Mice in the control group received no adoptive transfer of cells. (A) Allo antibody response after the intravenous injection. Anti-Balb/c IgM or <t>IgG</t> was evaluated by FCM assay, and MFI was calculated (error bars, SEM). CD19 + B cells of the Balb/c splenocytes were excluded from the analyses, because they included surface IgM or IgG expressing cells. (B) Subclasses of anti-Balb/c IgGs produced after the intravenous injection. <t>Anti-IgG1,</t> <t>anti-IgG2a/2b,</t> or <t>anti-IgG3</t> was used to detect the production of allo-IgG subclasses and evaluated by FCM assay. Data are presented as MFI ± SEM; * p
    Mn Normal Mouse Igg2 B, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Becton Dickinson rat igg 2b isotype control
    Allo-Ab assay after intravenous injection of allogeneic B cells. B cells of SPL or PerC, or non-B cells of PerC isolated from Balb/c mice were injected into B6 mice trough the tail vein. To evaluate allo-Ab production, sera of recipient B6 mice were obtained every week after the adoptive transfer, and were incubated together with splenocytes from Balb/c mice as target cells. Mice in the control group received no adoptive transfer of cells. (A) Allo antibody response after the intravenous injection. Anti-Balb/c IgM or <t>IgG</t> was evaluated by FCM assay, and MFI was calculated (error bars, SEM). CD19 + B cells of the Balb/c splenocytes were excluded from the analyses, because they included surface IgM or IgG expressing cells. (B) Subclasses of anti-Balb/c IgGs produced after the intravenous injection. <t>Anti-IgG1,</t> <t>anti-IgG2a/2b,</t> or <t>anti-IgG3</t> was used to detect the production of allo-IgG subclasses and evaluated by FCM assay. Data are presented as MFI ± SEM; * p
    Rat Igg 2b Isotype Control, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher igg2b apc
    Allo-Ab assay after intravenous injection of allogeneic B cells. B cells of SPL or PerC, or non-B cells of PerC isolated from Balb/c mice were injected into B6 mice trough the tail vein. To evaluate allo-Ab production, sera of recipient B6 mice were obtained every week after the adoptive transfer, and were incubated together with splenocytes from Balb/c mice as target cells. Mice in the control group received no adoptive transfer of cells. (A) Allo antibody response after the intravenous injection. Anti-Balb/c IgM or <t>IgG</t> was evaluated by FCM assay, and MFI was calculated (error bars, SEM). CD19 + B cells of the Balb/c splenocytes were excluded from the analyses, because they included surface IgM or IgG expressing cells. (B) Subclasses of anti-Balb/c IgGs produced after the intravenous injection. <t>Anti-IgG1,</t> <t>anti-IgG2a/2b,</t> or <t>anti-IgG3</t> was used to detect the production of allo-IgG subclasses and evaluated by FCM assay. Data are presented as MFI ± SEM; * p
    Igg2b Apc, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Pharmingen igg2b
    Serum anti-Fx1A Ab IgG isotype switching and subclass production in HN were not altered by CD8 + T cell depletion. IgG subclasses were detected in similar proportions at 4 and 8 wk in CD8-depleted HN, ATx/HN, and normal HN groups. CFA controls did not mount an Fx1A Ab response. At 4 wk, only the Th2-mediated IgG1 response was significantly raised above controls. By 8 wk the IgG1 response had increased further, and a significant Th1 mediated IgG2a response was identified compared with CFA controls. There was no <t>significant-IgG2b</t> response. Values represent mean ± SEM ( n = 5) of anti-Fx1A Ab levels, expressed as percentage binding of known strongly positive serum (Fx1A Ab titer 1:250), measured by ELISA. * P
    Igg2b, supplied by Pharmingen, used in various techniques. Bioz Stars score: 94/100, based on 294 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    BioLegend igg2b
    CC induce PTF1.2 dependent on the TF pathway. ( A ) PTF1.2 induction over time (60, 120, and 240 min) upon exposure to CC 2000 μg/ml, PBS/HSA, E. coli (1 × 10 7 particles/ml), or glass ( n = 7 donors) in human whole blood. ( B ) Effect of TF inhibition following exposure of CC (500, 1000, and 2000 μg/ml) or E.coli after addition of the functional-grade inhibitory Ab against TF (anti-TF) or the corresponding Ultra-LEAF purified IgG1κ control Ab (Ctr <t>IgG)</t> for 240 min in human whole blood ( n = 6 donors). ( C ) Effect of factor XII inhibition by CTI (40 μg/ml) in PFP following incubation with CC (2000 μg/ml) or glass for 60 min ( n = 5). Effect of CC on platelet aggregation in human whole blood measured by electrical impedance aggregometry in ( D ) by CC (2000 μg/ml) or its controls, HSA/PBS, or TRAP ( n = 6), ( E ) by additional stimulation with ADP ( n = 3), and in ( F ) with TRAP following 6 min of incubation ( n = 3), given as arbitrary units × min. In all experiments, thrombin was inhibited with lepirudin. All data are given as means ± SEM. * p
    Igg2b, supplied by BioLegend, used in various techniques. Bioz Stars score: 90/100, based on 390 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore igg2a
    Figure 6. Immune responses of infected mouse groups. ( A–E ) correspond to the humoral response. Results are expressed as the mean O.D. 490 ± standard deviation at different times pi. ( A ) IgG1 antibody results, ( B ) <t>IgG2a,</t> ( C ) IgG2b,
    Igg2a, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1392 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Miltenyi Biotec igg2a
    Figure 6. Immune responses of infected mouse groups. ( A–E ) correspond to the humoral response. Results are expressed as the mean O.D. 490 ± standard deviation at different times pi. ( A ) IgG1 antibody results, ( B ) <t>IgG2a,</t> ( C ) IgG2b,
    Igg2a, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 92/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson igg1
    Heterogeneity of switched memory B cells is differentially represented in spleen and bone marrow. Cells for single cell sequencing were FACSorted as <t>IgG-expressing</t> CD19+CD38+CD138-GL7-small lymphocytes. a) Six transcriptionally defined clusters were identified by shared nearest neighbor (SNN) modularity optimization based clustering algorithm mapped to tSNE representation of spleen and BM cells. tSNE coordinates and clustering was computed for 4754 from spleen and 2947 from BM cells, presentation is separated by organ. b) Percentage of cells per cluster in each organ by mouse. c) Distribution of IgG subclass mapped on tSNE. d) Signature genes for each cluster, area under curve (AUC) of responder-operator characteristics (ROC) of > 0.7. e) Distribution of transcription levels for representative genes mapped on tSNE. Cells for single cell sequencing were FACSorted as IgG-expressing CD19+CD38+CD138-GL7-small lymphocytes. f) Flow-cytometric evaluation of expression of representative markers to identify clusters observed by transcriptional profiles in IgG+ memory B cells <t>(IgG1+</t> or <t>IgG2b+CD19+CD38+IgM-IgD-CD138-GL7-CD93-Zombie</t> Aqua-small lymphocytes). Dotplots represent one of three mice.
    Igg1, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 3105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    GeneTex igg2a
    Trial 2 VapA specific antibody responses. Mean and SD of VapA total IgG triplicate OD values at a 1:100 dilution of mouse sera. A representation of time at which sera was collected at day 0 (pre vaccination), day 14 (boost vaccination), day 28 (pre challenge) and day 35 (1 week post challenge, pre euthanasia) of each vaccine group; R . equi , Empty vector negative control, HAdV- vapA high dose and HAdV- vapA low dose. Top left total IgG antibody, top right IgG1 antibody, bottom left <t>IgG2a</t> antibody, bottom right IgG2b antibody.
    Igg2a, supplied by GeneTex, used in various techniques. Bioz Stars score: 90/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore igg2b isotype
    P2X receptor expression on human monocytes. Freshly isolated monocytes were analyzed by FACS analysis. a After blocking the cells with 1% goat serum, the surface antigen expression was analyzed using an anti-P2X 1 Ab ( dotted line ) or the respective <t>IgG</t>
    Igg2b Isotype, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher mouse igg1
    Effects of adsorption of LPS or MPLA onto TT/alum sensitization on serum isotype production. Protocol as shown in Figure 2(a) . Concentration of serum isotypes: (a) total IgE, (b) TT-specific <t>IgG1,</t> and (c) TT-specific IgG2a. Data shown as mean ± SE, one-way ANOVA: # P
    Mouse Igg1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 1175 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    SouthernBiotech igg1
    Antibody response to peptide vaccination. Serum <t>IgG</t> was measured in the serum of vaccinated animals at day 49 post vaccination (Study 1, n = 5). IgG against each respective G peptide ( A ), CX3C peptide ( B ) or RSV A2 ( C ) were detected by indirect ELISA assay. Experiments were independently repeated at least 3 times and analyzed by one-way ANOVA test. Ns, no significant; ** p
    Igg1, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 94/100, based on 4380 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher mouse igg2b κ
    FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) are all involved in ADE of ZIKV infection by DENV immune sera. PBMCs from healthy donors (n = 3) were treated with monoclonal antibodies (1 μg/ml, 3 μg/ml or 10 μg/ml) against each FcγR types or combined (1 μg/ml, 3 μg/ml or 10 μg/ml in total), or with <t>IgG</t> control antibody prior to performing the boosted ZIKV ADE assay with DENV-1 immune serum (D1-E01, 1/2,500 dilution). The percentage of anti-E positive Vero cells in the control group, where no anti-FcγR antibodies were applied at the ADE condition was defined as 100%. The mean and standard deviation of results from three independent experiments without blocking antibody treatment (A) or with blocking antibody treatment (B) were presented. Statistical significances were determined by comparison of the relative proportion of infected cells with blocking antibody treatment to that of the control IgG antibody treatment under the same antibody concentration using unpaired two-tailed Student’s t test and designated as “*”for p
    Mouse Igg2b κ, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Boehringer Mannheim murine antiphysarum β tubulin igg 2b
    Concentration-dependent effect of TSP on tyrosine phosphorylation of EC proteins in the presence of PTP inhibition. ECs were exposed for 1 h to TSP or media alone with or without vanadate (200 μM), PAO (1.0 μM), or both. Cell lysates were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes, and the blots were probed with antiphosphotyrosine <t>IgG.</t> (A) Phosphotyrosine-containing proteins in extracts of ECs exposed to TSP (20 μg/ml, 1 h) or media alone in the presence and absence of vanadate, PAO, or both. Arrows indicate bands with apparent M r of 240,000, 205,000, 185,000, 135,000, 110,000, 95,000, and 66,000. (B) Phosphotyrosine-containing proteins in EC extracts obtained from monolayers exposed for 1 h to increasing concentrations of TSP in the presence of fixed concentrations of both vanadate (200 μM) and PAO (1.0 μM). To confirm equivalent protein loading, each blot was stripped and reprobed with <t>anti-β-tubulin</t> antibody. Molecular weights in kilodaltons are indicated on the left. Each is a representative blot of three experiments.
    Murine Antiphysarum β Tubulin Igg 2b, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 80/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher igg2a fitc
    Concentration-dependent effect of TSP on tyrosine phosphorylation of EC proteins in the presence of PTP inhibition. ECs were exposed for 1 h to TSP or media alone with or without vanadate (200 μM), PAO (1.0 μM), or both. Cell lysates were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes, and the blots were probed with antiphosphotyrosine <t>IgG.</t> (A) Phosphotyrosine-containing proteins in extracts of ECs exposed to TSP (20 μg/ml, 1 h) or media alone in the presence and absence of vanadate, PAO, or both. Arrows indicate bands with apparent M r of 240,000, 205,000, 185,000, 135,000, 110,000, 95,000, and 66,000. (B) Phosphotyrosine-containing proteins in EC extracts obtained from monolayers exposed for 1 h to increasing concentrations of TSP in the presence of fixed concentrations of both vanadate (200 μM) and PAO (1.0 μM). To confirm equivalent protein loading, each blot was stripped and reprobed with <t>anti-β-tubulin</t> antibody. Molecular weights in kilodaltons are indicated on the left. Each is a representative blot of three experiments.
    Igg2a Fitc, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 144 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Total and virus-specific antibodies in class II-deficient (Ab°) and class I-deficient β 2 m [−/−]) mice. Total and TMEV-specific IgG and IgM in serum of chronically TMEV-infected Ab° (N = 6), β 2 m (−/−) (N = 4), and nonmutant C57BL/6 (N = 3) mice were determined by indirect ELISA. (A) Total IgG in Ab° was comparable to nonmutant C57BL/6 whereas (B) IgM antibodies were higher in Ab° mice than in C57BL/6 and β 2 m (−/−) mice. (C) TMEV-specific IgG was not present in Ab° mice but was detected in C57BL/6 and β 2 m (−/−) mice. (D) Ab° mice showed TMEV-specific IgM which was not observed in nonmutant C57BL/6 and β 2 m (−/−) mice. Because sera from Ab° mice did not neutralize TMEV in vitro (Insert in D), whereas sera from C57BL/6 and β 2 m (−/−) did, this indicated that the TMEV-neutralizing antibodies are predominantly IgG. Pooled serum from noninfected C57BL/6 mice was used as a negative control.

