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  • 99
    Name:
    Goat F ab 2 IgG BIOT
    Description:

    Catalog Number:
    0110-08
    Price:
    None
    Source:
    Pepsin digest of Goat IgG (SB Cat. No. 0109)
    Applications:
    Quality tested applications for relevant formats include -Flow Cytometry 1-3ELISAFLISAOther referenced applications for relevant formats include -Immunocytochemistry 4
    Format:
    BIOT (Biotin)
    Buy from Supplier


    Structured Review

    SouthernBiotech igg2b
    Goat F ab 2 IgG BIOT

    https://www.bioz.com/result/igg2b/product/SouthernBiotech
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    igg2b - by Bioz Stars, 2021-06
    99/100 stars

    Images

    1) Product Images from "Distinct B-cell populations contribute to vaccine antigen-specific antibody production in a transgenic mouse model"

    Article Title: Distinct B-cell populations contribute to vaccine antigen-specific antibody production in a transgenic mouse model

    Journal: Immunology

    doi: 10.1111/imm.12287

    Immunization of ROSA yellow fluorescent protein transgenic (YFP Tg) mice induces vaccine-specific IgG antibodies (a) Total IgG and (b) IgM antibodies specific for inactivated influenza (A/PR8) virus antigen day 9 post prime and boost immunization ( n =
    Figure Legend Snippet: Immunization of ROSA yellow fluorescent protein transgenic (YFP Tg) mice induces vaccine-specific IgG antibodies (a) Total IgG and (b) IgM antibodies specific for inactivated influenza (A/PR8) virus antigen day 9 post prime and boost immunization ( n =

    Techniques Used: Transgenic Assay, Mouse Assay

    2) Product Images from "Phosphatidylcholine as a metabolic cue for determining B cell fate and function"

    Article Title: Phosphatidylcholine as a metabolic cue for determining B cell fate and function

    Journal: Cellular immunology

    doi: 10.1016/j.cellimm.2016.08.002

    The frequency of germinal center-resident NP-specific IgG1 + B cells is reduced in Cγ1 Cre/Cre mice NP-specific germinal center B cells were identified using flow cytometry. Briefly, in (a), single lymphocytes (upper left and middle-left plots) were gated on B cells (middle-right plot) in germinal centers (far right plot), then assessed for frequency of NP-binding IgG1 B cells (lower plots). Minus 1 staining negative controls (not shown) were used to establish gates for NP-binding. Representative data from multiple wild-type, Cγ1 Cre/wt and Cγ1 Cre/Cre mice are shown. The frequency and number of IgM + (b and d) and IgG1 + (c and e) NP-binding splenic germinal center B cells. Statistical differences were determined with 1-way ANOVA using Tukey’s multiple comparison test (* p
    Figure Legend Snippet: The frequency of germinal center-resident NP-specific IgG1 + B cells is reduced in Cγ1 Cre/Cre mice NP-specific germinal center B cells were identified using flow cytometry. Briefly, in (a), single lymphocytes (upper left and middle-left plots) were gated on B cells (middle-right plot) in germinal centers (far right plot), then assessed for frequency of NP-binding IgG1 B cells (lower plots). Minus 1 staining negative controls (not shown) were used to establish gates for NP-binding. Representative data from multiple wild-type, Cγ1 Cre/wt and Cγ1 Cre/Cre mice are shown. The frequency and number of IgM + (b and d) and IgG1 + (c and e) NP-binding splenic germinal center B cells. Statistical differences were determined with 1-way ANOVA using Tukey’s multiple comparison test (* p

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Binding Assay, Staining

    Changes in relative antibody affinity in immunized Cγ1-CCTα mice Sera from individual mice were titrated in ELISA to determine the amount of high-affinity (NP 2 -BSA) and total (NP 20 -BSA) NP-specific IgG (a) and IgM (b) for wild-type (filled circles), Cγ1 Cre/wt (filled squares) and Cγ1 Cre/Cre (filled triangles) mice. The ratio of NP2:NP20 titers is expressed as mean ratios ± SEM and are compiled from three independent experiments.
    Figure Legend Snippet: Changes in relative antibody affinity in immunized Cγ1-CCTα mice Sera from individual mice were titrated in ELISA to determine the amount of high-affinity (NP 2 -BSA) and total (NP 20 -BSA) NP-specific IgG (a) and IgM (b) for wild-type (filled circles), Cγ1 Cre/wt (filled squares) and Cγ1 Cre/Cre (filled triangles) mice. The ratio of NP2:NP20 titers is expressed as mean ratios ± SEM and are compiled from three independent experiments.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Germinal center recall responses are impaired in Cγ1-Cre-expressing mice NP-specific germinal center B cells were identified using flow cytometry as described earlier. Representative data from multiple wild-type, Cγ1 Cre/wt and Cγ1 Cre/Cre mice are shown in (a). The frequency (b, c) and number (d, e) of IgM + (b, d) and IgG1 + (c, e) NP-binding splenic germinal center B cells for multiple mice are shown. Statistical differences are indicated (* p
    Figure Legend Snippet: Germinal center recall responses are impaired in Cγ1-Cre-expressing mice NP-specific germinal center B cells were identified using flow cytometry as described earlier. Representative data from multiple wild-type, Cγ1 Cre/wt and Cγ1 Cre/Cre mice are shown in (a). The frequency (b, c) and number (d, e) of IgM + (b, d) and IgG1 + (c, e) NP-binding splenic germinal center B cells for multiple mice are shown. Statistical differences are indicated (* p

    Techniques Used: Expressing, Mouse Assay, Flow Cytometry, Cytometry, Binding Assay

    Antibody levels in naïve Cγ1-Cre-expressing mice Ig titers for IgM and IgG subclasses in serum from naïve 2 month-old wild-type (n=5, black bars), Cγ1 Cre/wt (n=5, gray) and Cγ1 Cre/Cre (n=5, white bars) mice were measured by ELISA. Data shown are means, with standard error indicated (** p
    Figure Legend Snippet: Antibody levels in naïve Cγ1-Cre-expressing mice Ig titers for IgM and IgG subclasses in serum from naïve 2 month-old wild-type (n=5, black bars), Cγ1 Cre/wt (n=5, gray) and Cγ1 Cre/Cre (n=5, white bars) mice were measured by ELISA. Data shown are means, with standard error indicated (** p

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

    Impaired antigen-specific IgG response and ASC formation in Cγ1-Cre-expressing mice Mice were immunized i.p. using 50 μg NP 5 -KLH emulsified in alum. Three weeks after immunization, ELISA (a) and ELIspot assays (b and c) were used to measure antigen-specific responses. In (a), IgM and IgG primary antibody responses (mean + standard deviation) in wild-type mice (black bars) and Cγ1-Cre-expressing mice (gray bars). NP-specific IgM (left panels) and IgG (total, right panels) antibody-secreting cells (ASC) in spleen (b) and bone marrow (c); filled circles represent data from individual wild-type mice, filled squares are data from Cγ1 Cre/wt mice, and filled triangles are data from Cγ1 Cre/Cre mice (all mice were CCTα flox/flox ). Statistics were calculated using 1-way ANOVA with Tukey’s multiple comparison test (* p
    Figure Legend Snippet: Impaired antigen-specific IgG response and ASC formation in Cγ1-Cre-expressing mice Mice were immunized i.p. using 50 μg NP 5 -KLH emulsified in alum. Three weeks after immunization, ELISA (a) and ELIspot assays (b and c) were used to measure antigen-specific responses. In (a), IgM and IgG primary antibody responses (mean + standard deviation) in wild-type mice (black bars) and Cγ1-Cre-expressing mice (gray bars). NP-specific IgM (left panels) and IgG (total, right panels) antibody-secreting cells (ASC) in spleen (b) and bone marrow (c); filled circles represent data from individual wild-type mice, filled squares are data from Cγ1 Cre/wt mice, and filled triangles are data from Cγ1 Cre/Cre mice (all mice were CCTα flox/flox ). Statistics were calculated using 1-way ANOVA with Tukey’s multiple comparison test (* p

    Techniques Used: Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Standard Deviation

    CCTα is required for IgG but not IgM antibody and ASC during secondary immune responses Mice were immunized i.p. using 50 μg NP 5 -KLH emulsified in alum. Five days after booster immunization, ELISA (a) and ELIspot assays (b) were used to measure antigen-specific responses. In (a), IgM (left panel) and IgG (right panel) primary antibody responses (mean + standard deviation) in wild-type (black bars) and Cγ1-Cre-expressing mice (gray bars). In (b and c), NP-specific IgM (left panels) and IgG (right panels) antibody-secreting cells (ASC) in spleen (b) and bone marrow (c); filled circles represent data from individual wild-type mice, filled squares are data from Cγ1 Cre/wt mice, and filled triangles are data from Cγ1 Cre/Cre mice (all mice were CCTα flox/flox ). Statistical differences were determined with 1-way ANOVA using Tukey’s multiple comparison test (* p
    Figure Legend Snippet: CCTα is required for IgG but not IgM antibody and ASC during secondary immune responses Mice were immunized i.p. using 50 μg NP 5 -KLH emulsified in alum. Five days after booster immunization, ELISA (a) and ELIspot assays (b) were used to measure antigen-specific responses. In (a), IgM (left panel) and IgG (right panel) primary antibody responses (mean + standard deviation) in wild-type (black bars) and Cγ1-Cre-expressing mice (gray bars). In (b and c), NP-specific IgM (left panels) and IgG (right panels) antibody-secreting cells (ASC) in spleen (b) and bone marrow (c); filled circles represent data from individual wild-type mice, filled squares are data from Cγ1 Cre/wt mice, and filled triangles are data from Cγ1 Cre/Cre mice (all mice were CCTα flox/flox ). Statistical differences were determined with 1-way ANOVA using Tukey’s multiple comparison test (* p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Standard Deviation, Expressing

    3) Product Images from "Selective regulation of autoreactive B cells by Fc?RIIB"

    Article Title: Selective regulation of autoreactive B cells by Fc?RIIB

    Journal: Journal of autoimmunity

    doi: 10.1016/j.jaut.2009.02.009

    Flow cytometry of B cells in R4A × FcγRIIB -/- BALB/c mice A) Splenocytes from 5 to 10 month-old R4A mice ( n = 7) and R4A × FcγRIIB -/- mice ( n = 7) mice were analyzed for the percentage of Tg + B cells using an anti-IgG2b antibody. Analysis of B220 + /Tg + cells revealed no difference by Mann-Whitney in this study, and the analysis of a second set of mice. B) Splenocytes from R4A mice ( n = 17) and R4A × FcγRIIB -/- mice ( n = 17) were stained with B220 and CD138 to determine the percentage of B220 lo /CD138 + plasma cells. Representative contour plots from each mouse are shown. The percentage of B220 lo /CD138 + cells were significantly increased in R4A × FcγRIIB -/- mice (* p
    Figure Legend Snippet: Flow cytometry of B cells in R4A × FcγRIIB -/- BALB/c mice A) Splenocytes from 5 to 10 month-old R4A mice ( n = 7) and R4A × FcγRIIB -/- mice ( n = 7) mice were analyzed for the percentage of Tg + B cells using an anti-IgG2b antibody. Analysis of B220 + /Tg + cells revealed no difference by Mann-Whitney in this study, and the analysis of a second set of mice. B) Splenocytes from R4A mice ( n = 17) and R4A × FcγRIIB -/- mice ( n = 17) were stained with B220 and CD138 to determine the percentage of B220 lo /CD138 + plasma cells. Representative contour plots from each mouse are shown. The percentage of B220 lo /CD138 + cells were significantly increased in R4A × FcγRIIB -/- mice (* p

    Techniques Used: Flow Cytometry, Cytometry, Mouse Assay, MANN-WHITNEY, Staining

    Spontaneous production of anti-DNA antibody titers in R4A × FcγRIIB -/- BALB/c mice A) Serum was collected once a month beginning at 1 month of age from R4A BALB/c ( n = 5) and R4A × FcγRIIB -/- BALB/c ( n = 5) mice and IgG2b anti-DNA antibodies were measured by ELISA using a 1:500 dilution of serum. IgG2b anti-DNA antibody levels significantly increased in R4A × FcγRIIB -/- mice with age (* p
    Figure Legend Snippet: Spontaneous production of anti-DNA antibody titers in R4A × FcγRIIB -/- BALB/c mice A) Serum was collected once a month beginning at 1 month of age from R4A BALB/c ( n = 5) and R4A × FcγRIIB -/- BALB/c ( n = 5) mice and IgG2b anti-DNA antibodies were measured by ELISA using a 1:500 dilution of serum. IgG2b anti-DNA antibody levels significantly increased in R4A × FcγRIIB -/- mice with age (* p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    4) Product Images from "Loss of immune tolerance to IL-2 in type 1 diabetes"

    Article Title: Loss of immune tolerance to IL-2 in type 1 diabetes

    Journal: Nature Communications

    doi: 10.1038/ncomms13027

    Anti-mIL-2-autoantibodies in NOD mice. ( a – c ) Serum samples were obtained from different mouse strains: NSG (NOD scid gamma), B6, Balb/c, pre-diabetic NOD (NOD Pre-diabetic) and diabetic NOD (NOD Diabetic). ( a , b ) Serum titres of anti-mIL-2 IgG ( a ), IgG isotypes (IgG1, 2b, 2c and 3) and IgA ( b ) in the different strains. ( c ) Proliferation of CTLL-2 cells cultured for 3 days with 1 ng ml −1 mIL-2 and different concentrations of B6 (closed circles) or NOD (open circles) sera. Proliferation is expressed as percentage of control (CTLL-2 cultured for 3 days with 1 ng ml −1 mIL-2 without mouse serum, mean c.p.m. of 84,590). ( d – f ) Sera were obtained at different ages after birth and at disease onset (Onset) in two independent cohorts of female NOD mice ( n =13 and 6, respectively) and one cohort of male NOD mice ( n =5). ( d , e ) Serum anti-mIL-2 IgG titres in NOD mice in function of the age ( d ) or of the sex ( e ). Dashed line indicates the mean value given by B6 mouse sera in the corresponding ELISA. ( f ) Correlation between anti-mIL-2 IgG titres at 6 weeks and time to onset of diabetes in female NOD mice (non-parametric Spearman correlation test). ( g , h ) Serum samples were obtained from different mouse strains (all females and age-matched): B6, wild-type NOD, NOD. Idd3 B6 , Il2 -hemizygous NOD: NOD. Idd3 NOD/NOD-IL-2null (NOD. Il2 +/− ), NOD. Idd6 C3H and their corresponding controls NOD. Idd6 NOD , as well as NOR. Serum titres of anti-mIL-2 IgG ( g ) and IL-2/anti-rhIL-2-autoantibodies complex ( h ) in the different mouse strains. Symbols and curves represent individual mice, horizontal bars are the medians and error bars represent the s.e.m. Data are cumulative of at least two independent experiments. ns, not significant. * P
    Figure Legend Snippet: Anti-mIL-2-autoantibodies in NOD mice. ( a – c ) Serum samples were obtained from different mouse strains: NSG (NOD scid gamma), B6, Balb/c, pre-diabetic NOD (NOD Pre-diabetic) and diabetic NOD (NOD Diabetic). ( a , b ) Serum titres of anti-mIL-2 IgG ( a ), IgG isotypes (IgG1, 2b, 2c and 3) and IgA ( b ) in the different strains. ( c ) Proliferation of CTLL-2 cells cultured for 3 days with 1 ng ml −1 mIL-2 and different concentrations of B6 (closed circles) or NOD (open circles) sera. Proliferation is expressed as percentage of control (CTLL-2 cultured for 3 days with 1 ng ml −1 mIL-2 without mouse serum, mean c.p.m. of 84,590). ( d – f ) Sera were obtained at different ages after birth and at disease onset (Onset) in two independent cohorts of female NOD mice ( n =13 and 6, respectively) and one cohort of male NOD mice ( n =5). ( d , e ) Serum anti-mIL-2 IgG titres in NOD mice in function of the age ( d ) or of the sex ( e ). Dashed line indicates the mean value given by B6 mouse sera in the corresponding ELISA. ( f ) Correlation between anti-mIL-2 IgG titres at 6 weeks and time to onset of diabetes in female NOD mice (non-parametric Spearman correlation test). ( g , h ) Serum samples were obtained from different mouse strains (all females and age-matched): B6, wild-type NOD, NOD. Idd3 B6 , Il2 -hemizygous NOD: NOD. Idd3 NOD/NOD-IL-2null (NOD. Il2 +/− ), NOD. Idd6 C3H and their corresponding controls NOD. Idd6 NOD , as well as NOR. Serum titres of anti-mIL-2 IgG ( g ) and IL-2/anti-rhIL-2-autoantibodies complex ( h ) in the different mouse strains. Symbols and curves represent individual mice, horizontal bars are the medians and error bars represent the s.e.m. Data are cumulative of at least two independent experiments. ns, not significant. * P

    Techniques Used: Mouse Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    Circulating anti-IL-2-specific B and T lymphocytes in T1D patients. ( a ) (Left) Relative frequency and (right) absolute numbers of antigen-specific IgG memory B cells across four T1D patients. Error bars represent s.d., P -values calculated using an unpaired t -test. ( b ) IL-2-specific heavy and light chain V gene segment usage, CDR3 amino acid sequences, and rhIL-2 solution equilibrium titration (SET) affinity for recombinant IgG obtained from a single T1D patient, ( c ) IFN-γ production by peripheral blood mononuclear cells (PBMCs) from healthy donors (HD) ( n =14, closed circles) or T1D patients ( n =13, open circles) quantified by ELISPOT after stimulation with rhIL-2 (Proleukin, Pro) or Pro peptides (10 μM per each), intracellular IA-2, adenovirus lysate (AdV), or PHA. The number of IFN-γ spot-forming cells (SFC)/10 6 PBMCs is depicted, the dashed line indicates the positive cut-off threshold, and the grey shaded area shows undetectable responses (that is, identical to spontaneous background responses; see material and methods for threshold determination). The percent of positive T1D (top number) and HD (bottom number) is indicated for each condition, with antigens yielding responses significantly different between HD and T1D patients in bold ( P
    Figure Legend Snippet: Circulating anti-IL-2-specific B and T lymphocytes in T1D patients. ( a ) (Left) Relative frequency and (right) absolute numbers of antigen-specific IgG memory B cells across four T1D patients. Error bars represent s.d., P -values calculated using an unpaired t -test. ( b ) IL-2-specific heavy and light chain V gene segment usage, CDR3 amino acid sequences, and rhIL-2 solution equilibrium titration (SET) affinity for recombinant IgG obtained from a single T1D patient, ( c ) IFN-γ production by peripheral blood mononuclear cells (PBMCs) from healthy donors (HD) ( n =14, closed circles) or T1D patients ( n =13, open circles) quantified by ELISPOT after stimulation with rhIL-2 (Proleukin, Pro) or Pro peptides (10 μM per each), intracellular IA-2, adenovirus lysate (AdV), or PHA. The number of IFN-γ spot-forming cells (SFC)/10 6 PBMCs is depicted, the dashed line indicates the positive cut-off threshold, and the grey shaded area shows undetectable responses (that is, identical to spontaneous background responses; see material and methods for threshold determination). The percent of positive T1D (top number) and HD (bottom number) is indicated for each condition, with antigens yielding responses significantly different between HD and T1D patients in bold ( P

    Techniques Used: Titration, Recombinant, Enzyme-linked Immunospot, IA

    T1D patients present a humoural autoimmune response against IL-2. ( a ) Percentage of anti-rhIL-2 positive subjects among five different cohorts: serum samples were obtained from healthy donors (HD, n =249) and patients diagnosed with type 2 (T2D; n =24) or type 1 diabetes ( n =39 in cohort 1, n =15 in cohort 2 and n =21 in cohort 3). P -values were calculated using pairwise Fisher exact tests. ( b ) IgG subclass-specific anti-IL2 ELISA in T1D patients, absorbance OD measured at 450 after incubation of sera diluted 1:50. ( c ) T1D patient samples were divided into three groups based on EC 50 values for an IL-2 direct ELISA from total sera (weak, moderate and strong binders) and are plotted relative to the anti-IL-2 EC 50 value of IgG purified from the respective serum sample. Mean and s.d. overlay individual data points, and P -values were calculated using Welch's t -test. ( d ) (upper panel) rhIL-2 competition ELISA using IgG purified from the sera of T1D patients, with the control (lower panel) of an anti-influenza ELISA with competing soluble rhIL-2.
    Figure Legend Snippet: T1D patients present a humoural autoimmune response against IL-2. ( a ) Percentage of anti-rhIL-2 positive subjects among five different cohorts: serum samples were obtained from healthy donors (HD, n =249) and patients diagnosed with type 2 (T2D; n =24) or type 1 diabetes ( n =39 in cohort 1, n =15 in cohort 2 and n =21 in cohort 3). P -values were calculated using pairwise Fisher exact tests. ( b ) IgG subclass-specific anti-IL2 ELISA in T1D patients, absorbance OD measured at 450 after incubation of sera diluted 1:50. ( c ) T1D patient samples were divided into three groups based on EC 50 values for an IL-2 direct ELISA from total sera (weak, moderate and strong binders) and are plotted relative to the anti-IL-2 EC 50 value of IgG purified from the respective serum sample. Mean and s.d. overlay individual data points, and P -values were calculated using Welch's t -test. ( d ) (upper panel) rhIL-2 competition ELISA using IgG purified from the sera of T1D patients, with the control (lower panel) of an anti-influenza ELISA with competing soluble rhIL-2.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Incubation, Direct ELISA, Purification

    High frequencies of anti-IL-2 autoantibodies are present in different autoimmune diseases. ( a ) Serum samples were obtained from healthy donors (HD, n =249), T1D ( n =75 in the three pooled cohorts), multiple sclerosis (MS; n =33), Sjögren syndrome (SJO; n =22), anti-JO1 positive polymyositis (JO1; n =16), rheumatoid arthritis (RA; n =33), systemic lupus erythematosus (SLE; n =20), chronic inflammatory demyelinating neuropathy (CIPD; n =51) and cancer (Cancer; n =128) patients. Cancer patients were used as controls for a non-autoimmune disease. Left panel: serum titres of anti-rhIL-2 IgG in the different cohorts. Dashed line indicates the threshold of positivity. Right panel: percentage of anti-rhIL-2 positive subjects among the different cohorts. Symbols represent individual subjects and horizontal bars are the medians. * P
    Figure Legend Snippet: High frequencies of anti-IL-2 autoantibodies are present in different autoimmune diseases. ( a ) Serum samples were obtained from healthy donors (HD, n =249), T1D ( n =75 in the three pooled cohorts), multiple sclerosis (MS; n =33), Sjögren syndrome (SJO; n =22), anti-JO1 positive polymyositis (JO1; n =16), rheumatoid arthritis (RA; n =33), systemic lupus erythematosus (SLE; n =20), chronic inflammatory demyelinating neuropathy (CIPD; n =51) and cancer (Cancer; n =128) patients. Cancer patients were used as controls for a non-autoimmune disease. Left panel: serum titres of anti-rhIL-2 IgG in the different cohorts. Dashed line indicates the threshold of positivity. Right panel: percentage of anti-rhIL-2 positive subjects among the different cohorts. Symbols represent individual subjects and horizontal bars are the medians. * P