    Journal: Journal of neuropathology and experimental neurology

    Article Title: Absence of Spontaneous Central Nervous System Remyelination in Class II-deficient Mice Infected with Theiler’s Virus

    doi:

    Figure Lengend Snippet: Total and virus-specific antibodies in class II-deficient (Ab°) and class I-deficient β 2 m [−/−]) mice. Total and TMEV-specific IgG and IgM in serum of chronically TMEV-infected Ab° (N = 6), β 2 m (−/−) (N = 4), and nonmutant C57BL/6 (N = 3) mice were determined by indirect ELISA. (A) Total IgG in Ab° was comparable to nonmutant C57BL/6 whereas (B) IgM antibodies were higher in Ab° mice than in C57BL/6 and β 2 m (−/−) mice. (C) TMEV-specific IgG was not present in Ab° mice but was detected in C57BL/6 and β 2 m (−/−) mice. (D) Ab° mice showed TMEV-specific IgM which was not observed in nonmutant C57BL/6 and β 2 m (−/−) mice. Because sera from Ab° mice did not neutralize TMEV in vitro (Insert in D), whereas sera from C57BL/6 and β 2 m (−/−) did, this indicated that the TMEV-neutralizing antibodies are predominantly IgG. Pooled serum from noninfected C57BL/6 mice was used as a negative control.

    Article Snippet: To ensure detection of all isotypes, bound IgGs were detected by incubating plates with a mixture of rabbit anti-mouse IgG1, IgG2a, IgG2b, and IgG3 (Zymed) at 37°C for 2 hours.

    Techniques: Mouse Assay, Infection, Indirect ELISA, In Vitro, Negative Control

    Vacc-4x Ig antibody levels. Changes (Δ) from baseline to end of study in rectal IgA, nasal IgA and serum IgG antibody levels in the four dose groups. Adj = adjuvant, LD = low, MD = median and HD = high dose. IgA antibody levels are adjusted to total IgA in rectal and nasal samples (please note different scales). Data are given as medians, interquartile and overall ranges. Differences between dose groups and the adjuvant group with p-values less than 0.10 are indicated (Mann-Whitney U test). Kruskal-Wallis ANOVA analysis for all four groups yielded p = 0.044 (rectal IgA), p = 0.093 (nasal IgA) and p = 0.067 (serum IgG).

    Journal: PLoS ONE

    Article Title: Intranasal Administration of a Therapeutic HIV Vaccine (Vacc-4x) Induces Dose-Dependent Systemic and Mucosal Immune Responses in a Randomized Controlled Trial

    doi: 10.1371/journal.pone.0112556

    Figure Lengend Snippet: Vacc-4x Ig antibody levels. Changes (Δ) from baseline to end of study in rectal IgA, nasal IgA and serum IgG antibody levels in the four dose groups. Adj = adjuvant, LD = low, MD = median and HD = high dose. IgA antibody levels are adjusted to total IgA in rectal and nasal samples (please note different scales). Data are given as medians, interquartile and overall ranges. Differences between dose groups and the adjuvant group with p-values less than 0.10 are indicated (Mann-Whitney U test). Kruskal-Wallis ANOVA analysis for all four groups yielded p = 0.044 (rectal IgA), p = 0.093 (nasal IgA) and p = 0.067 (serum IgG).

    Article Snippet: Purified human IgA (dimer) and IgG (pooled serum) (Nordic Immunological Laboratories, Tilburg, the Netherlands) were used as standards.

    Techniques: MANN-WHITNEY

    Allo-Ab assay after intravenous injection of allogeneic B cells. B cells of SPL or PerC, or non-B cells of PerC isolated from Balb/c mice were injected into B6 mice trough the tail vein. To evaluate allo-Ab production, sera of recipient B6 mice were obtained every week after the adoptive transfer, and were incubated together with splenocytes from Balb/c mice as target cells. Mice in the control group received no adoptive transfer of cells. (A) Allo antibody response after the intravenous injection. Anti-Balb/c IgM or IgG was evaluated by FCM assay, and MFI was calculated (error bars, SEM). CD19 + B cells of the Balb/c splenocytes were excluded from the analyses, because they included surface IgM or IgG expressing cells. (B) Subclasses of anti-Balb/c IgGs produced after the intravenous injection. Anti-IgG1, anti-IgG2a/2b, or anti-IgG3 was used to detect the production of allo-IgG subclasses and evaluated by FCM assay. Data are presented as MFI ± SEM; * p

    Journal: PLoS ONE

    Article Title: PD-L1/PD-L2-expressing B-1 cells inhibit alloreactive T cells in mice

    doi: 10.1371/journal.pone.0178765

    Figure Lengend Snippet: Allo-Ab assay after intravenous injection of allogeneic B cells. B cells of SPL or PerC, or non-B cells of PerC isolated from Balb/c mice were injected into B6 mice trough the tail vein. To evaluate allo-Ab production, sera of recipient B6 mice were obtained every week after the adoptive transfer, and were incubated together with splenocytes from Balb/c mice as target cells. Mice in the control group received no adoptive transfer of cells. (A) Allo antibody response after the intravenous injection. Anti-Balb/c IgM or IgG was evaluated by FCM assay, and MFI was calculated (error bars, SEM). CD19 + B cells of the Balb/c splenocytes were excluded from the analyses, because they included surface IgM or IgG expressing cells. (B) Subclasses of anti-Balb/c IgGs produced after the intravenous injection. Anti-IgG1, anti-IgG2a/2b, or anti-IgG3 was used to detect the production of allo-IgG subclasses and evaluated by FCM assay. Data are presented as MFI ± SEM; * p

    Article Snippet: Rat immunoglobulin G (IgG) 2b (R35-38, BD Pharmingen) and IgG2a (R35-95, BD Pharmingen) were used as isotype-matched control IgGs for anti-PD-L1 and anti-PD-L2 mAbs, respectively.

    Techniques: Injection, Isolation, Mouse Assay, Adoptive Transfer Assay, Incubation, Expressing, Produced

    Phenotypic analysis of B cells in the organs of Balb/c mice. We investigated the phenotypic characteristics of naïve B cells isolated from the PerC, SPL, liver, BM, PB, and CLN of Balb/c mice by performing anatomical screening of immune-regulatory markers PD-L1, PD-L2, FasL, and TRAIL and co-stimulatory membrane markers CD80, CD86, and I-A/I-E (MHC class II) by performing FCM analysis. Anti-CD19 mAb was used as a B cell marker. (A) Histograms indicating representative FCM results of phenotypic analysis of B cells. Dashed lines indicate the isotype-matched control IgG. (B) Percentage (mean ± SEM) expression of each membrane marker expressed on B cells from each organ. * p

    Journal: PLoS ONE

    Article Title: PD-L1/PD-L2-expressing B-1 cells inhibit alloreactive T cells in mice

    doi: 10.1371/journal.pone.0178765

    Figure Lengend Snippet: Phenotypic analysis of B cells in the organs of Balb/c mice. We investigated the phenotypic characteristics of naïve B cells isolated from the PerC, SPL, liver, BM, PB, and CLN of Balb/c mice by performing anatomical screening of immune-regulatory markers PD-L1, PD-L2, FasL, and TRAIL and co-stimulatory membrane markers CD80, CD86, and I-A/I-E (MHC class II) by performing FCM analysis. Anti-CD19 mAb was used as a B cell marker. (A) Histograms indicating representative FCM results of phenotypic analysis of B cells. Dashed lines indicate the isotype-matched control IgG. (B) Percentage (mean ± SEM) expression of each membrane marker expressed on B cells from each organ. * p

    Article Snippet: Rat immunoglobulin G (IgG) 2b (R35-38, BD Pharmingen) and IgG2a (R35-95, BD Pharmingen) were used as isotype-matched control IgGs for anti-PD-L1 and anti-PD-L2 mAbs, respectively.

    Techniques: Mouse Assay, Isolation, Marker, Expressing

    CFSE-MLR assay after the intravenous injection of PerC B cells pre-incubated with anti-PD-L1 and/or anti-PD-L2 mAbs. PerC B cells from Balb/c mice were isolated, pre-incubated with anti-PD-L1 and/or anti-PD-L2 mAb for 30 min, and injected to B6 mice through the tail vein. Isotype-matched control IgGs were used instead of anti-PD-L1 or anti-PD-L2 mAb. Rat IgG2b was used instead of anti-PD-L1 mAb, and Rat IgG2a was used instead of anti-PD-L2 mAb. Mice in the control group did not receive adoptive transfer of cells or mAbs. T-cell alloreactivity was determined by performing the CFSE-MLR assay 2 weeks after the intravenous injection. Upper row shows the mean SIs of CD4 + and CD8 + T cells, and lower row shows the mean PFs of CD4 + and CD8 + T cells; * p

    Journal: PLoS ONE

    Article Title: PD-L1/PD-L2-expressing B-1 cells inhibit alloreactive T cells in mice

    doi: 10.1371/journal.pone.0178765

    Figure Lengend Snippet: CFSE-MLR assay after the intravenous injection of PerC B cells pre-incubated with anti-PD-L1 and/or anti-PD-L2 mAbs. PerC B cells from Balb/c mice were isolated, pre-incubated with anti-PD-L1 and/or anti-PD-L2 mAb for 30 min, and injected to B6 mice through the tail vein. Isotype-matched control IgGs were used instead of anti-PD-L1 or anti-PD-L2 mAb. Rat IgG2b was used instead of anti-PD-L1 mAb, and Rat IgG2a was used instead of anti-PD-L2 mAb. Mice in the control group did not receive adoptive transfer of cells or mAbs. T-cell alloreactivity was determined by performing the CFSE-MLR assay 2 weeks after the intravenous injection. Upper row shows the mean SIs of CD4 + and CD8 + T cells, and lower row shows the mean PFs of CD4 + and CD8 + T cells; * p

    Article Snippet: Rat immunoglobulin G (IgG) 2b (R35-38, BD Pharmingen) and IgG2a (R35-95, BD Pharmingen) were used as isotype-matched control IgGs for anti-PD-L1 and anti-PD-L2 mAbs, respectively.

    Techniques: Mlr Assay, Injection, Incubation, Mouse Assay, Isolation, Adoptive Transfer Assay

    Serum anti-Fx1A Ab IgG isotype switching and subclass production in HN were not altered by CD8 + T cell depletion. IgG subclasses were detected in similar proportions at 4 and 8 wk in CD8-depleted HN, ATx/HN, and normal HN groups. CFA controls did not mount an Fx1A Ab response. At 4 wk, only the Th2-mediated IgG1 response was significantly raised above controls. By 8 wk the IgG1 response had increased further, and a significant Th1 mediated IgG2a response was identified compared with CFA controls. There was no significant-IgG2b response. Values represent mean ± SEM ( n = 5) of anti-Fx1A Ab levels, expressed as percentage binding of known strongly positive serum (Fx1A Ab titer 1:250), measured by ELISA. * P

    Journal: The Journal of Experimental Medicine

    Article Title: Permanent CD8+ T Cell Depletion Prevents Proteinuria in Active Heymann Nephritis

    doi:

    Figure Lengend Snippet: Serum anti-Fx1A Ab IgG isotype switching and subclass production in HN were not altered by CD8 + T cell depletion. IgG subclasses were detected in similar proportions at 4 and 8 wk in CD8-depleted HN, ATx/HN, and normal HN groups. CFA controls did not mount an Fx1A Ab response. At 4 wk, only the Th2-mediated IgG1 response was significantly raised above controls. By 8 wk the IgG1 response had increased further, and a significant Th1 mediated IgG2a response was identified compared with CFA controls. There was no significant-IgG2b response. Values represent mean ± SEM ( n = 5) of anti-Fx1A Ab levels, expressed as percentage binding of known strongly positive serum (Fx1A Ab titer 1:250), measured by ELISA. * P

    Article Snippet: Anti-Fx1A Ab titers were determined by ELISA as previously described , using alkaline phosphatase–conjugated Abs: sheep anti–rat Ig Fab fragments ( Boehringer Mannheim, GmbH , Mannheim, Germany) and mouse anti–rat IgG1, IgG2a, and IgG2b ( PharMingen , San Diego, CA).

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

    CD8 + T cell depletion in HN did not prevent glomerular deposition of IgG or C3, but abrogated the infiltration of mononuclear cells including CD8 + cells. ( a–c ) Glomerular IgG and C3 deposition were not different between normal HN and CD8-depleted HN groups. Note the amorphous staining of IgG and C3 within tubular lumina in the proteinuric normal HN rat. ( d ) The glomerular infiltrate of CD8 + cells in normal HN was absent in CD8-depleted HN hosts.

    Journal: The Journal of Experimental Medicine

    Article Title: Permanent CD8+ T Cell Depletion Prevents Proteinuria in Active Heymann Nephritis

    doi:

    Figure Lengend Snippet: CD8 + T cell depletion in HN did not prevent glomerular deposition of IgG or C3, but abrogated the infiltration of mononuclear cells including CD8 + cells. ( a–c ) Glomerular IgG and C3 deposition were not different between normal HN and CD8-depleted HN groups. Note the amorphous staining of IgG and C3 within tubular lumina in the proteinuric normal HN rat. ( d ) The glomerular infiltrate of CD8 + cells in normal HN was absent in CD8-depleted HN hosts.

    Article Snippet: Anti-Fx1A Ab titers were determined by ELISA as previously described , using alkaline phosphatase–conjugated Abs: sheep anti–rat Ig Fab fragments ( Boehringer Mannheim, GmbH , Mannheim, Germany) and mouse anti–rat IgG1, IgG2a, and IgG2b ( PharMingen , San Diego, CA).

    Techniques: Staining

    CD8 + T cell depletion in HN prevented proteinuria in the presence of elevated serum anti-Fx1A Ab levels and glomerular IgG deposition. (a ) Proteinuria in early or late CD8-depleted HN was not significantly greater than that in CFA controls. In normal HN and ATx/HN, proteinuria was detected from 8 wk, and was not different between these groups at 8 and 12 wk. Proteinuria was significantly greater in normal HN and ATx/HN than in early or late CD8-depleted HN, or CFA controls, at 8 ( P

    Journal: The Journal of Experimental Medicine

    Article Title: Permanent CD8+ T Cell Depletion Prevents Proteinuria in Active Heymann Nephritis

    doi:

    Figure Lengend Snippet: CD8 + T cell depletion in HN prevented proteinuria in the presence of elevated serum anti-Fx1A Ab levels and glomerular IgG deposition. (a ) Proteinuria in early or late CD8-depleted HN was not significantly greater than that in CFA controls. In normal HN and ATx/HN, proteinuria was detected from 8 wk, and was not different between these groups at 8 and 12 wk. Proteinuria was significantly greater in normal HN and ATx/HN than in early or late CD8-depleted HN, or CFA controls, at 8 ( P

    Article Snippet: Anti-Fx1A Ab titers were determined by ELISA as previously described , using alkaline phosphatase–conjugated Abs: sheep anti–rat Ig Fab fragments ( Boehringer Mannheim, GmbH , Mannheim, Germany) and mouse anti–rat IgG1, IgG2a, and IgG2b ( PharMingen , San Diego, CA).