    Techniques Used: Mass Spectrometry

    High-doses rhIL-2 injection in NOD induce neutralizing anti-rhIL-2 antibodies. ( a – f ) Five-to-14-week-old male or female NOD mice were daily treated with PBS or high-doses rhIL-2 (250,000; 500,000 or 1,000,000 IU) over 30 days. ( a , b ) Kaplan-Meier survival curves of treated female ( a , top panel) or male ( b , top panel) mice; and diabetes incidence in female ( a , bottom panel) or male ( b , bottom panel) mice. ( c ) Percentage of dead, diabetic or alive and non-diabetic NOD mice after 30 days of treatment; IL-2-treated: pool of (250,000; 500,000 and 1,000,000 IU IL-2 treated mice. ( d ) Percentage of Foxp3 + among CD3 + CD4 + splenocytes of NOD mice treated for 5 to 30 days with high-doses IL-2 or PBS. ( e ) Serum anti-rhIL-2 IgG titres of untreated B6 mice and pre-diabetic NOD mice treated for 0, 7 or 30 days with high-dose IL-2. ( f ) Proliferation of CTLL-2 cells cultured for 3 days with 3 IU ml −1 rhIL-2 and serially diluted serum from B6 (closed circles) or NOD mice treated for 30 days with high-dose rhIL-2 (open circles). Proliferation is expressed as percentage of control (CTLL-2 cultured for 3 days with 3 IU ml −1 rhIL-2 without mouse serum). Data are cumulative of at least two independent experiments. ns, not significant. *** P
    Figure Legend Snippet: High-doses rhIL-2 injection in NOD induce neutralizing anti-rhIL-2 antibodies. ( a – f ) Five-to-14-week-old male or female NOD mice were daily treated with PBS or high-doses rhIL-2 (250,000; 500,000 or 1,000,000 IU) over 30 days. ( a , b ) Kaplan-Meier survival curves of treated female ( a , top panel) or male ( b , top panel) mice; and diabetes incidence in female ( a , bottom panel) or male ( b , bottom panel) mice. ( c ) Percentage of dead, diabetic or alive and non-diabetic NOD mice after 30 days of treatment; IL-2-treated: pool of (250,000; 500,000 and 1,000,000 IU IL-2 treated mice. ( d ) Percentage of Foxp3 + among CD3 + CD4 + splenocytes of NOD mice treated for 5 to 30 days with high-doses IL-2 or PBS. ( e ) Serum anti-rhIL-2 IgG titres of untreated B6 mice and pre-diabetic NOD mice treated for 0, 7 or 30 days with high-dose IL-2. ( f ) Proliferation of CTLL-2 cells cultured for 3 days with 3 IU ml −1 rhIL-2 and serially diluted serum from B6 (closed circles) or NOD mice treated for 30 days with high-dose rhIL-2 (open circles). Proliferation is expressed as percentage of control (CTLL-2 cultured for 3 days with 3 IU ml −1 rhIL-2 without mouse serum). Data are cumulative of at least two independent experiments. ns, not significant. *** P

    Techniques Used: Injection, Mouse Assay, Cell Culture

    5) Product Images from "XRCC1 suppresses somatic hypermutation and promotes alternative nonhomologous end joining in Igh genes"

    Article Title: XRCC1 suppresses somatic hypermutation and promotes alternative nonhomologous end joining in Igh genes

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20111135

    Xrcc1 +/− cells have shorter overlaps in switch junctions. (A) DNA synthesis in Xrcc1 +/+ and Xrcc1 +/− splenic B cells. Cells were cultured with LPS and IL-4 in the presence of EdU. Error bars represent the SD of values from two independent experiments with one mouse per genotype per experiment. (B) Percentage of switching to the indicated isotypes by Xrcc1 +/− cells relative to Xrcc1 +/+ cells after 4 d in culture. Wild-type (WT) levels (dotted line) were 37% for IgG1, 17% for IgG2a, 32% for IgG2b, and 3% for IgG3. Error bars signify the SD of values from three independent experiments with two mice per genotype per experiment. (C) Microhomology in S μ -S γ 3 switch joins from cells stimulated with LPS. Lengths of overlaps are depicted on the x-axis, and mean overlap for number ( n ) of sequences from two independent experiments with two mice per genotype per experiment is shown in the inset. Statistical difference was determined by the Mann-Whitney test.
    Figure Legend Snippet: Xrcc1 +/− cells have shorter overlaps in switch junctions. (A) DNA synthesis in Xrcc1 +/+ and Xrcc1 +/− splenic B cells. Cells were cultured with LPS and IL-4 in the presence of EdU. Error bars represent the SD of values from two independent experiments with one mouse per genotype per experiment. (B) Percentage of switching to the indicated isotypes by Xrcc1 +/− cells relative to Xrcc1 +/+ cells after 4 d in culture. Wild-type (WT) levels (dotted line) were 37% for IgG1, 17% for IgG2a, 32% for IgG2b, and 3% for IgG3. Error bars signify the SD of values from three independent experiments with two mice per genotype per experiment. (C) Microhomology in S μ -S γ 3 switch joins from cells stimulated with LPS. Lengths of overlaps are depicted on the x-axis, and mean overlap for number ( n ) of sequences from two independent experiments with two mice per genotype per experiment is shown in the inset. Statistical difference was determined by the Mann-Whitney test.

    Techniques Used: DNA Synthesis, Cell Culture, Mouse Assay, MANN-WHITNEY

    Xrcc1 +/− cells have increased double-strand breaks. (A) Localization of XRCC1 to the S μ region as assessed by ChIP in Xrcc1 +/+ , Xrcc1 +/− , and Aid −/− cells stimulated with LPS and IL-4 for 2 d. Numbers are graphed relative to input DNA. Both S μ and control IgG signals are standardized to C µ signals. For Xrcc1 +/+ and Aid −/− cells, error bars represent the SD of values from three independent experiments with one mouse per genotype per experiment. For Xrcc1 +/− cells, error bars represent the SD of values from two independent experiments with one mouse per genotype per experiment. Significance was determined by the Student’s t test. (B) LM-PCR was performed on dilutions of DNA from cells stimulated with LPS plus IL-4, using Gapdh amplification as a loading control. After electrophoresis, bands were identified by hybridization to S μ . Representative blots are shown. (C) Quantification of breaks from the LM-PCR assay was performed using the highest dilution of DNA and was calculated from 10 independent PCR reactions each from two Xrcc1 +/− and two Aid −/− mice and 30 independent reactions from six Xrcc1 +/+ mice. Horizontal bars show the mean intensity of hybridization. Statistical analysis was performed using a two-tailed Student’s t test.
    Figure Legend Snippet: Xrcc1 +/− cells have increased double-strand breaks. (A) Localization of XRCC1 to the S μ region as assessed by ChIP in Xrcc1 +/+ , Xrcc1 +/− , and Aid −/− cells stimulated with LPS and IL-4 for 2 d. Numbers are graphed relative to input DNA. Both S μ and control IgG signals are standardized to C µ signals. For Xrcc1 +/+ and Aid −/− cells, error bars represent the SD of values from three independent experiments with one mouse per genotype per experiment. For Xrcc1 +/− cells, error bars represent the SD of values from two independent experiments with one mouse per genotype per experiment. Significance was determined by the Student’s t test. (B) LM-PCR was performed on dilutions of DNA from cells stimulated with LPS plus IL-4, using Gapdh amplification as a loading control. After electrophoresis, bands were identified by hybridization to S μ . Representative blots are shown. (C) Quantification of breaks from the LM-PCR assay was performed using the highest dilution of DNA and was calculated from 10 independent PCR reactions each from two Xrcc1 +/− and two Aid −/− mice and 30 independent reactions from six Xrcc1 +/+ mice. Horizontal bars show the mean intensity of hybridization. Statistical analysis was performed using a two-tailed Student’s t test.

    Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Electrophoresis, Hybridization, Mouse Assay, Two Tailed Test

    6) Product Images from "Tuning of in vivo cognate B-T cell interactions by Intersectin 2 is required for effective anti-viral B cell immunity"

    Article Title: Tuning of in vivo cognate B-T cell interactions by Intersectin 2 is required for effective anti-viral B cell immunity

    Journal: eLife

    doi: 10.7554/eLife.26556

    Defective humoral responses in Itsn2 -/- animals ( A ) WT and Itsn2 -/- littermates were immunised with NP-KLH precipitated in Alum, and spleens of immunised animals analysed by flow cytometry 13d after immunisation. Tfh cells (CD4 + CXCR5 + PD-1 + ). Panels on the right show the show percentage of cells in the indicated gates (each dot represents a different animal). ( B ) WT and Itsn2 -/- littermates were immunised with NP-KLH precipitated in Alum, and 13 days post-immunisation, NP-specific antibody secreting cells were detected in the spleen by ELISPOT. ( F ) WT and Itsn2 -/- littermates were immunised with NP-LPS and titres of NP 23 -specific IgM and IgG3 were measured by ELISA. Data are representative of at least 2 independent experiments with more than three animals in each group. Student’s t-test, ns p > 0.05, *p
    Figure Legend Snippet: Defective humoral responses in Itsn2 -/- animals ( A ) WT and Itsn2 -/- littermates were immunised with NP-KLH precipitated in Alum, and spleens of immunised animals analysed by flow cytometry 13d after immunisation. Tfh cells (CD4 + CXCR5 + PD-1 + ). Panels on the right show the show percentage of cells in the indicated gates (each dot represents a different animal). ( B ) WT and Itsn2 -/- littermates were immunised with NP-KLH precipitated in Alum, and 13 days post-immunisation, NP-specific antibody secreting cells were detected in the spleen by ELISPOT. ( F ) WT and Itsn2 -/- littermates were immunised with NP-LPS and titres of NP 23 -specific IgM and IgG3 were measured by ELISA. Data are representative of at least 2 independent experiments with more than three animals in each group. Student’s t-test, ns p > 0.05, *p

    Techniques Used: Flow Cytometry, Cytometry, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay

    7) Product Images from "Xenobiotic metal-induced autoimmunity: mercury and silver differentially induce antinucleolar autoantibody production in susceptible H-2s, H-2q and H-2f mice"

    Article Title: Xenobiotic metal-induced autoimmunity: mercury and silver differentially induce antinucleolar autoantibody production in susceptible H-2s, H-2q and H-2f mice

    Journal: Clinical and Experimental Immunology

    doi: 10.1046/j.1365-2249.2003.02085.x

    F 1 hybrid crossetween a silver-resistant (H-2 f ) and -susceptible (H-2 s ) genotype are resistant to silver, but not to mercury. Groups of F 1 hybrid crosses between silver-susceptible, A.SW (H-2 s ) and its H-2 congenic silver-resistant A.CA (H-2 f ) mice [(A.SW × A.CA) F 1 and/or (A.CA × A.SW) F 1 hybrids] were treated continuously with mercury (filled circles), silver (filled squares) or as controls, with saline (open squares) for 4 weeks. Thereafter, the mice were bled and killed. The sera were tested for the presence of IgG1 (a) and IgG2a (b) ANolA by using an indirect immunofluorescence (IIF) method. Hep-2 cell line was used as the substrate. Data are shown as mean + 1 s.e. Significant differences between the parameters in mercury-, silver- and saline-injected mice were calculated by Wilcoxon–Mann–Whitney test. ** P
    Figure Legend Snippet: F 1 hybrid crossetween a silver-resistant (H-2 f ) and -susceptible (H-2 s ) genotype are resistant to silver, but not to mercury. Groups of F 1 hybrid crosses between silver-susceptible, A.SW (H-2 s ) and its H-2 congenic silver-resistant A.CA (H-2 f ) mice [(A.SW × A.CA) F 1 and/or (A.CA × A.SW) F 1 hybrids] were treated continuously with mercury (filled circles), silver (filled squares) or as controls, with saline (open squares) for 4 weeks. Thereafter, the mice were bled and killed. The sera were tested for the presence of IgG1 (a) and IgG2a (b) ANolA by using an indirect immunofluorescence (IIF) method. Hep-2 cell line was used as the substrate. Data are shown as mean + 1 s.e. Significant differences between the parameters in mercury-, silver- and saline-injected mice were calculated by Wilcoxon–Mann–Whitney test. ** P