    Techniques:

    CC induce PTF1.2 dependent on the TF pathway. ( A ) PTF1.2 induction over time (60, 120, and 240 min) upon exposure to CC 2000 μg/ml, PBS/HSA, E. coli (1 × 10 7 particles/ml), or glass ( n = 7 donors) in human whole blood. ( B ) Effect of TF inhibition following exposure of CC (500, 1000, and 2000 μg/ml) or E.coli after addition of the functional-grade inhibitory Ab against TF (anti-TF) or the corresponding Ultra-LEAF purified IgG1κ control Ab (Ctr IgG) for 240 min in human whole blood ( n = 6 donors). ( C ) Effect of factor XII inhibition by CTI (40 μg/ml) in PFP following incubation with CC (2000 μg/ml) or glass for 60 min ( n = 5). Effect of CC on platelet aggregation in human whole blood measured by electrical impedance aggregometry in ( D ) by CC (2000 μg/ml) or its controls, HSA/PBS, or TRAP ( n = 6), ( E ) by additional stimulation with ADP ( n = 3), and in ( F ) with TRAP following 6 min of incubation ( n = 3), given as arbitrary units × min. In all experiments, thrombin was inhibited with lepirudin. All data are given as means ± SEM. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Cholesterol Crystals Induce Coagulation Activation through Complement-Dependent Expression of Monocytic Tissue Factor

    doi: 10.4049/jimmunol.1900503

    Figure Lengend Snippet: CC induce PTF1.2 dependent on the TF pathway. ( A ) PTF1.2 induction over time (60, 120, and 240 min) upon exposure to CC 2000 μg/ml, PBS/HSA, E. coli (1 × 10 7 particles/ml), or glass ( n = 7 donors) in human whole blood. ( B ) Effect of TF inhibition following exposure of CC (500, 1000, and 2000 μg/ml) or E.coli after addition of the functional-grade inhibitory Ab against TF (anti-TF) or the corresponding Ultra-LEAF purified IgG1κ control Ab (Ctr IgG) for 240 min in human whole blood ( n = 6 donors). ( C ) Effect of factor XII inhibition by CTI (40 μg/ml) in PFP following incubation with CC (2000 μg/ml) or glass for 60 min ( n = 5). Effect of CC on platelet aggregation in human whole blood measured by electrical impedance aggregometry in ( D ) by CC (2000 μg/ml) or its controls, HSA/PBS, or TRAP ( n = 6), ( E ) by additional stimulation with ADP ( n = 3), and in ( F ) with TRAP following 6 min of incubation ( n = 3), given as arbitrary units × min. In all experiments, thrombin was inhibited with lepirudin. All data are given as means ± SEM. * p

    Article Snippet: Isotype controls were purified mouse Igs IgG1 (clone MOPC-21), IgG2a (clone MOPC-172), and IgG2b (clone MPC-11) from BioLegend.

    Techniques: Inhibition, Functional Assay, Purification, Incubation

    Figure 6. Immune responses of infected mouse groups. ( A–E ) correspond to the humoral response. Results are expressed as the mean O.D. 490 ± standard deviation at different times pi. ( A ) IgG1 antibody results, ( B ) IgG2a, ( C ) IgG2b,

    Journal: Virulence

    Article Title: The invA gene of Brucella melitensis is involved in intracellular invasion and is required to establish infection in a mouse model

    doi: 10.4161/viru.28589

    Figure Lengend Snippet: Figure 6. Immune responses of infected mouse groups. ( A–E ) correspond to the humoral response. Results are expressed as the mean O.D. 490 ± standard deviation at different times pi. ( A ) IgG1 antibody results, ( B ) IgG2a, ( C ) IgG2b,

    Article Snippet: IgG1, IgG2a, IgG2b, IgG3, and IgM antibodies were analyzed with Mouse Monoclonal Antibody Isotyping Reagents (Sigma Aldrich, ISO2-1) following the manufacturer’s instructions.

    Techniques: Infection, Standard Deviation

    Heterogeneity of switched memory B cells is differentially represented in spleen and bone marrow. Cells for single cell sequencing were FACSorted as IgG-expressing CD19+CD38+CD138-GL7-small lymphocytes. a) Six transcriptionally defined clusters were identified by shared nearest neighbor (SNN) modularity optimization based clustering algorithm mapped to tSNE representation of spleen and BM cells. tSNE coordinates and clustering was computed for 4754 from spleen and 2947 from BM cells, presentation is separated by organ. b) Percentage of cells per cluster in each organ by mouse. c) Distribution of IgG subclass mapped on tSNE. d) Signature genes for each cluster, area under curve (AUC) of responder-operator characteristics (ROC) of > 0.7. e) Distribution of transcription levels for representative genes mapped on tSNE. Cells for single cell sequencing were FACSorted as IgG-expressing CD19+CD38+CD138-GL7-small lymphocytes. f) Flow-cytometric evaluation of expression of representative markers to identify clusters observed by transcriptional profiles in IgG+ memory B cells (IgG1+ or IgG2b+CD19+CD38+IgM-IgD-CD138-GL7-CD93-Zombie Aqua-small lymphocytes). Dotplots represent one of three mice.

    Journal: bioRxiv

    Article Title: Discrete populations of isotype-switched memory B lymphocytes are maintained in murine spleen and bone marrow

    doi: 10.1101/825224

    Figure Lengend Snippet: Heterogeneity of switched memory B cells is differentially represented in spleen and bone marrow. Cells for single cell sequencing were FACSorted as IgG-expressing CD19+CD38+CD138-GL7-small lymphocytes. a) Six transcriptionally defined clusters were identified by shared nearest neighbor (SNN) modularity optimization based clustering algorithm mapped to tSNE representation of spleen and BM cells. tSNE coordinates and clustering was computed for 4754 from spleen and 2947 from BM cells, presentation is separated by organ. b) Percentage of cells per cluster in each organ by mouse. c) Distribution of IgG subclass mapped on tSNE. d) Signature genes for each cluster, area under curve (AUC) of responder-operator characteristics (ROC) of > 0.7. e) Distribution of transcription levels for representative genes mapped on tSNE. Cells for single cell sequencing were FACSorted as IgG-expressing CD19+CD38+CD138-GL7-small lymphocytes. f) Flow-cytometric evaluation of expression of representative markers to identify clusters observed by transcriptional profiles in IgG+ memory B cells (IgG1+ or IgG2b+CD19+CD38+IgM-IgD-CD138-GL7-CD93-Zombie Aqua-small lymphocytes). Dotplots represent one of three mice.

    Article Snippet: Antibodies against following murine antigens were used: Ki-67 (B56, BD Biosciences), CD11c (N418, DRFZ), CD19 (1D3, DRFZ and 6D5, BioLegend), CD38 (90, BioLegend), CD138 (281-2, BD Biosciences), GL7 (GL7, DRFZ), IgA (C10-3, BD Biosciences), IgD (11.26c, DRFZ), IgG1 (A85-1, BD Biosciences and X-56, Miltenyi), IgG2a/b (R2-40, BD Biosciences and X-57, Miltenyi), IgG2b (A95-1, BD Biosciences and MRG2b-85, BioLegend), IgM (M41, DRFZ and RMM-1, BioLegend), CD93 (AA4.1, BioLegend and DRFZ), CD21/35 (7G6, DRFZ and 7E9, BioLegend), CD23 (B3B4, BioLegend), CD29 (HMß1-1, Miltenyi), CD49d (R1-2, BioLegend), CD49f (GoH3, eBioscience), CD62L (MEL-14, DRFZ).

    Techniques: Sequencing, Expressing, Mouse Assay

    The bone marrow contains a major population of isotype-switched non-proliferating memory B cells. a) Quantification of NP-specific IgG2a/b+ spleen, peripheral lymph nodes, blood and BM memory B cells. C57BL/6 mice were immunized with NP-KLH/LPS SC. Numbers of NP-binding+ IgG2b+ cells in Spleen, BM, blood, and peripheral lymph nodes (pLN) were determined by flow-cytometry on d421 or d426 post immunization; data pooled from 2 independent experiments. OVA ctrl indicates staining controls from mice immunized with the irrelevant antigen ovalbumin (OVA). Gated for IgG2b+CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI-small lymphocytes. Lines connect samples from one individual, p value of paired t test for spleen and BM samples is indicated. b) Flow-cytometric quantification of Ki-67 expression in IgG2b+ Bsm (IgG2b+CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI-small lymphocytes) splenic naïve (IgM+IgD+IgG2b-CD19+CD38+CD138-GL7-CD11c-PI-small lymphocytes) and germinal center (GC) (CD19+CD38loGL7+CD11c--PI-lymphocytes) B cells. Indicated are the frequencies of Ki-67+ cells within the subset indicated, data in right graph from 2 independent experiments using pooled cells from 4-20 C57BL/6 mice, p value (paired t test) as indicated. c) Flow-cytometric quantification of CD19+ B cells and IgG2b+ memory B cells in mice treated with Cyclophosphamide (CyP) or untreated controls (PBS) after immunization with 3x NP-CGG/IFA. Anaylsis was performed after 7 days of CyP. IgG2b+ B cells were quantified as IgG2b+CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI-small lymphocytes, CD19+ B cells as CD19+CD138-PI-lymphocytes, p value (Welch’s test). Representative data shown for one out of two independent experiments. d) IgG2b+ B memory cells (Ki-67-IgD-Blimp1-GFP-) are dispersed as single cells throughout the bone marrow. Arrows indicate IgG2b+DAPI+ cells. Scale bar: 20µm. e) Co-localization of IgG2b+GFP-IgD-IgG2b+ cells (arrows) with mesenchymal stromal cells. Arrows indicate IgG2b+DAPI+ cells. Representative micrograph. Scale bars: 10µm. f) Co-localization of IgG2b+ cells to mesenchymal stromal cells. Graph shows frequency of IgG2b+ cells in direct contact (black) or within 10mm (grey) of a cell stained for the molecule indicated. g) Flow cytometric quantification of surface expression of the VLA-4 and VLA-6 components CD29, CD49d, CD49f in spleen and BM IgG2b+ Bsm. Gated for IgG2b+CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI-small lymphocytes, histogram plots are representative of three biological replicates.