    Techniques Used: Mouse Assay, Immunofluorescence, Injection, MANN-WHITNEY

    Induction of antinucleolar autoantibodies (ANolAs) of various IgG isotypes in mercury- and silver-treated A.SW (H-2 s ), FVB/N (H-2 q ) and A.CA (H-2 f ) mice. Groups of female A.SW (H-2 s ) (solid and open circles), FVB/N (H-2 q ) (solid and open squares) and A.CA (H-2 f ) (solid and open polygons) mice were injected repeatedly subcutaneously (s.c.) with HgCl 2 (solid symbols, upper panel) or AgNO 3 (solid symbols, lower panel) and/or NaCl (open symbols upper and lower panels) for 4 weeks. At the end of each experiment the mice were bled and killed. The sera were tested for the presence of ANolA using an indirect immunofluorescence (IIF) method. Hep-2 cell line was used as the substrate. Data are shown as mean + 1 s.e. Significant differences between the parameters in mercury- or silver- and saline-injected mice were calculated by Wilcoxon–Mann–Whitney test. * P
    Figure Legend Snippet: Induction of antinucleolar autoantibodies (ANolAs) of various IgG isotypes in mercury- and silver-treated A.SW (H-2 s ), FVB/N (H-2 q ) and A.CA (H-2 f ) mice. Groups of female A.SW (H-2 s ) (solid and open circles), FVB/N (H-2 q ) (solid and open squares) and A.CA (H-2 f ) (solid and open polygons) mice were injected repeatedly subcutaneously (s.c.) with HgCl 2 (solid symbols, upper panel) or AgNO 3 (solid symbols, lower panel) and/or NaCl (open symbols upper and lower panels) for 4 weeks. At the end of each experiment the mice were bled and killed. The sera were tested for the presence of ANolA using an indirect immunofluorescence (IIF) method. Hep-2 cell line was used as the substrate. Data are shown as mean + 1 s.e. Significant differences between the parameters in mercury- or silver- and saline-injected mice were calculated by Wilcoxon–Mann–Whitney test. * P

    Techniques Used: Mouse Assay, Injection, Immunofluorescence, MANN-WHITNEY

    Induction of antinucleolar autoantibodies (ANolAs) of various IgG isotypes in H-2 congenic B10 mice injected with mercuric chloride (HgCl 2 ) and silver nitrate (AgNO 3 ). Groups of female B10.S (H-2 s ), B10.G (H-2 q ) and B10.M (H-2 f ) mice were injected repeatedly subcutaneously (s.c.) with HgCl 2 (solid bars, upper panel) or AgNO 3 (solid bars, lower panel) and/or NaCl (open bars) for 4 weeks. At the end of each experiment the mice were bled and killed. The sera were tested for the presence of ANolA by using an indirect immunofluorescence (IIF) method. Rat liver sections were used as substrates. Data are shown as mean + 1 se. Significant differences between the parameters in mercury- or silver- and saline-injected mice were calculated by Wilcoxon–Mann–Whitney test. * P
    Figure Legend Snippet: Induction of antinucleolar autoantibodies (ANolAs) of various IgG isotypes in H-2 congenic B10 mice injected with mercuric chloride (HgCl 2 ) and silver nitrate (AgNO 3 ). Groups of female B10.S (H-2 s ), B10.G (H-2 q ) and B10.M (H-2 f ) mice were injected repeatedly subcutaneously (s.c.) with HgCl 2 (solid bars, upper panel) or AgNO 3 (solid bars, lower panel) and/or NaCl (open bars) for 4 weeks. At the end of each experiment the mice were bled and killed. The sera were tested for the presence of ANolA by using an indirect immunofluorescence (IIF) method. Rat liver sections were used as substrates. Data are shown as mean + 1 se. Significant differences between the parameters in mercury- or silver- and saline-injected mice were calculated by Wilcoxon–Mann–Whitney test. * P

    Techniques Used: Mouse Assay, Injection, Immunofluorescence, MANN-WHITNEY

    8) Product Images from "Mice Deficient in Nuclear Factor (NF)-?B/p52 Present with Defects in Humoral Responses, Germinal Center Reactions, and Splenic Microarchitecture "

    Article Title: Mice Deficient in Nuclear Factor (NF)-?B/p52 Present with Defects in Humoral Responses, Germinal Center Reactions, and Splenic Microarchitecture

    Journal: The Journal of Experimental Medicine

    doi:

    Reduction in the B/T cell ratio and reduced levels of total serum antibodies in p52 / p100 (−/−) mice. ( A ) Representative FCM analysis of splenocytes from 16-wk-old p52 / p100 (−/−) and (+/−) mice. IgM–Red 670 versus IgD-FITC two-color profiles are displayed in the top panels. Single-color profiles depict B220-PE and CD3-FITC staining on total splenocytes ( middle and bottom , respectively). Numbers reflect the percentage of positively stained spleen cells. ( B ) Reduced levels of IgG1, IgG2b, and IgA in sera of p52 / p100 (−/−) mice. Titers of total immunoglobulin isotypes from 7–10-wk-old mice of each genotype are shown as indicated. IgG1, IgG2a, and IgA titers differed significantly ( P
    Figure Legend Snippet: Reduction in the B/T cell ratio and reduced levels of total serum antibodies in p52 / p100 (−/−) mice. ( A ) Representative FCM analysis of splenocytes from 16-wk-old p52 / p100 (−/−) and (+/−) mice. IgM–Red 670 versus IgD-FITC two-color profiles are displayed in the top panels. Single-color profiles depict B220-PE and CD3-FITC staining on total splenocytes ( middle and bottom , respectively). Numbers reflect the percentage of positively stained spleen cells. ( B ) Reduced levels of IgG1, IgG2b, and IgA in sera of p52 / p100 (−/−) mice. Titers of total immunoglobulin isotypes from 7–10-wk-old mice of each genotype are shown as indicated. IgG1, IgG2a, and IgA titers differed significantly ( P

    Techniques Used: Mouse Assay, Staining

    Defective antibody response to T-dependent antigens in p52-deficient mice. 5–30-wk-old mice of both sexes were injected intraperitoneally with ( A ) 100 μg of TNP (2,4,6 trinitro-phenyl)–KLH (TD), ( B ) 25 μg of TNP-Ficoll (TI-2) or ( C ) 50 μg of TNP-LPS (TI-1). Serum levels of anti-TNP specific antibodies were determined by isotype-specific ELISA 14 d after injection of the TNP conjugates. Antibody levels, expressed as OD, are shown for each genotype, as indicated. Anti-TNP antibodies were undetectable in sera of animals before challenge (data not shown). OD values differed significantly among the following groups of animals by one-way analysis of variance. ( A ) (−/−) versus (+/+) all isotypes tested; (−/−) versus (+/−), IgM, IgG1, IgG2b and IgG3; (+/−) versus (+/+), IgG1, IgG2a, IgG2b. ( C ) (−/−) versus (+/−) and (+/+), IgM, and IgG1. All other groups were not significantly different.
    Figure Legend Snippet: Defective antibody response to T-dependent antigens in p52-deficient mice. 5–30-wk-old mice of both sexes were injected intraperitoneally with ( A ) 100 μg of TNP (2,4,6 trinitro-phenyl)–KLH (TD), ( B ) 25 μg of TNP-Ficoll (TI-2) or ( C ) 50 μg of TNP-LPS (TI-1). Serum levels of anti-TNP specific antibodies were determined by isotype-specific ELISA 14 d after injection of the TNP conjugates. Antibody levels, expressed as OD, are shown for each genotype, as indicated. Anti-TNP antibodies were undetectable in sera of animals before challenge (data not shown). OD values differed significantly among the following groups of animals by one-way analysis of variance. ( A ) (−/−) versus (+/+) all isotypes tested; (−/−) versus (+/−), IgM, IgG1, IgG2b and IgG3; (+/−) versus (+/+), IgG1, IgG2a, IgG2b. ( C ) (−/−) versus (+/−) and (+/+), IgM, and IgG1. All other groups were not significantly different.

    Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay

    9) Product Images from "AP-1 Transcription Factor JunD Confers Protection from Accelerated Nephrotoxic Nephritis and Control Podocyte-Specific Vegfa Expression"

    Article Title: AP-1 Transcription Factor JunD Confers Protection from Accelerated Nephrotoxic Nephritis and Control Podocyte-Specific Vegfa Expression

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2011.03.006

    Deposited glomerular IgG and humoral immune response assessment in WT and Jund -/- mice 10 days after accelerated NTN induction. Quantitative immunofluorescence for sheep IgG ( A ) and mouse IgG ( B ). Representative glomeruli showing immunofluorescence for sheep and mouse IgG 10 days after the induction of accelerated NTN ( C ). Serum levels of mouse total IgG specific for sheep IgG ( D ). Ten mice were used in each group. AFU indicates arbitrary fluorescence unit; AEU, arbitrary enzyme-linked immunosorbent assay unit; ns, nonsignificant.
    Figure Legend Snippet: Deposited glomerular IgG and humoral immune response assessment in WT and Jund -/- mice 10 days after accelerated NTN induction. Quantitative immunofluorescence for sheep IgG ( A ) and mouse IgG ( B ). Representative glomeruli showing immunofluorescence for sheep and mouse IgG 10 days after the induction of accelerated NTN ( C ). Serum levels of mouse total IgG specific for sheep IgG ( D ). Ten mice were used in each group. AFU indicates arbitrary fluorescence unit; AEU, arbitrary enzyme-linked immunosorbent assay unit; ns, nonsignificant.

    Techniques Used: Mouse Assay, Immunofluorescence, Fluorescence, Enzyme-linked Immunosorbent Assay

    10) Product Images from "Genetic Variation in the Magnitude and Longevity of the IgG Subclass Response to a Diphtheria-Tetanus-Acellular Pertussis (DTaP) Vaccine in Mice"

    Article Title: Genetic Variation in the Magnitude and Longevity of the IgG Subclass Response to a Diphtheria-Tetanus-Acellular Pertussis (DTaP) Vaccine in Mice

    Journal: Vaccines

    doi: 10.3390/vaccines7040124

    IgG1/IgG3 ratios for different vaccine antigens. ( a ) IgG1 to IgG3 ratios were calculated as log(IgG1/IgG3) for titers measured at wk14 of age. The calculated Pearson correlation coefficients of IgG1/IgG3 ratio (average value of each strain) between the four DTaP antigens were all significant as indicated by the p -values. ( b ) Number of genes identified by genome-wide association mapping algorithm for IgG1/IgG3 ratio were plotted by Venn diagram.
    Figure Legend Snippet: IgG1/IgG3 ratios for different vaccine antigens. ( a ) IgG1 to IgG3 ratios were calculated as log(IgG1/IgG3) for titers measured at wk14 of age. The calculated Pearson correlation coefficients of IgG1/IgG3 ratio (average value of each strain) between the four DTaP antigens were all significant as indicated by the p -values. ( b ) Number of genes identified by genome-wide association mapping algorithm for IgG1/IgG3 ratio were plotted by Venn diagram.

    Techniques Used: GWAS

    Correlations between antigen-specific responses among IgG1, IgG2b, and IgG3. The correlations between antibody response to different vaccine antigens for IgG1, IgG2b, and IgG3 were plotted for antibody magnitude ( a ) and longevity ( b ). The correlations were presented by Pearson r values for each pair of antigen-specific antibody within the same IgG subclass. Significant values ( p
    Figure Legend Snippet: Correlations between antigen-specific responses among IgG1, IgG2b, and IgG3. The correlations between antibody response to different vaccine antigens for IgG1, IgG2b, and IgG3 were plotted for antibody magnitude ( a ) and longevity ( b ). The correlations were presented by Pearson r values for each pair of antigen-specific antibody within the same IgG subclass. Significant values ( p

    Techniques Used:

    Effect of TLR4 on the magnitude antigen-specific IgG subclass titers in mice immunized with DTaP. Antibody titers at wk14 of age were compared between C3H/HeJ (TLR4-deficient) and C3H/HeOuJ (TRL4-sufficient) mice. * p
    Figure Legend Snippet: Effect of TLR4 on the magnitude antigen-specific IgG subclass titers in mice immunized with DTaP. Antibody titers at wk14 of age were compared between C3H/HeJ (TLR4-deficient) and C3H/HeOuJ (TRL4-sufficient) mice. * p

    Techniques Used: Mouse Assay

    IgG1, IgG2b, and IgG3 titers to Diphtheria-Tetanus-Acellular Pertussis (DTaP) vaccine antigens in inbred mouse strains. Titers were determined at week 14 (two weeks after the third vaccination) against four antigens in the DTaP vaccine by ELISA. There was significant variation ( p
    Figure Legend Snippet: IgG1, IgG2b, and IgG3 titers to Diphtheria-Tetanus-Acellular Pertussis (DTaP) vaccine antigens in inbred mouse strains. Titers were determined at week 14 (two weeks after the third vaccination) against four antigens in the DTaP vaccine by ELISA. There was significant variation ( p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Ranking of mouse strains based on the mean of the IgG1/IgG3 ratios of the four vaccine antigens. A low IgG1/IgG3 ratio indicates Th1-prone and a high ratio indicates Th2-prone. NA: Antibodies induced against Prn in PWK/PhJ mice were below the cutoff and this was eliminated from the analysis.
    Figure Legend Snippet: Ranking of mouse strains based on the mean of the IgG1/IgG3 ratios of the four vaccine antigens. A low IgG1/IgG3 ratio indicates Th1-prone and a high ratio indicates Th2-prone. NA: Antibodies induced against Prn in PWK/PhJ mice were below the cutoff and this was eliminated from the analysis.