    Journal: bioRxiv

    Article Title: Discrete populations of isotype-switched memory B lymphocytes are maintained in murine spleen and bone marrow

    doi: 10.1101/825224

    Figure Lengend Snippet: The bone marrow contains a major population of isotype-switched non-proliferating memory B cells. a) Quantification of NP-specific IgG2a/b+ spleen, peripheral lymph nodes, blood and BM memory B cells. C57BL/6 mice were immunized with NP-KLH/LPS SC. Numbers of NP-binding+ IgG2b+ cells in Spleen, BM, blood, and peripheral lymph nodes (pLN) were determined by flow-cytometry on d421 or d426 post immunization; data pooled from 2 independent experiments. OVA ctrl indicates staining controls from mice immunized with the irrelevant antigen ovalbumin (OVA). Gated for IgG2b+CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI-small lymphocytes. Lines connect samples from one individual, p value of paired t test for spleen and BM samples is indicated. b) Flow-cytometric quantification of Ki-67 expression in IgG2b+ Bsm (IgG2b+CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI-small lymphocytes) splenic naïve (IgM+IgD+IgG2b-CD19+CD38+CD138-GL7-CD11c-PI-small lymphocytes) and germinal center (GC) (CD19+CD38loGL7+CD11c--PI-lymphocytes) B cells. Indicated are the frequencies of Ki-67+ cells within the subset indicated, data in right graph from 2 independent experiments using pooled cells from 4-20 C57BL/6 mice, p value (paired t test) as indicated. c) Flow-cytometric quantification of CD19+ B cells and IgG2b+ memory B cells in mice treated with Cyclophosphamide (CyP) or untreated controls (PBS) after immunization with 3x NP-CGG/IFA. Anaylsis was performed after 7 days of CyP. IgG2b+ B cells were quantified as IgG2b+CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI-small lymphocytes, CD19+ B cells as CD19+CD138-PI-lymphocytes, p value (Welch’s test). Representative data shown for one out of two independent experiments. d) IgG2b+ B memory cells (Ki-67-IgD-Blimp1-GFP-) are dispersed as single cells throughout the bone marrow. Arrows indicate IgG2b+DAPI+ cells. Scale bar: 20µm. e) Co-localization of IgG2b+GFP-IgD-IgG2b+ cells (arrows) with mesenchymal stromal cells. Arrows indicate IgG2b+DAPI+ cells. Representative micrograph. Scale bars: 10µm. f) Co-localization of IgG2b+ cells to mesenchymal stromal cells. Graph shows frequency of IgG2b+ cells in direct contact (black) or within 10mm (grey) of a cell stained for the molecule indicated. g) Flow cytometric quantification of surface expression of the VLA-4 and VLA-6 components CD29, CD49d, CD49f in spleen and BM IgG2b+ Bsm. Gated for IgG2b+CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI-small lymphocytes, histogram plots are representative of three biological replicates.

    Article Snippet: Antibodies against following murine antigens were used: Ki-67 (B56, BD Biosciences), CD11c (N418, DRFZ), CD19 (1D3, DRFZ and 6D5, BioLegend), CD38 (90, BioLegend), CD138 (281-2, BD Biosciences), GL7 (GL7, DRFZ), IgA (C10-3, BD Biosciences), IgD (11.26c, DRFZ), IgG1 (A85-1, BD Biosciences and X-56, Miltenyi), IgG2a/b (R2-40, BD Biosciences and X-57, Miltenyi), IgG2b (A95-1, BD Biosciences and MRG2b-85, BioLegend), IgM (M41, DRFZ and RMM-1, BioLegend), CD93 (AA4.1, BioLegend and DRFZ), CD21/35 (7G6, DRFZ and 7E9, BioLegend), CD23 (B3B4, BioLegend), CD29 (HMß1-1, Miltenyi), CD49d (R1-2, BioLegend), CD49f (GoH3, eBioscience), CD62L (MEL-14, DRFZ).

    Techniques: Mouse Assay, Binding Assay, Flow Cytometry, Staining, Expressing, Immunofluorescence

    a) Six transcriptionally defined Clusters were identified by shared nearest neighbor (SNN) modularity optimization based clustering algorithm mapped to tSNE representation of spleen and bone marrow (BM) cells. tSNE coordinates and clustering was computed for 4754 from spleen and 2947 from BM cells, representation combined for spleen and BM (left). Distribution of expression for Cd19 , Ptprc (CD45), Pax5 , Cd38 , Sdc1 (CD138), and Bcl6 genes mapped on tSNE representation. b) Distribution of expression for Itgax (CD11c), Itgam (CD11b), Cd5 genes mapped on tSNE representation. c) Single cell gene set enrichment analysis of expression of the Reactome and Gene ontology enrichment (GO) mapped on tSNE representation. Cells for single cell sequencing were FACSorted as IgG-expressing CD19+CD38+CD138-GL7-small lymphocytes.

    Journal: bioRxiv

    Article Title: Discrete populations of isotype-switched memory B lymphocytes are maintained in murine spleen and bone marrow

    doi: 10.1101/825224

    Figure Lengend Snippet: a) Six transcriptionally defined Clusters were identified by shared nearest neighbor (SNN) modularity optimization based clustering algorithm mapped to tSNE representation of spleen and bone marrow (BM) cells. tSNE coordinates and clustering was computed for 4754 from spleen and 2947 from BM cells, representation combined for spleen and BM (left). Distribution of expression for Cd19 , Ptprc (CD45), Pax5 , Cd38 , Sdc1 (CD138), and Bcl6 genes mapped on tSNE representation. b) Distribution of expression for Itgax (CD11c), Itgam (CD11b), Cd5 genes mapped on tSNE representation. c) Single cell gene set enrichment analysis of expression of the Reactome and Gene ontology enrichment (GO) mapped on tSNE representation. Cells for single cell sequencing were FACSorted as IgG-expressing CD19+CD38+CD138-GL7-small lymphocytes.

    Article Snippet: Antibodies against following murine antigens were used: Ki-67 (B56, BD Biosciences), CD11c (N418, DRFZ), CD19 (1D3, DRFZ and 6D5, BioLegend), CD38 (90, BioLegend), CD138 (281-2, BD Biosciences), GL7 (GL7, DRFZ), IgA (C10-3, BD Biosciences), IgD (11.26c, DRFZ), IgG1 (A85-1, BD Biosciences and X-56, Miltenyi), IgG2a/b (R2-40, BD Biosciences and X-57, Miltenyi), IgG2b (A95-1, BD Biosciences and MRG2b-85, BioLegend), IgM (M41, DRFZ and RMM-1, BioLegend), CD93 (AA4.1, BioLegend and DRFZ), CD21/35 (7G6, DRFZ and 7E9, BioLegend), CD23 (B3B4, BioLegend), CD29 (HMß1-1, Miltenyi), CD49d (R1-2, BioLegend), CD49f (GoH3, eBioscience), CD62L (MEL-14, DRFZ).

    Techniques: Expressing, Sequencing

    a) Percentage of shared clones between clusters of spleen and BM. Clones were defined according to IgG heavy and light chain sequences. Overlap significantly higher than expected for random overlap (p

    Journal: bioRxiv

    Article Title: Discrete populations of isotype-switched memory B lymphocytes are maintained in murine spleen and bone marrow

    doi: 10.1101/825224

    Figure Lengend Snippet: a) Percentage of shared clones between clusters of spleen and BM. Clones were defined according to IgG heavy and light chain sequences. Overlap significantly higher than expected for random overlap (p

    Article Snippet: Antibodies against following murine antigens were used: Ki-67 (B56, BD Biosciences), CD11c (N418, DRFZ), CD19 (1D3, DRFZ and 6D5, BioLegend), CD38 (90, BioLegend), CD138 (281-2, BD Biosciences), GL7 (GL7, DRFZ), IgA (C10-3, BD Biosciences), IgD (11.26c, DRFZ), IgG1 (A85-1, BD Biosciences and X-56, Miltenyi), IgG2a/b (R2-40, BD Biosciences and X-57, Miltenyi), IgG2b (A95-1, BD Biosciences and MRG2b-85, BioLegend), IgM (M41, DRFZ and RMM-1, BioLegend), CD93 (AA4.1, BioLegend and DRFZ), CD21/35 (7G6, DRFZ and 7E9, BioLegend), CD23 (B3B4, BioLegend), CD29 (HMß1-1, Miltenyi), CD49d (R1-2, BioLegend), CD49f (GoH3, eBioscience), CD62L (MEL-14, DRFZ).

    Techniques: Clone Assay

    Spleen and bone marrow isotype-switched memory B cells are distinct in Ig heavy chain repertoire. a) and c) Observed overlap between the IgG1/2+ or IgA+ heavy chain CDR3 repertoire of switched memory B cells from spleen and bone marrow or b and d) random distribution (upper panels). Venn diagrams represent clonotype presence in a given sample: numbers indicate clonotypes present in one organ exclusively or in both (overlap). Random distributions (b and d) show median values for 1000 random distributions drawn while retaining the number of clones initially observed in the sample. Histograms (b and d, lower panels) represent number of clonotypes in spleen and BM in 1000 randomized distributions of observed clonotypes to spleen and BM, dashed red line indicates experimentally observed number of clonotypes present in both spleen and BM. P value of one-sided t test for difference of randomized overlap against observed. e) VH gene recruitment to spleen and bone marrow IgG1/2+ (upper panel) and IgA+ (lower panel) memory B cells, represented as frequency of a particular VH gene among total CDR3s per organ. Bars show relative abundance of the 10 most abundant VH genes, error bars indicate SEM. Significance of difference in VH gene distribution to Spleen and BM assessed by MANOVA, p values corrected for multiple testing (Benjamini-Hochberg), * indicates significant difference in means for a particular VH gene (Welch’s test). BM: bone marrow, M1-M3: replicate samples of three C57BL/6 mice immunized 3x NP-CGG/IFA. Only clones consistently found in technical replicates were considered.

    Journal: bioRxiv

    Article Title: Discrete populations of isotype-switched memory B lymphocytes are maintained in murine spleen and bone marrow

    doi: 10.1101/825224

    Figure Lengend Snippet: Spleen and bone marrow isotype-switched memory B cells are distinct in Ig heavy chain repertoire. a) and c) Observed overlap between the IgG1/2+ or IgA+ heavy chain CDR3 repertoire of switched memory B cells from spleen and bone marrow or b and d) random distribution (upper panels). Venn diagrams represent clonotype presence in a given sample: numbers indicate clonotypes present in one organ exclusively or in both (overlap). Random distributions (b and d) show median values for 1000 random distributions drawn while retaining the number of clones initially observed in the sample. Histograms (b and d, lower panels) represent number of clonotypes in spleen and BM in 1000 randomized distributions of observed clonotypes to spleen and BM, dashed red line indicates experimentally observed number of clonotypes present in both spleen and BM. P value of one-sided t test for difference of randomized overlap against observed. e) VH gene recruitment to spleen and bone marrow IgG1/2+ (upper panel) and IgA+ (lower panel) memory B cells, represented as frequency of a particular VH gene among total CDR3s per organ. Bars show relative abundance of the 10 most abundant VH genes, error bars indicate SEM. Significance of difference in VH gene distribution to Spleen and BM assessed by MANOVA, p values corrected for multiple testing (Benjamini-Hochberg), * indicates significant difference in means for a particular VH gene (Welch’s test). BM: bone marrow, M1-M3: replicate samples of three C57BL/6 mice immunized 3x NP-CGG/IFA. Only clones consistently found in technical replicates were considered.

    Article Snippet: Antibodies against following murine antigens were used: Ki-67 (B56, BD Biosciences), CD11c (N418, DRFZ), CD19 (1D3, DRFZ and 6D5, BioLegend), CD38 (90, BioLegend), CD138 (281-2, BD Biosciences), GL7 (GL7, DRFZ), IgA (C10-3, BD Biosciences), IgD (11.26c, DRFZ), IgG1 (A85-1, BD Biosciences and X-56, Miltenyi), IgG2a/b (R2-40, BD Biosciences and X-57, Miltenyi), IgG2b (A95-1, BD Biosciences and MRG2b-85, BioLegend), IgM (M41, DRFZ and RMM-1, BioLegend), CD93 (AA4.1, BioLegend and DRFZ), CD21/35 (7G6, DRFZ and 7E9, BioLegend), CD23 (B3B4, BioLegend), CD29 (HMß1-1, Miltenyi), CD49d (R1-2, BioLegend), CD49f (GoH3, eBioscience), CD62L (MEL-14, DRFZ).

    Techniques: Clone Assay, Mouse Assay, Immunofluorescence

    Trial 2 VapA specific antibody responses. Mean and SD of VapA total IgG triplicate OD values at a 1:100 dilution of mouse sera. A representation of time at which sera was collected at day 0 (pre vaccination), day 14 (boost vaccination), day 28 (pre challenge) and day 35 (1 week post challenge, pre euthanasia) of each vaccine group; R . equi , Empty vector negative control, HAdV- vapA high dose and HAdV- vapA low dose. Top left total IgG antibody, top right IgG1 antibody, bottom left IgG2a antibody, bottom right IgG2b antibody.

    Journal: PLoS ONE

    Article Title: An Adenoviral Vector Based Vaccine for Rhodococcus equi

    doi: 10.1371/journal.pone.0152149

    Figure Lengend Snippet: Trial 2 VapA specific antibody responses. Mean and SD of VapA total IgG triplicate OD values at a 1:100 dilution of mouse sera. A representation of time at which sera was collected at day 0 (pre vaccination), day 14 (boost vaccination), day 28 (pre challenge) and day 35 (1 week post challenge, pre euthanasia) of each vaccine group; R . equi , Empty vector negative control, HAdV- vapA high dose and HAdV- vapA low dose. Top left total IgG antibody, top right IgG1 antibody, bottom left IgG2a antibody, bottom right IgG2b antibody.

    Article Snippet: Secondary antibody concentrations were Total IgG- 1:10 000 (Sigma, Australia), IgG1- 1:5 000 (Genetex, USA), IgG2a- 1:5000 (Genetex, USA) and IgG2b- 1:5000 (Genetex, USA).

    Techniques: Plasmid Preparation, Negative Control

    HAdV Capsid specific Total IgG antibody response in mice (tested at different time points) and titre. Diluted HAdV capsid antibody levels over the 35 day duration of the trial. Mouse sera pooled per vaccine group, diluted in a 2 fold fashion from 100–1600. Sera was collected at day 0 (pre vaccination), prime vaccine administered, day 14 (boost vaccination), day 28 (pre challenge) and day 35 (1 week post challenge, pre euthanasia) of each vaccine group; R . equi positive control, Empty vector negative control, HAdV- vapA high dose and HAdV- vapA low dose.

    Journal: PLoS ONE

    Article Title: An Adenoviral Vector Based Vaccine for Rhodococcus equi

    doi: 10.1371/journal.pone.0152149

    Figure Lengend Snippet: HAdV Capsid specific Total IgG antibody response in mice (tested at different time points) and titre. Diluted HAdV capsid antibody levels over the 35 day duration of the trial. Mouse sera pooled per vaccine group, diluted in a 2 fold fashion from 100–1600. Sera was collected at day 0 (pre vaccination), prime vaccine administered, day 14 (boost vaccination), day 28 (pre challenge) and day 35 (1 week post challenge, pre euthanasia) of each vaccine group; R . equi positive control, Empty vector negative control, HAdV- vapA high dose and HAdV- vapA low dose.

    Article Snippet: Secondary antibody concentrations were Total IgG- 1:10 000 (Sigma, Australia), IgG1- 1:5 000 (Genetex, USA), IgG2a- 1:5000 (Genetex, USA) and IgG2b- 1:5000 (Genetex, USA).

    Techniques: Mouse Assay, Positive Control, Plasmid Preparation, Negative Control

    Trial 1 HAdV- vapA prime/Boost regime VapA total IgG ELISA response. Mean and SD VapA total IgG antibody levels at a 1:100 dilution of mouse sera pooled in the HAdV- vapA prime/boost vaccine regime per group over the duration of trial 1. Sera was collected at day 0 pre vaccination; day 14, 2 weeks post prime; day 28, 2 weeks post boost; day 42, 4 weeks post boost; and day 49 5 weeks post boost.

    Journal: PLoS ONE

    Article Title: An Adenoviral Vector Based Vaccine for Rhodococcus equi

    doi: 10.1371/journal.pone.0152149

    Figure Lengend Snippet: Trial 1 HAdV- vapA prime/Boost regime VapA total IgG ELISA response. Mean and SD VapA total IgG antibody levels at a 1:100 dilution of mouse sera pooled in the HAdV- vapA prime/boost vaccine regime per group over the duration of trial 1. Sera was collected at day 0 pre vaccination; day 14, 2 weeks post prime; day 28, 2 weeks post boost; day 42, 4 weeks post boost; and day 49 5 weeks post boost.

    Article Snippet: Secondary antibody concentrations were Total IgG- 1:10 000 (Sigma, Australia), IgG1- 1:5 000 (Genetex, USA), IgG2a- 1:5000 (Genetex, USA) and IgG2b- 1:5000 (Genetex, USA).

    Techniques: Enzyme-linked Immunosorbent Assay

    P2X receptor expression on human monocytes. Freshly isolated monocytes were analyzed by FACS analysis. a After blocking the cells with 1% goat serum, the surface antigen expression was analyzed using an anti-P2X 1 Ab ( dotted line ) or the respective IgG

    Journal:

    Article Title: Involvement of P2X receptors in the NAD+-induced rise in [Ca2+]i in human monocytes

    doi: 10.1007/s11302-009-9144-4

    Figure Lengend Snippet: P2X receptor expression on human monocytes. Freshly isolated monocytes were analyzed by FACS analysis. a After blocking the cells with 1% goat serum, the surface antigen expression was analyzed using an anti-P2X 1 Ab ( dotted line ) or the respective IgG

    Article Snippet: The polyclonal goat anti-human P2X1 receptor Ab (A-15, sc-31489), the goat IgG isotype (sc-2028), and the FITC-labeled donkey-anti-goat antibody (sc-2024) were from Santa Cruz Biotechnology (Heidelberg, Germany); the rabbit-anti-human P2X4 receptor was from Alomone Labs (Jerusalem, Israel); the FITC-labeled goat anti-mouse antibody was from DiaMak (Leipzig, Germany); the IgG2b isotype, the anti-β-actin antibody (clone AC-74), and the POD-conjugated goat anti-mouse antibody were from Sigma-Aldrich (Taufkirchen, Germany); and the monoclonal mouse anti-human P2X7 receptor Ab was kindly provided by GlaxoSmithKline (Harlow, UK).