    Techniques Used: Mouse Assay

    Correlations between IgG1, IgG2b, and IgG3 against the same vaccine antigen. The correlations between IgG1, IgG2b, and IgG3 were plotted against the same vaccine antigen for antibody magnitude ( a ) and longevity ( b ). Mean value of the longevity of each strain was used to calculate for Pearson’s correlation coefficient (r) for the correlations between the magnitude and longevity of IgG1 and IgG2b, IgG1 and IgG3, IgG2b and IgG3. Significant values ( p
    Figure Legend Snippet: Correlations between IgG1, IgG2b, and IgG3 against the same vaccine antigen. The correlations between IgG1, IgG2b, and IgG3 were plotted against the same vaccine antigen for antibody magnitude ( a ) and longevity ( b ). Mean value of the longevity of each strain was used to calculate for Pearson’s correlation coefficient (r) for the correlations between the magnitude and longevity of IgG1 and IgG2b, IgG1 and IgG3, IgG2b and IgG3. Significant values ( p

    Techniques Used:

    11) Product Images from "Genetic Variation in the Magnitude and Longevity of the IgG Subclass Response to a Diphtheria-Tetanus-Acellular Pertussis (DTaP) Vaccine in Mice"

    Article Title: Genetic Variation in the Magnitude and Longevity of the IgG Subclass Response to a Diphtheria-Tetanus-Acellular Pertussis (DTaP) Vaccine in Mice

    Journal: Vaccines

    doi: 10.3390/vaccines7040124

    IgG1/IgG3 ratios for different vaccine antigens. ( a ) IgG1 to IgG3 ratios were calculated as log(IgG1/IgG3) for titers measured at wk14 of age. The calculated Pearson correlation coefficients of IgG1/IgG3 ratio (average value of each strain) between the four DTaP antigens were all significant as indicated by the p -values. ( b ) Number of genes identified by genome-wide association mapping algorithm for IgG1/IgG3 ratio were plotted by Venn diagram.
    Figure Legend Snippet: IgG1/IgG3 ratios for different vaccine antigens. ( a ) IgG1 to IgG3 ratios were calculated as log(IgG1/IgG3) for titers measured at wk14 of age. The calculated Pearson correlation coefficients of IgG1/IgG3 ratio (average value of each strain) between the four DTaP antigens were all significant as indicated by the p -values. ( b ) Number of genes identified by genome-wide association mapping algorithm for IgG1/IgG3 ratio were plotted by Venn diagram.

    Techniques Used: GWAS

    Correlations between antigen-specific responses among IgG1, IgG2b, and IgG3. The correlations between antibody response to different vaccine antigens for IgG1, IgG2b, and IgG3 were plotted for antibody magnitude ( a ) and longevity ( b ). The correlations were presented by Pearson r values for each pair of antigen-specific antibody within the same IgG subclass. Significant values ( p
    Figure Legend Snippet: Correlations between antigen-specific responses among IgG1, IgG2b, and IgG3. The correlations between antibody response to different vaccine antigens for IgG1, IgG2b, and IgG3 were plotted for antibody magnitude ( a ) and longevity ( b ). The correlations were presented by Pearson r values for each pair of antigen-specific antibody within the same IgG subclass. Significant values ( p

    Techniques Used:

    Effect of TLR4 on the magnitude antigen-specific IgG subclass titers in mice immunized with DTaP. Antibody titers at wk14 of age were compared between C3H/HeJ (TLR4-deficient) and C3H/HeOuJ (TRL4-sufficient) mice. * p
    Figure Legend Snippet: Effect of TLR4 on the magnitude antigen-specific IgG subclass titers in mice immunized with DTaP. Antibody titers at wk14 of age were compared between C3H/HeJ (TLR4-deficient) and C3H/HeOuJ (TRL4-sufficient) mice. * p

    Techniques Used: Mouse Assay

    IgG1, IgG2b, and IgG3 titers to Diphtheria-Tetanus-Acellular Pertussis (DTaP) vaccine antigens in inbred mouse strains. Titers were determined at week 14 (two weeks after the third vaccination) against four antigens in the DTaP vaccine by ELISA. There was significant variation ( p
    Figure Legend Snippet: IgG1, IgG2b, and IgG3 titers to Diphtheria-Tetanus-Acellular Pertussis (DTaP) vaccine antigens in inbred mouse strains. Titers were determined at week 14 (two weeks after the third vaccination) against four antigens in the DTaP vaccine by ELISA. There was significant variation ( p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Ranking of mouse strains based on the mean of the IgG1/IgG3 ratios of the four vaccine antigens. A low IgG1/IgG3 ratio indicates Th1-prone and a high ratio indicates Th2-prone. NA: Antibodies induced against Prn in PWK/PhJ mice were below the cutoff and this was eliminated from the analysis.
    Figure Legend Snippet: Ranking of mouse strains based on the mean of the IgG1/IgG3 ratios of the four vaccine antigens. A low IgG1/IgG3 ratio indicates Th1-prone and a high ratio indicates Th2-prone. NA: Antibodies induced against Prn in PWK/PhJ mice were below the cutoff and this was eliminated from the analysis.

    Techniques Used: Mouse Assay

    Correlations between IgG1, IgG2b, and IgG3 against the same vaccine antigen. The correlations between IgG1, IgG2b, and IgG3 were plotted against the same vaccine antigen for antibody magnitude ( a ) and longevity ( b ). Mean value of the longevity of each strain was used to calculate for Pearson’s correlation coefficient (r) for the correlations between the magnitude and longevity of IgG1 and IgG2b, IgG1 and IgG3, IgG2b and IgG3. Significant values ( p
    Figure Legend Snippet: Correlations between IgG1, IgG2b, and IgG3 against the same vaccine antigen. The correlations between IgG1, IgG2b, and IgG3 were plotted against the same vaccine antigen for antibody magnitude ( a ) and longevity ( b ). Mean value of the longevity of each strain was used to calculate for Pearson’s correlation coefficient (r) for the correlations between the magnitude and longevity of IgG1 and IgG2b, IgG1 and IgG3, IgG2b and IgG3. Significant values ( p

    Techniques Used:

    12) Product Images from "p53 Represses Class Switch Recombination to IgG2a through its Antioxidant Function"

    Article Title: p53 Represses Class Switch Recombination to IgG2a through its Antioxidant Function

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

    doi: 10.4049/jimmunol.0904085

    Increased ROS in switching p53−/− splenic B cells; NAC inhibits CSR and abolishes increased IgG2a switching in p53−/− splenic B cells (A) Shown are overlay histograms of wild-type (red profile) and p53−/− (blue profile) splenic B cells loaded with the CM-H 2 -DCFDA fluorescent ROS probe after activation with LPS + anti-δ-dextran + IFNγ for 48 h. Treatment with 10 mM (blue profile) and 20 mM NAC (green profile) decreases intracellular ROS in activated wild-type and p53−/− splenic B cells. (B) CFSE-loaded cells were activated for IgG2a switching and treated with 10 mM NAC. IgG2a switching was assessed by flow cytometry for surface Ig staining after 2.5 days. Representative FACS plots of 3 independent experiments are shown. Percentages of IgG2a switched cells are given within the gates. (C) CFSE-loaded wild-type splenic B cells were activated with LPS + cytokines and/or anti-δ-dextran to induce CSR to the indicated Ig isotypes. CSR was assessed by flow cytometry for surface Ig staining. Percentages of switched cells are indicated within the gates. Top row shows untreated control cultures, middle row shows cultures treated with 10 mM NAC. Bottom row shows overlay histograms of CSFE fluorescence in control cultures (red profiles) and 10 mM NAC treated cultures (blue profiles). Representative FACS plots of three experiments are shown. (D) Bar graph shows data from the 3 experiments normalized to untreated cells, shown as 100%. Means (+SEM) of percentages of switched cells in NAC-treated cutures relative to untreated cultures are shown. Significance was determined by the one-sample t test. (E) Sμ LM-PCR was performed on threefold dilutions of Gapdh-normalized DNA from wild-type and aid−/− splenic B cells activated with LPS + anti-δ-dextran with or without 10 mM NAC for 2 days. LM-PCR products were blotted and hybridized with an internal Sμ probe. Densitometry scanning was performed on all three lanes representing the threefold dilutions. Untreated sample was set at 1.0 to determine relative density. Representative result of 2 experiments is shown.
    Figure Legend Snippet: Increased ROS in switching p53−/− splenic B cells; NAC inhibits CSR and abolishes increased IgG2a switching in p53−/− splenic B cells (A) Shown are overlay histograms of wild-type (red profile) and p53−/− (blue profile) splenic B cells loaded with the CM-H 2 -DCFDA fluorescent ROS probe after activation with LPS + anti-δ-dextran + IFNγ for 48 h. Treatment with 10 mM (blue profile) and 20 mM NAC (green profile) decreases intracellular ROS in activated wild-type and p53−/− splenic B cells. (B) CFSE-loaded cells were activated for IgG2a switching and treated with 10 mM NAC. IgG2a switching was assessed by flow cytometry for surface Ig staining after 2.5 days. Representative FACS plots of 3 independent experiments are shown. Percentages of IgG2a switched cells are given within the gates. (C) CFSE-loaded wild-type splenic B cells were activated with LPS + cytokines and/or anti-δ-dextran to induce CSR to the indicated Ig isotypes. CSR was assessed by flow cytometry for surface Ig staining. Percentages of switched cells are indicated within the gates. Top row shows untreated control cultures, middle row shows cultures treated with 10 mM NAC. Bottom row shows overlay histograms of CSFE fluorescence in control cultures (red profiles) and 10 mM NAC treated cultures (blue profiles). Representative FACS plots of three experiments are shown. (D) Bar graph shows data from the 3 experiments normalized to untreated cells, shown as 100%. Means (+SEM) of percentages of switched cells in NAC-treated cutures relative to untreated cultures are shown. Significance was determined by the one-sample t test. (E) Sμ LM-PCR was performed on threefold dilutions of Gapdh-normalized DNA from wild-type and aid−/− splenic B cells activated with LPS + anti-δ-dextran with or without 10 mM NAC for 2 days. LM-PCR products were blotted and hybridized with an internal Sμ probe. Densitometry scanning was performed on all three lanes representing the threefold dilutions. Untreated sample was set at 1.0 to determine relative density. Representative result of 2 experiments is shown.