    Techniques: Expressing, Isolation, FACS, Blocking Assay

    Engagement of P2X 7 receptors. Monocytes were preincubated with KN62 (2.5 μM) or DMSO (0.5%; control; a ), oxATP (300 μM) or medium (control; b ), and an anti-P2X 7 mAb (5 μg/ml) or the respective IgG2b isotype

    Journal:

    Article Title: Involvement of P2X receptors in the NAD+-induced rise in [Ca2+]i in human monocytes

    doi: 10.1007/s11302-009-9144-4

    Figure Lengend Snippet: Engagement of P2X 7 receptors. Monocytes were preincubated with KN62 (2.5 μM) or DMSO (0.5%; control; a ), oxATP (300 μM) or medium (control; b ), and an anti-P2X 7 mAb (5 μg/ml) or the respective IgG2b isotype

    Article Snippet: The polyclonal goat anti-human P2X1 receptor Ab (A-15, sc-31489), the goat IgG isotype (sc-2028), and the FITC-labeled donkey-anti-goat antibody (sc-2024) were from Santa Cruz Biotechnology (Heidelberg, Germany); the rabbit-anti-human P2X4 receptor was from Alomone Labs (Jerusalem, Israel); the FITC-labeled goat anti-mouse antibody was from DiaMak (Leipzig, Germany); the IgG2b isotype, the anti-β-actin antibody (clone AC-74), and the POD-conjugated goat anti-mouse antibody were from Sigma-Aldrich (Taufkirchen, Germany); and the monoclonal mouse anti-human P2X7 receptor Ab was kindly provided by GlaxoSmithKline (Harlow, UK).

    Techniques:

    Effects of adsorption of LPS or MPLA onto TT/alum sensitization on serum isotype production. Protocol as shown in Figure 2(a) . Concentration of serum isotypes: (a) total IgE, (b) TT-specific IgG1, and (c) TT-specific IgG2a. Data shown as mean ± SE, one-way ANOVA: # P

    Journal: Journal of Immunology Research

    Article Title: Adsorption of Toll-Like Receptor 4 Agonist to Alum-Based Tetanus Toxoid Vaccine Dampens Pro-T Helper 2 Activities and Enhances Antibody Responses

    doi: 10.1155/2015/280238

    Figure Lengend Snippet: Effects of adsorption of LPS or MPLA onto TT/alum sensitization on serum isotype production. Protocol as shown in Figure 2(a) . Concentration of serum isotypes: (a) total IgE, (b) TT-specific IgG1, and (c) TT-specific IgG2a. Data shown as mean ± SE, one-way ANOVA: # P

    Article Snippet: Purified mouse IgG1 or IgG2a (Invitrogen) was used as standard.

    Techniques: Adsorption, Concentration Assay

    Effects of adsorption of LPS onto OVA/TT/alum sensitization on OVA- or TT-induced isotype production. Protocol shown in Figure 4(a) . ((a) and (c)) Concentration of OVA-specific isotypes: (a) PCA titers anaphylactic IgE, (b) IgG1, and (c) IgG2a. ((d) and (f)) Concentration of TT-specific isotypes: (d) PCA levels of anaphylactic IgE, (e) IgG1, and (c) IgG2a. Data shown as mean ± SE, one-way ANOVA: # P

    Journal: Journal of Immunology Research

    Article Title: Adsorption of Toll-Like Receptor 4 Agonist to Alum-Based Tetanus Toxoid Vaccine Dampens Pro-T Helper 2 Activities and Enhances Antibody Responses

    doi: 10.1155/2015/280238

    Figure Lengend Snippet: Effects of adsorption of LPS onto OVA/TT/alum sensitization on OVA- or TT-induced isotype production. Protocol shown in Figure 4(a) . ((a) and (c)) Concentration of OVA-specific isotypes: (a) PCA titers anaphylactic IgE, (b) IgG1, and (c) IgG2a. ((d) and (f)) Concentration of TT-specific isotypes: (d) PCA levels of anaphylactic IgE, (e) IgG1, and (c) IgG2a. Data shown as mean ± SE, one-way ANOVA: # P

    Article Snippet: Purified mouse IgG1 or IgG2a (Invitrogen) was used as standard.

    Techniques: Adsorption, Concentration Assay

    Effects of adsorption of PIC (TLR3) or LPS (TLR4) agonists onto OVA/alum sensitization on OVA-induced cellular and humoral responses. (a) Protocol: C57BL/6 WT mice sensitized with s.c. OVA/alum in the presence or not of PIC (10 μ g) or LPS (10 μ g) on days 0 and 7 and challenged with OVA i.n. on days 14 and 21. Samples obtained on day 22. (b) Total number of cells and (c) eosinophils in BAL; ((d), (e), and (f)) levels of IL-5, IL-13, and IFN- g in BAL. ((g), (h), and (i)) Levels of serum isotypes: total IgE, OVA-specific IgG1, and OVA-specific IgG2a. Data shown as mean ± SE, one-way ANOVA: # P

    Journal: Journal of Immunology Research

    Article Title: Adsorption of Toll-Like Receptor 4 Agonist to Alum-Based Tetanus Toxoid Vaccine Dampens Pro-T Helper 2 Activities and Enhances Antibody Responses

    doi: 10.1155/2015/280238

    Figure Lengend Snippet: Effects of adsorption of PIC (TLR3) or LPS (TLR4) agonists onto OVA/alum sensitization on OVA-induced cellular and humoral responses. (a) Protocol: C57BL/6 WT mice sensitized with s.c. OVA/alum in the presence or not of PIC (10 μ g) or LPS (10 μ g) on days 0 and 7 and challenged with OVA i.n. on days 14 and 21. Samples obtained on day 22. (b) Total number of cells and (c) eosinophils in BAL; ((d), (e), and (f)) levels of IL-5, IL-13, and IFN- g in BAL. ((g), (h), and (i)) Levels of serum isotypes: total IgE, OVA-specific IgG1, and OVA-specific IgG2a. Data shown as mean ± SE, one-way ANOVA: # P

    Article Snippet: Purified mouse IgG1 or IgG2a (Invitrogen) was used as standard.

    Techniques: Adsorption, Mouse Assay

    Antibody response to peptide vaccination. Serum IgG was measured in the serum of vaccinated animals at day 49 post vaccination (Study 1, n = 5). IgG against each respective G peptide ( A ), CX3C peptide ( B ) or RSV A2 ( C ) were detected by indirect ELISA assay. Experiments were independently repeated at least 3 times and analyzed by one-way ANOVA test. Ns, no significant; ** p

    Journal: Vaccines

    Article Title: Layer-By-Layer Nanoparticle Vaccines Carrying the G Protein CX3C Motif Protect against RSV Infection and Disease

    doi: 10.3390/vaccines3040829

    Figure Lengend Snippet: Antibody response to peptide vaccination. Serum IgG was measured in the serum of vaccinated animals at day 49 post vaccination (Study 1, n = 5). IgG against each respective G peptide ( A ), CX3C peptide ( B ) or RSV A2 ( C ) were detected by indirect ELISA assay. Experiments were independently repeated at least 3 times and analyzed by one-way ANOVA test. Ns, no significant; ** p

    Article Snippet: Vaccination with LbL-NP induced both IgG1 and IgG2a isotypes, with IgG1 being more predominant then IgG2a ( ).

    Techniques: Indirect ELISA

    Humoral and cellular responses induced by LbL nanoparticle vaccination. Mice were vaccinated with LbL nanoparticles on day 0 and 21. At day 35 post vaccination serum was collected and RSV-specific IgG response was measured via indirect ELISA assay ( A ). At the same time point spleens were harvested, splenocytes were stimulated with G peptide 183–197 for 24 h and IFN-γ and IL-4 secretion detected by ELISPOT assay ( B ). Experiments were independently repeated at least 3 times and analyzed by one-way ANOVA test. * p

    Journal: Vaccines

    Article Title: Layer-By-Layer Nanoparticle Vaccines Carrying the G Protein CX3C Motif Protect against RSV Infection and Disease

    doi: 10.3390/vaccines3040829

    Figure Lengend Snippet: Humoral and cellular responses induced by LbL nanoparticle vaccination. Mice were vaccinated with LbL nanoparticles on day 0 and 21. At day 35 post vaccination serum was collected and RSV-specific IgG response was measured via indirect ELISA assay ( A ). At the same time point spleens were harvested, splenocytes were stimulated with G peptide 183–197 for 24 h and IFN-γ and IL-4 secretion detected by ELISPOT assay ( B ). Experiments were independently repeated at least 3 times and analyzed by one-way ANOVA test. * p

    Article Snippet: Vaccination with LbL-NP induced both IgG1 and IgG2a isotypes, with IgG1 being more predominant then IgG2a ( ).

    Techniques: Mouse Assay, Indirect ELISA, Enzyme-linked Immunospot

    Lung virus titers following RSV challenge of mice passively transferred with purified IgG from mice vaccinated with LbL nanoparticles. Mice were challenged i.n. with 10 6 PFU RSV A2, and passively transferred with purified IgG from group A (Study 2) on day 2 and 4 post challenge. At day 5 post challenge lung virus titers were determined by immunostaining plaque assay. Data are presented as total PFU/whole lung tissue. Values shown as the mean ± SEM (one-way ANOVA); ns, non-significant; * p

    Journal: Vaccines

    Article Title: Layer-By-Layer Nanoparticle Vaccines Carrying the G Protein CX3C Motif Protect against RSV Infection and Disease

    doi: 10.3390/vaccines3040829

    Figure Lengend Snippet: Lung virus titers following RSV challenge of mice passively transferred with purified IgG from mice vaccinated with LbL nanoparticles. Mice were challenged i.n. with 10 6 PFU RSV A2, and passively transferred with purified IgG from group A (Study 2) on day 2 and 4 post challenge. At day 5 post challenge lung virus titers were determined by immunostaining plaque assay. Data are presented as total PFU/whole lung tissue. Values shown as the mean ± SEM (one-way ANOVA); ns, non-significant; * p

    Article Snippet: Vaccination with LbL-NP induced both IgG1 and IgG2a isotypes, with IgG1 being more predominant then IgG2a ( ).

    Techniques: Mouse Assay, Purification, Immunostaining, Plaque Assay

    RSV G protein CX3C-CX3CR1 blocking antibody studies. Total IgG was purified from serum derived from mice vaccinated with CX3C peptides and evaluated for the ability to prevent native RSV G protein binding to CX3CR1 expressed on HEK293-CX3CR1 cells. Data are presented as the percent inhibition of RSV G protein binding to HEK293-CX3CR1 cells. Ns, non-significant; * p

    Journal: Vaccines

    Article Title: Layer-By-Layer Nanoparticle Vaccines Carrying the G Protein CX3C Motif Protect against RSV Infection and Disease

    doi: 10.3390/vaccines3040829

    Figure Lengend Snippet: RSV G protein CX3C-CX3CR1 blocking antibody studies. Total IgG was purified from serum derived from mice vaccinated with CX3C peptides and evaluated for the ability to prevent native RSV G protein binding to CX3CR1 expressed on HEK293-CX3CR1 cells. Data are presented as the percent inhibition of RSV G protein binding to HEK293-CX3CR1 cells. Ns, non-significant; * p

    Article Snippet: Vaccination with LbL-NP induced both IgG1 and IgG2a isotypes, with IgG1 being more predominant then IgG2a ( ).

    Techniques: Blocking Assay, Purification, Derivative Assay, Mouse Assay, Protein Binding, Inhibition

    Pulmonary infiltration following therapeutic antibody treatment. BAL leukocytes (CD45 + cells), T cells (CD45 + CD3 + ), Macrophages (CD45 + SiglecF + CD11c + ), Neutrophils (CD45 + Gr1 + ) and Eosinophils (CD45 + SiglecF + CD11c-) numbers in the lungs of mice treated at day 2 and 4 p.i. with nIg, anti-RSV G mAb 131-2G or purified IgG from LbL nanoparticle were determined at day 5 p.i. Data shown are the means ± SEM. Asterisks indicate a significant difference ( p

    Journal: Vaccines

    Article Title: Layer-By-Layer Nanoparticle Vaccines Carrying the G Protein CX3C Motif Protect against RSV Infection and Disease

    doi: 10.3390/vaccines3040829

    Figure Lengend Snippet: Pulmonary infiltration following therapeutic antibody treatment. BAL leukocytes (CD45 + cells), T cells (CD45 + CD3 + ), Macrophages (CD45 + SiglecF + CD11c + ), Neutrophils (CD45 + Gr1 + ) and Eosinophils (CD45 + SiglecF + CD11c-) numbers in the lungs of mice treated at day 2 and 4 p.i. with nIg, anti-RSV G mAb 131-2G or purified IgG from LbL nanoparticle were determined at day 5 p.i. Data shown are the means ± SEM. Asterisks indicate a significant difference ( p

    Article Snippet: Vaccination with LbL-NP induced both IgG1 and IgG2a isotypes, with IgG1 being more predominant then IgG2a ( ).

    Techniques: Mouse Assay, Purification

    FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) are all involved in ADE of ZIKV infection by DENV immune sera. PBMCs from healthy donors (n = 3) were treated with monoclonal antibodies (1 μg/ml, 3 μg/ml or 10 μg/ml) against each FcγR types or combined (1 μg/ml, 3 μg/ml or 10 μg/ml in total), or with IgG control antibody prior to performing the boosted ZIKV ADE assay with DENV-1 immune serum (D1-E01, 1/2,500 dilution). The percentage of anti-E positive Vero cells in the control group, where no anti-FcγR antibodies were applied at the ADE condition was defined as 100%. The mean and standard deviation of results from three independent experiments without blocking antibody treatment (A) or with blocking antibody treatment (B) were presented. Statistical significances were determined by comparison of the relative proportion of infected cells with blocking antibody treatment to that of the control IgG antibody treatment under the same antibody concentration using unpaired two-tailed Student’s t test and designated as “*”for p

    Journal: PLoS ONE

    Article Title: Dengue immune sera enhance Zika virus infection in human peripheral blood monocytes through Fc gamma receptors

    doi: 10.1371/journal.pone.0200478

    Figure Lengend Snippet: FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) are all involved in ADE of ZIKV infection by DENV immune sera. PBMCs from healthy donors (n = 3) were treated with monoclonal antibodies (1 μg/ml, 3 μg/ml or 10 μg/ml) against each FcγR types or combined (1 μg/ml, 3 μg/ml or 10 μg/ml in total), or with IgG control antibody prior to performing the boosted ZIKV ADE assay with DENV-1 immune serum (D1-E01, 1/2,500 dilution). The percentage of anti-E positive Vero cells in the control group, where no anti-FcγR antibodies were applied at the ADE condition was defined as 100%. The mean and standard deviation of results from three independent experiments without blocking antibody treatment (A) or with blocking antibody treatment (B) were presented. Statistical significances were determined by comparison of the relative proportion of infected cells with blocking antibody treatment to that of the control IgG antibody treatment under the same antibody concentration using unpaired two-tailed Student’s t test and designated as “*”for p

    Article Snippet: Isotype control murine immunoglobulin IgG1 κ (FITC; APC; clone P3.6.2.8.1), mouse IgG2a κ (PE; PE-Cy7; Alexa Fluor 488; clone eBM2a), mouse IgG2b κ (PE; clone eBMG2b) were purchased from eBioscience.

    Techniques: Infection, Standard Deviation, Blocking Assay, Concentration Assay, Two Tailed Test

    Concentration-dependent effect of TSP on tyrosine phosphorylation of EC proteins in the presence of PTP inhibition. ECs were exposed for 1 h to TSP or media alone with or without vanadate (200 μM), PAO (1.0 μM), or both. Cell lysates were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes, and the blots were probed with antiphosphotyrosine IgG. (A) Phosphotyrosine-containing proteins in extracts of ECs exposed to TSP (20 μg/ml, 1 h) or media alone in the presence and absence of vanadate, PAO, or both. Arrows indicate bands with apparent M r of 240,000, 205,000, 185,000, 135,000, 110,000, 95,000, and 66,000. (B) Phosphotyrosine-containing proteins in EC extracts obtained from monolayers exposed for 1 h to increasing concentrations of TSP in the presence of fixed concentrations of both vanadate (200 μM) and PAO (1.0 μM). To confirm equivalent protein loading, each blot was stripped and reprobed with anti-β-tubulin antibody. Molecular weights in kilodaltons are indicated on the left. Each is a representative blot of three experiments.

    Journal: Molecular Biology of the Cell

    Article Title: Thrombospondin-1 Induces Tyrosine Phosphorylation of Adherens Junction Proteins and Regulates an Endothelial Paracellular Pathway

    doi:

    Figure Lengend Snippet: Concentration-dependent effect of TSP on tyrosine phosphorylation of EC proteins in the presence of PTP inhibition. ECs were exposed for 1 h to TSP or media alone with or without vanadate (200 μM), PAO (1.0 μM), or both. Cell lysates were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes, and the blots were probed with antiphosphotyrosine IgG. (A) Phosphotyrosine-containing proteins in extracts of ECs exposed to TSP (20 μg/ml, 1 h) or media alone in the presence and absence of vanadate, PAO, or both. Arrows indicate bands with apparent M r of 240,000, 205,000, 185,000, 135,000, 110,000, 95,000, and 66,000. (B) Phosphotyrosine-containing proteins in EC extracts obtained from monolayers exposed for 1 h to increasing concentrations of TSP in the presence of fixed concentrations of both vanadate (200 μM) and PAO (1.0 μM). To confirm equivalent protein loading, each blot was stripped and reprobed with anti-β-tubulin antibody. Molecular weights in kilodaltons are indicated on the left. Each is a representative blot of three experiments.

    Article Snippet: To confirm equivalent protein loading, blots were stripped with 100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl, pH 6.7, and incubated with 0.5 μg/ml murine antiphysarum β-tubulin IgG 2b (Boehringer Mannheim, Indianapolis, IN) followed by HRP-conjugated anti-mouse IgG (Transduction Laboratories, Lexington, KY), and developed with ECL.

    Techniques: Concentration Assay, Inhibition, SDS Page