    Techniques Used: Activation Assay, Flow Cytometry, Cytometry, Staining, FACS, Fluorescence, Polymerase Chain Reaction

    Polyoma virus infected p53−/− mice show increased in vivo IgG2a switching p53−/− , aid−/− , and wild-type littermate mice were inoculated i.p. with 2×10 6 pfu of polyoma virus strain A2. Splenic germinal center B cells were analyzed for surface isotype expression by flow cytometry 12–20 days after inoculation. (A) FACS plots show surface Ig expression on B220+ GL7+ splenic B cells from an experiment in which cells were harvested 14 days after infection. Percentages of switched cells are depicted next to the gates. (B) Data points and bars represent individual mice and means (n=7 for wild-type, n=5 for p53−/− ) of the percentages of isotype switched cells within the B220+ GL7+ splenic germinal center B-cell subset. Three independent experiments were performed using: 3, 2, and 2 wild-type mice, 1, 2, and 2 p53−/− mice, and in the second experiment (shown) 2 aid−/− mice were included. Significance was determined by the Mann-Whitney U test.
    Figure Legend Snippet: Polyoma virus infected p53−/− mice show increased in vivo IgG2a switching p53−/− , aid−/− , and wild-type littermate mice were inoculated i.p. with 2×10 6 pfu of polyoma virus strain A2. Splenic germinal center B cells were analyzed for surface isotype expression by flow cytometry 12–20 days after inoculation. (A) FACS plots show surface Ig expression on B220+ GL7+ splenic B cells from an experiment in which cells were harvested 14 days after infection. Percentages of switched cells are depicted next to the gates. (B) Data points and bars represent individual mice and means (n=7 for wild-type, n=5 for p53−/− ) of the percentages of isotype switched cells within the B220+ GL7+ splenic germinal center B-cell subset. Three independent experiments were performed using: 3, 2, and 2 wild-type mice, 1, 2, and 2 p53−/− mice, and in the second experiment (shown) 2 aid−/− mice were included. Significance was determined by the Mann-Whitney U test.

    Techniques Used: Infection, Mouse Assay, In Vivo, Expressing, Flow Cytometry, Cytometry, FACS, MANN-WHITNEY

    S region DSBs are increased p53−/− splenic B cells induced for IgG2a switching Sμ and Sγ2a LM-PCR were performed on threefold dilutions of GAPDH-normalized DNA isolated from wild-type (WT), p53−/− , and aid−/− splenic B cells that had been stimulated for 2 days with LPS+IL4 or LPS+IFNγ. Blunt DSBs in Sμ and Sγ2a were assessed by use of 5’ Sμ or 5’ Sγ2a primer, respectively, in combination with a linker specific primer. PCR products were blotted and hybridized with an internal Sμ or Sγ2a probe, respectively. (A) Sμ DSBs in cells activated for IgG1 switching. (B) Sμ DSBs in cells activated for IgG2a switching. (C) Sγ2a DSBs in cells activated for IgG2a switching. (D) Bar graph depicts means (+SEM) of densitometry measurements of autoradiographic films, normalized to WT, which was set at 1.0 (n=4 for all experiments, except for Sγ2a DSBs under IgG2a conditions, n=3.) All 3 titration lanes were scanned together. Each replicate experiment was performed on material from a separate set of mice. Two independent experiments using two sets of mice were performed. (E) Mutations in Sμ-Sγ3 junctions from WT and p53−/− splenic B cells activated to undergo IgG3 switching. Mutation frequency per 50-nucleotide segment is plotted for WT (black bars) and p53−/− (white bars). Overall mutation frequency is 3.1-fold increased in p53−/− versus WT (p = 0.00001). Total mutations/nucleotides analyzed for WT: 31/10,231; for p53: 30/3,182.
    Figure Legend Snippet: S region DSBs are increased p53−/− splenic B cells induced for IgG2a switching Sμ and Sγ2a LM-PCR were performed on threefold dilutions of GAPDH-normalized DNA isolated from wild-type (WT), p53−/− , and aid−/− splenic B cells that had been stimulated for 2 days with LPS+IL4 or LPS+IFNγ. Blunt DSBs in Sμ and Sγ2a were assessed by use of 5’ Sμ or 5’ Sγ2a primer, respectively, in combination with a linker specific primer. PCR products were blotted and hybridized with an internal Sμ or Sγ2a probe, respectively. (A) Sμ DSBs in cells activated for IgG1 switching. (B) Sμ DSBs in cells activated for IgG2a switching. (C) Sγ2a DSBs in cells activated for IgG2a switching. (D) Bar graph depicts means (+SEM) of densitometry measurements of autoradiographic films, normalized to WT, which was set at 1.0 (n=4 for all experiments, except for Sγ2a DSBs under IgG2a conditions, n=3.) All 3 titration lanes were scanned together. Each replicate experiment was performed on material from a separate set of mice. Two independent experiments using two sets of mice were performed. (E) Mutations in Sμ-Sγ3 junctions from WT and p53−/− splenic B cells activated to undergo IgG3 switching. Mutation frequency per 50-nucleotide segment is plotted for WT (black bars) and p53−/− (white bars). Overall mutation frequency is 3.1-fold increased in p53−/− versus WT (p = 0.00001). Total mutations/nucleotides analyzed for WT: 31/10,231; for p53: 30/3,182.

    Techniques Used: Polymerase Chain Reaction, Isolation, Titration, Mouse Assay, Mutagenesis

    p53 Ser18 phosphorylation regulates IgG2a switching CFSE-loaded splenic B cells from p53S18A and wild-type control mice were cultured for 2.5 days with LPS, cytokines and/or anti-δ-dextran to induce Ig class switching to the indicated isotypes. Class switching was determined by flow cytometry for surface Ig staining. (A) Representative FACS plots are shown. Upper panel shows equal CFSE loading in wild-type mice from the same colony and p53S18A splenic B cells. Percentages of switched cells are indicated within the gates. (B) Bar graph depicts data from 4 sets of mice, which were all analyzed in one experiment. Percent CSR in p53S18A relative to WT (+SEM) for the different isotypes are shown. Significance was determined by the one-sample t test.
    Figure Legend Snippet: p53 Ser18 phosphorylation regulates IgG2a switching CFSE-loaded splenic B cells from p53S18A and wild-type control mice were cultured for 2.5 days with LPS, cytokines and/or anti-δ-dextran to induce Ig class switching to the indicated isotypes. Class switching was determined by flow cytometry for surface Ig staining. (A) Representative FACS plots are shown. Upper panel shows equal CFSE loading in wild-type mice from the same colony and p53S18A splenic B cells. Percentages of switched cells are indicated within the gates. (B) Bar graph depicts data from 4 sets of mice, which were all analyzed in one experiment. Percent CSR in p53S18A relative to WT (+SEM) for the different isotypes are shown. Significance was determined by the one-sample t test.

    Techniques Used: Mouse Assay, Cell Culture, Flow Cytometry, Cytometry, Staining, FACS

    p53-deficient splenic B cells show increased IgG2a switching in culture CFSE-loaded splenic B cells from p53−/− and wild-type littermate mice were cultured for 2.5 days with LPS, cytokines and/or anti-δ-dextran to induce Ig class switching to the indicated isotypes. (A) Cells were analyzed for CSFE-dilution and surface Ig by flow cytometry; representative FACS plots are shown; percentages of switched cells are indicated within the gates. (B) Data from 8 experiments (8 sets of mice) were normalized to the percent of wild-type littermate (WT) switching, indicated as 100%. Mean percentages of switching in p53−/− relative to WT (+SEM) for the different isotypes are shown. Significance was determined by the one-sample t test.
    Figure Legend Snippet: p53-deficient splenic B cells show increased IgG2a switching in culture CFSE-loaded splenic B cells from p53−/− and wild-type littermate mice were cultured for 2.5 days with LPS, cytokines and/or anti-δ-dextran to induce Ig class switching to the indicated isotypes. (A) Cells were analyzed for CSFE-dilution and surface Ig by flow cytometry; representative FACS plots are shown; percentages of switched cells are indicated within the gates. (B) Data from 8 experiments (8 sets of mice) were normalized to the percent of wild-type littermate (WT) switching, indicated as 100%. Mean percentages of switching in p53−/− relative to WT (+SEM) for the different isotypes are shown. Significance was determined by the one-sample t test.

    Techniques Used: Mouse Assay, Cell Culture, Flow Cytometry, Cytometry, FACS

    13) Product Images from "Accelerated Pathological and Clinical Nephritis in Systemic Lupus Erythematosus-Prone New Zealand Mixed 2328 Mice Doubly Deficient in TNF Receptor 1 and TNF Receptor 2 via a Th17-Associated Pathway"

    Article Title: Accelerated Pathological and Clinical Nephritis in Systemic Lupus Erythematosus-Prone New Zealand Mixed 2328 Mice Doubly Deficient in TNF Receptor 1 and TNF Receptor 2 via a Th17-Associated Pathway

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

    doi: 10.4049/jimmunol.0802948

    Representative H E staining (original magnification ×200) and IgG1, IgG2b, and C3 immunofluorescence staining (original magnification ×40) of kidney sections from female NZM 2328 mice with the indicated genotypes and ages. Increased
    Figure Legend Snippet: Representative H E staining (original magnification ×200) and IgG1, IgG2b, and C3 immunofluorescence staining (original magnification ×40) of kidney sections from female NZM 2328 mice with the indicated genotypes and ages. Increased

    Techniques Used: Staining, Immunofluorescence, Mouse Assay

    IgG anti-dsDNA autoantibodies in the different lines of mice at 5–5.5 and 7.5–9 mo of age. n = 7–13 mice in the various groups. Each symbol represents an individual mouse. The composite results are plotted as box plots. The lines
    Figure Legend Snippet: IgG anti-dsDNA autoantibodies in the different lines of mice at 5–5.5 and 7.5–9 mo of age. n = 7–13 mice in the various groups. Each symbol represents an individual mouse. The composite results are plotted as box plots. The lines

    Techniques Used: Mouse Assay

    14) Product Images from "Chronic Inhibition of Cyclooxygenase-2 Attenuates Antibody Responses against Vaccinia Infection"

    Article Title: Chronic Inhibition of Cyclooxygenase-2 Attenuates Antibody Responses against Vaccinia Infection

    Journal:

    doi: 10.1016/j.vaccine.2009.11.005

    Chronic exposure to the Cox-2 inhibitor, SC-58125, impairs production of VV-specific neutralizing IgG. C57BL/6 mice (n = 4) were administered vehicle or SC-58125 (5 mg/kg) starting seven days before infection with VV (1×10 6 PFU) and ending on
    Figure Legend Snippet: Chronic exposure to the Cox-2 inhibitor, SC-58125, impairs production of VV-specific neutralizing IgG. C57BL/6 mice (n = 4) were administered vehicle or SC-58125 (5 mg/kg) starting seven days before infection with VV (1×10 6 PFU) and ending on

    Techniques Used: Mouse Assay, Infection

    Cox-2 deficient mice have impaired VV-specific IgG responses. Wild-type and Cox-2 −/− mice were infected with 1×10 6 PFU VV (n = 4). VV-specific IgG2a antibody titers (plasma) were assessed by ELISA 28 days after infection. (A) IgG2a
    Figure Legend Snippet: Cox-2 deficient mice have impaired VV-specific IgG responses. Wild-type and Cox-2 −/− mice were infected with 1×10 6 PFU VV (n = 4). VV-specific IgG2a antibody titers (plasma) were assessed by ELISA 28 days after infection. (A) IgG2a

    Techniques Used: Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay

    15) Product Images from "IL-21 Receptor Is Required for the Systemic Accumulation of Activated B and T Lymphocytes in MRL/MpJ-Faslpr/lpr/J Mice"

    Article Title: IL-21 Receptor Is Required for the Systemic Accumulation of Activated B and T Lymphocytes in MRL/MpJ-Faslpr/lpr/J Mice

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

    doi: 10.4049/jimmunol.1003871

    Autoantibody production and hypergammaglobulinemia develop in an IL-21R–dependent manner in MRL lpr mice. A , Circulating levels of IgG1 Ab present in serum obtained from WT (black bars) and KO (white bars) mice at 6, 12, and 20 wk of age. Bars
    Figure Legend Snippet: Autoantibody production and hypergammaglobulinemia develop in an IL-21R–dependent manner in MRL lpr mice. A , Circulating levels of IgG1 Ab present in serum obtained from WT (black bars) and KO (white bars) mice at 6, 12, and 20 wk of age. Bars

    Techniques Used: Mouse Assay

    16) Product Images from "Murine B Cell Development and Antibody Responses to Model Antigens Are Not Impaired in the Absence of the TNF Receptor GITR"

    Article Title: Murine B Cell Development and Antibody Responses to Model Antigens Are Not Impaired in the Absence of the TNF Receptor GITR

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0031632

    GITR-deficient mice mount relatively normal antibody responses to model antigens. (A) Total serum levels of IgM, IgG1, IgG2b, IgG2a and IgE in 4 month-old GITR +/+ (129S1 strain, white bars, empty circles) and GITR −/− mice (gray bars, filled circles). n = 7. (B) GITR +/+ (129S1, white bars, empty circles) and GITR −/− (grey bars, filled circles) mice ( n = 5 per group) were immunized i.p. with 5 µg NP 28 -FICOLL. Sera were collected before (day 0) and 4 and 7 days after immunization, and NP-specific IgM and IgG3 were measured by ELISA. (C) GITR +/+ (129S1, white bars, empty circles) and GITR −/− (grey bars, filled circles) mice ( n = 5 per group) were immunized i.p. with 100 µg NP 36 -CGG in Alum. Sera were collected at indicated time points and NP-specific IgM, IgG1, IgG2b and IgG2a were measured by ELISA. (D) Mice described in (C) were boosted with 5 µg/ml NP 36 -CGG injected i.p. 178 days after primary immunization, and sera were collected at indicated times after boost. NP-specific IgG1, IgG2b and IgG2a were measured by ELISA ( n = 4 per group). Bars and circles represent geometric means and individual mice, respectively. * p
    Figure Legend Snippet: GITR-deficient mice mount relatively normal antibody responses to model antigens. (A) Total serum levels of IgM, IgG1, IgG2b, IgG2a and IgE in 4 month-old GITR +/+ (129S1 strain, white bars, empty circles) and GITR −/− mice (gray bars, filled circles). n = 7. (B) GITR +/+ (129S1, white bars, empty circles) and GITR −/− (grey bars, filled circles) mice ( n = 5 per group) were immunized i.p. with 5 µg NP 28 -FICOLL. Sera were collected before (day 0) and 4 and 7 days after immunization, and NP-specific IgM and IgG3 were measured by ELISA. (C) GITR +/+ (129S1, white bars, empty circles) and GITR −/− (grey bars, filled circles) mice ( n = 5 per group) were immunized i.p. with 100 µg NP 36 -CGG in Alum. Sera were collected at indicated time points and NP-specific IgM, IgG1, IgG2b and IgG2a were measured by ELISA. (D) Mice described in (C) were boosted with 5 µg/ml NP 36 -CGG injected i.p. 178 days after primary immunization, and sera were collected at indicated times after boost. NP-specific IgG1, IgG2b and IgG2a were measured by ELISA ( n = 4 per group). Bars and circles represent geometric means and individual mice, respectively. * p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Injection

    B cells express GITR starting at the transitional stage of development. (A) Bone marrow and spleen cells from wild-type (129S1) mice were analyzed ex vivo by flow cytometry for the expression of indicated markers. Live cells in dot plots were gated as indicated on top. Histograms depict GITR expression on B cells in subsets gated as shown in the dot plots in top panel. Expression of GITR on gated CD4 + T cells is shown as a positive control for GITR staining. Rat IgG2b was used as an isotype control for GITR staining. Data are representative of 3 independent experiments. Similar GITR expression was observed on B cells from BALB/c mice (data not shown). (B) GITR +/+ (BALB/c) mice were immunized i.p. with 100 µg NP 39 -CGG in Alum. At days 4, 7, and 15 post immunization, spleen cells were harvested from 2 immunized and 2 nonimmunized (naïve) mice at each point and analyzed for GITR expression on germinal center B cells reactive with NP. The dot plots show NP and PNA binding on B220 + PNA high gated B cells from one naive (left panel) and one immunized (right panel) mouse at day 7. There were no NP + germinal center B cells at day 4, and reduced frequency at day 15 (data not shown). The histogram represents live, B220 + IgD − CD3 − CD11b − PNA high germinal center B cells. GITR expression on NP + germinal center B cells of one mouse at day 7 (black intact line) and one mouse at day 15 (gray intact line) following immunization are shown compared to those on total B220 + PNA − B cells (dark-shaded histogram and dashed black line) in respective animals and on lymphocytes of one GITR −/− mouse (light-shaded histogram).
    Figure Legend Snippet: B cells express GITR starting at the transitional stage of development. (A) Bone marrow and spleen cells from wild-type (129S1) mice were analyzed ex vivo by flow cytometry for the expression of indicated markers. Live cells in dot plots were gated as indicated on top. Histograms depict GITR expression on B cells in subsets gated as shown in the dot plots in top panel. Expression of GITR on gated CD4 + T cells is shown as a positive control for GITR staining. Rat IgG2b was used as an isotype control for GITR staining. Data are representative of 3 independent experiments. Similar GITR expression was observed on B cells from BALB/c mice (data not shown). (B) GITR +/+ (BALB/c) mice were immunized i.p. with 100 µg NP 39 -CGG in Alum. At days 4, 7, and 15 post immunization, spleen cells were harvested from 2 immunized and 2 nonimmunized (naïve) mice at each point and analyzed for GITR expression on germinal center B cells reactive with NP. The dot plots show NP and PNA binding on B220 + PNA high gated B cells from one naive (left panel) and one immunized (right panel) mouse at day 7. There were no NP + germinal center B cells at day 4, and reduced frequency at day 15 (data not shown). The histogram represents live, B220 + IgD − CD3 − CD11b − PNA high germinal center B cells. GITR expression on NP + germinal center B cells of one mouse at day 7 (black intact line) and one mouse at day 15 (gray intact line) following immunization are shown compared to those on total B220 + PNA − B cells (dark-shaded histogram and dashed black line) in respective animals and on lymphocytes of one GITR −/− mouse (light-shaded histogram).

    Techniques Used: Mouse Assay, Ex Vivo, Flow Cytometry, Cytometry, Expressing, Positive Control, Staining, Binding Assay

    17) Product Images from "Marginal zone B cells are naturally reactive to collagen type II and are involved in the initiation of the immune response in collagen-induced arthritis"

    Article Title: Marginal zone B cells are naturally reactive to collagen type II and are involved in the initiation of the immune response in collagen-induced arthritis

    Journal: Cellular and Molecular Immunology

    doi: 10.1038/cmi.2011.2

    Immunization with CII results in IgG1, IgG2a and IgG2b responses. ( a ) The number of CII-specific IgG1, IgG2a, IgG2b and IgG3 AFCs were analyzed in mice immunized with CII by ELISpot. The percentage of AFC of each subclass in the spleen and in lymph nodes
    Figure Legend Snippet: Immunization with CII results in IgG1, IgG2a and IgG2b responses. ( a ) The number of CII-specific IgG1, IgG2a, IgG2b and IgG3 AFCs were analyzed in mice immunized with CII by ELISpot. The percentage of AFC of each subclass in the spleen and in lymph nodes

    Techniques Used: Mouse Assay, Enzyme-linked Immunospot

    The B-cell response to CII is initiated in the spleen of CII-immunized mice. ( a–f ) The number of CII- and OVA-specific IgM and IgG AFCs were analyzed in mice immunized with CII ( n =6–21) or OVA ( n =6–14) by ELISpot. Data are presented
    Figure Legend Snippet: The B-cell response to CII is initiated in the spleen of CII-immunized mice. ( a–f ) The number of CII- and OVA-specific IgM and IgG AFCs were analyzed in mice immunized with CII ( n =6–21) or OVA ( n =6–14) by ELISpot. Data are presented

    Techniques Used: Mouse Assay, Enzyme-linked Immunospot

    18) Product Images from "The Small Rho GTPase TC10 Modulates B Cell Immune Responses"

    Article Title: The Small Rho GTPase TC10 Modulates B Cell Immune Responses

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.1602167

    Impaired B cell responses in TC10 KO animals. ( A ) Serum was collected from naive WT and TC10 KO animals, and baseline titers of IgM, IgG2b, IgG2c, IgG3, and IgA were measured by sandwich ELISA. ( B – E ) WT and TC10 KO animals were infected with 10
    Figure Legend Snippet: Impaired B cell responses in TC10 KO animals. ( A ) Serum was collected from naive WT and TC10 KO animals, and baseline titers of IgM, IgG2b, IgG2c, IgG3, and IgA were measured by sandwich ELISA. ( B – E ) WT and TC10 KO animals were infected with 10

    Techniques Used: Sandwich ELISA, Infection

    19) Product Images from "Immunization of Mice with Live-Attenuated Late Liver Stage-Arresting Plasmodium yoelii Parasites Generates Protective Antibody Responses to Preerythrocytic Stages of Malaria"

    Article Title: Immunization of Mice with Live-Attenuated Late Liver Stage-Arresting Plasmodium yoelii Parasites Generates Protective Antibody Responses to Preerythrocytic Stages of Malaria

    Journal: Infection and Immunity

    doi: 10.1128/IAI.02320-14

    Pyfabb/f − immunization of BALB/c mice and of C57BL/6 mice elicits different patterns of IgG isotypes. BALB/c (white bars, n = 5) or C57BL/6 (black bars, n = 5) mice were immunized with 2 doses of 50,000 Pyfabb/f − sporozoites or mock immunized.
    Figure Legend Snippet: Pyfabb/f − immunization of BALB/c mice and of C57BL/6 mice elicits different patterns of IgG isotypes. BALB/c (white bars, n = 5) or C57BL/6 (black bars, n = 5) mice were immunized with 2 doses of 50,000 Pyfabb/f − sporozoites or mock immunized.

    Techniques Used: Mouse Assay

    20) Product Images from "MyD88 Signaling Is Indispensable for Primary Influenza A Virus Infection but Dispensable for Secondary Infection ▿"

    Article Title: MyD88 Signaling Is Indispensable for Primary Influenza A Virus Infection but Dispensable for Secondary Infection ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01675-10

    Comparable systemic and airway antibody responses are induced in MyD88 −/− TRIF −/− mice. Wild-type B6 and MyD88 −/− TRIF −/− mice were infected with 5 × 10 2 PFU of PR8 virus. Four weeks after primary challenge, serum and BALF were prepared. (A) PR8-specific IgG in serum and BALF and IgG subclasses in BALF were determined by ELISA. (B) IgA levels of serum, BALF, nasal wash (NW), and saliva were determined by ELISA. (C) SIgA levels in BALF and NW samples were determined using pIgR-specific antibody. (D) Hemagglutination inhibition titer was assessed using BALF. *, P
    Figure Legend Snippet: Comparable systemic and airway antibody responses are induced in MyD88 −/− TRIF −/− mice. Wild-type B6 and MyD88 −/− TRIF −/− mice were infected with 5 × 10 2 PFU of PR8 virus. Four weeks after primary challenge, serum and BALF were prepared. (A) PR8-specific IgG in serum and BALF and IgG subclasses in BALF were determined by ELISA. (B) IgA levels of serum, BALF, nasal wash (NW), and saliva were determined by ELISA. (C) SIgA levels in BALF and NW samples were determined using pIgR-specific antibody. (D) Hemagglutination inhibition titer was assessed using BALF. *, P

    Techniques Used: Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay, HI Assay

    21) Product Images from "Class A scavenger receptors regulate tolerance against apoptotic cells, and autoantibodies against these receptors are predictive of systemic lupus"

    Article Title: Class A scavenger receptors regulate tolerance against apoptotic cells, and autoantibodies against these receptors are predictive of systemic lupus

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20070600

    SLE patients have circulating anti-MARCO antibodies. (A) IgG anti-MARCO reactivity is found in sera from SLE patients. sMARCO or blocking buffer–coated ELISA plates were incubated with sera from SLE patients ( n = 20) and healthy individuals ( n = 19). Data are shown as anti-MARCO data minus anti-block buffer data to reduce the level of binding to the block buffer. (B) IgG anti-DNA activity in SLE patients and healthy individuals was measured by ELISA. The patient indicated by an arrow had an OD 405 value of 1.44. (C) Correlation between anti-MARCO and anti-DNA levels in the patient group. The two measures do not correlate as deduced by linear regression analysis (R 2 = 0.038). (D) A model describing the proposed mechanism where anti-scavenger receptor antibodies influence SLE development by altering the marginal zone response to apoptotic cells. (1.) Anti-scavenger receptor autoantibodies alter the uptake of apoptotic cells by MZMs in the marginal zone leading to an increased local self-antigen load. (2.) Subsequently, autoreactive B cells have increased access to self-antigens in this area. (3.) In addition, engagement of the MARCO receptor by specific antibodies has the ability to send an activating signal to MZBs, suggesting a dual effect by the anti-scavenger receptor autoantibodies resulting in disease promotion.
    Figure Legend Snippet: SLE patients have circulating anti-MARCO antibodies. (A) IgG anti-MARCO reactivity is found in sera from SLE patients. sMARCO or blocking buffer–coated ELISA plates were incubated with sera from SLE patients ( n = 20) and healthy individuals ( n = 19). Data are shown as anti-MARCO data minus anti-block buffer data to reduce the level of binding to the block buffer. (B) IgG anti-DNA activity in SLE patients and healthy individuals was measured by ELISA. The patient indicated by an arrow had an OD 405 value of 1.44. (C) Correlation between anti-MARCO and anti-DNA levels in the patient group. The two measures do not correlate as deduced by linear regression analysis (R 2 = 0.038). (D) A model describing the proposed mechanism where anti-scavenger receptor antibodies influence SLE development by altering the marginal zone response to apoptotic cells. (1.) Anti-scavenger receptor autoantibodies alter the uptake of apoptotic cells by MZMs in the marginal zone leading to an increased local self-antigen load. (2.) Subsequently, autoreactive B cells have increased access to self-antigens in this area. (3.) In addition, engagement of the MARCO receptor by specific antibodies has the ability to send an activating signal to MZBs, suggesting a dual effect by the anti-scavenger receptor autoantibodies resulting in disease promotion.

    Techniques Used: Blocking Assay, Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay, Activity Assay

    Anti-MARCO antibodies affect TI-2 B cell responses in C57BL/6 mice. WT mice were injected with 5 μg of the TI-2 antigen TNP-Ficoll alone ( n = 7) or with 100 μg rat IgG anti-MARCO ( n = 7) or a rat isotype control ( n = 4). The data are shown for IgG (A), IgM (B), and IgG3 (C) anti-TNP response pre-immune (PI) at days 5 and 14. In A, data are presented as mean ± SEM, and in B and C, individual data and mean are shown. *, P
    Figure Legend Snippet: Anti-MARCO antibodies affect TI-2 B cell responses in C57BL/6 mice. WT mice were injected with 5 μg of the TI-2 antigen TNP-Ficoll alone ( n = 7) or with 100 μg rat IgG anti-MARCO ( n = 7) or a rat isotype control ( n = 4). The data are shown for IgG (A), IgM (B), and IgG3 (C) anti-TNP response pre-immune (PI) at days 5 and 14. In A, data are presented as mean ± SEM, and in B and C, individual data and mean are shown. *, P

    Techniques Used: Mouse Assay, Injection

    Anti-MARCO antibodies are found in SLE-prone mice. (A and C) IgG anti-DNA levels in 2–8-mo-old (NZB x NZW)F1 mice and in 3–14-mo-old FcγRIIB −/− mice. Control mice are 3-mo-old C57BL/6 mice. (B and D) IgG anti-MARCO reactivity in 2–8-mo-old (NZB x NZW)F1 mice, 3–14-mo-old FcγRIIB −/− mice, and 3-mo-old C57BL/6 mice. (E) Binding to MARCO in the anti-MARCO ELISA blocked with a rat monoclonal antibody (ED31) against MARCO, but not with an isotype control ( n = 2). (F) CHO cells transfected with mouse MARCO or SR-A stained with sera from (NZB x NZW)F1 mice and anti–mouse IgG-FITC. Arrows indicate transfected cells stained by the mouse sera. Bar, 20 μm.
    Figure Legend Snippet: Anti-MARCO antibodies are found in SLE-prone mice. (A and C) IgG anti-DNA levels in 2–8-mo-old (NZB x NZW)F1 mice and in 3–14-mo-old FcγRIIB −/− mice. Control mice are 3-mo-old C57BL/6 mice. (B and D) IgG anti-MARCO reactivity in 2–8-mo-old (NZB x NZW)F1 mice, 3–14-mo-old FcγRIIB −/− mice, and 3-mo-old C57BL/6 mice. (E) Binding to MARCO in the anti-MARCO ELISA blocked with a rat monoclonal antibody (ED31) against MARCO, but not with an isotype control ( n = 2). (F) CHO cells transfected with mouse MARCO or SR-A stained with sera from (NZB x NZW)F1 mice and anti–mouse IgG-FITC. Arrows indicate transfected cells stained by the mouse sera. Bar, 20 μm.

    Techniques Used: Mouse Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Transfection, Staining

    Class A scavenger receptors regulate tolerance against i.v. injected apoptotic cells. (A) 10 7 syngeneic apoptotic cells were injected i.v. four times weekly in WT and MARCO −/− /SR-A −/− DKO mice (C57BL/6 background). IgM and IgG anti-DNA responses in serum were measured pre-immune (PI) at days 12 and 19. Data are shown as mean ± SEM ( n = 8 per genotype). (B) The anti-DNA response in WT, MARCO −/− , SR-A −/− , and DKO mice at days 12 (IgM) and 19 (IgG). Individual data and mean are presented ( n = 8 per genotype). As a control in these experiments, a quantitative analysis of serial dilution of pooled MRL lpr sera that develop spontaneous disease revealed that the levels of IgM and IgG anti-DNA antibodies are ∼5–40 times that of WT mice injected with apoptotic cells. The PI values for MARCO −/− and SR-A −/− were not statistically different from WT controls (not depicted). (C) Subclass analysis of the anti-DNA response at day 26, after the fourth injection of apoptotic cells. Data are shown as mean ± SEM of the OD 405-nm ratio between IgG 2a /IgG 2b and IgG 1 ( n = 8 per genotype). (D) Representative ANA pattern from DKO and WT mice after the fourth injection (d26). Bar, 50 μm. *, P
    Figure Legend Snippet: Class A scavenger receptors regulate tolerance against i.v. injected apoptotic cells. (A) 10 7 syngeneic apoptotic cells were injected i.v. four times weekly in WT and MARCO −/− /SR-A −/− DKO mice (C57BL/6 background). IgM and IgG anti-DNA responses in serum were measured pre-immune (PI) at days 12 and 19. Data are shown as mean ± SEM ( n = 8 per genotype). (B) The anti-DNA response in WT, MARCO −/− , SR-A −/− , and DKO mice at days 12 (IgM) and 19 (IgG). Individual data and mean are presented ( n = 8 per genotype). As a control in these experiments, a quantitative analysis of serial dilution of pooled MRL lpr sera that develop spontaneous disease revealed that the levels of IgM and IgG anti-DNA antibodies are ∼5–40 times that of WT mice injected with apoptotic cells. The PI values for MARCO −/− and SR-A −/− were not statistically different from WT controls (not depicted). (C) Subclass analysis of the anti-DNA response at day 26, after the fourth injection of apoptotic cells. Data are shown as mean ± SEM of the OD 405-nm ratio between IgG 2a /IgG 2b and IgG 1 ( n = 8 per genotype). (D) Representative ANA pattern from DKO and WT mice after the fourth injection (d26). Bar, 50 μm. *, P

    Techniques Used: Injection, Mouse Assay, Serial Dilution

    22) Product Images from "The generation of influenza-specific humoral responses is impaired in ST6Gal I deficient mice"

    Article Title: The generation of influenza-specific humoral responses is impaired in ST6Gal I deficient mice

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

    doi: 10.4049/jimmunol.0802833

    Immune influenza-specific IgG responses
    Figure Legend Snippet: Immune influenza-specific IgG responses

    Techniques Used:

    23) Product Images from "Toll-like receptor 8 deletion accelerates autoimmunity in a mouse model of lupus through a Toll-like receptor 7-dependent mechanism"

    Article Title: Toll-like receptor 8 deletion accelerates autoimmunity in a mouse model of lupus through a Toll-like receptor 7-dependent mechanism

    Journal: Immunology

    doi: 10.1111/imm.12426

    Anti-dsDNA and antinuclear autoantibodies are detected in male Nba2. Yaa and Nba2.TLR8 − / Yaa . Staining of Crithidia luciliae or Hep2 cell coated slides for the presence of IgG anti-dsDNA or IgG antinuclear autoantibodies in the serum of Nba2. Yaa
    Figure Legend Snippet: Anti-dsDNA and antinuclear autoantibodies are detected in male Nba2. Yaa and Nba2.TLR8 − / Yaa . Staining of Crithidia luciliae or Hep2 cell coated slides for the presence of IgG anti-dsDNA or IgG antinuclear autoantibodies in the serum of Nba2. Yaa

    Techniques Used: Staining

    24) Product Images from "Distinct B-cell populations contribute to vaccine antigen-specific antibody production in a transgenic mouse model"

    Article Title: Distinct B-cell populations contribute to vaccine antigen-specific antibody production in a transgenic mouse model

    Journal: Immunology

    doi: 10.1111/imm.12287

    Immunization of ROSA yellow fluorescent protein transgenic (YFP Tg) mice induces vaccine-specific IgG antibodies (a) Total IgG and (b) IgM antibodies specific for inactivated influenza (A/PR8) virus antigen day 9 post prime and boost immunization ( n =
    Figure Legend Snippet: Immunization of ROSA yellow fluorescent protein transgenic (YFP Tg) mice induces vaccine-specific IgG antibodies (a) Total IgG and (b) IgM antibodies specific for inactivated influenza (A/PR8) virus antigen day 9 post prime and boost immunization ( n =

    Techniques Used: Transgenic Assay, Mouse Assay

    25) Product Images from "Engineered binding to erythrocytes induces immunological tolerance to E. coli asparaginase"

    Article Title: Engineered binding to erythrocytes induces immunological tolerance to E. coli asparaginase

    Journal: Science Advances

    doi: 10.1126/sciadv.1500112

    Erythrocyte-binding ASNase is nonimmunogenic and acts as a tolerogen that enables follow-on treatment with WT enzyme. ( A ) Time course of anti-ASNase IgG antibody development in plasma of mice administered with eight weekly 15-μg doses of either WT ASNase or ERY1-ASNase or with one or two tolerogenic doses of ERY1-ASNase followed by WT ASNase for the remaining doses (end-point IgG titers, Mann-Whitney U test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). ( B ) Time course of anti-ASNase IgG antibody development in plasma of mice administered a dose-sparing regimen of 15 μg of either WT ASNase or ERY1-ASNase every 3 weeks for a total of four doses (end-point IgG titers, Mann-Whitney U test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). ( C ) Immunogenicity incidence rates of the dosing regimens of ERY1-ASNase and WT ASNase (Mantel-Cox test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). ( D to G ) End-point plasma anti-ASNase antibody titers of subclass (D) IgG1, (E) IgG2a, (F) IgG2b, and (G) IgG3 (Mann-Whitney U test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). Initial dilutions were 100-fold for all samples; dotted lines indicate mean values for the naïve population ( n = 149); data are presented as means ± SEM ( n = 4 to 9 per group until 80 days; n = 3 to 9 from 80 days onward). ( H ) End-point IgG subclass profile, as calculated by normalization of the sum of all titers across subclasses ( n = 3 to 9).
    Figure Legend Snippet: Erythrocyte-binding ASNase is nonimmunogenic and acts as a tolerogen that enables follow-on treatment with WT enzyme. ( A ) Time course of anti-ASNase IgG antibody development in plasma of mice administered with eight weekly 15-μg doses of either WT ASNase or ERY1-ASNase or with one or two tolerogenic doses of ERY1-ASNase followed by WT ASNase for the remaining doses (end-point IgG titers, Mann-Whitney U test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). ( B ) Time course of anti-ASNase IgG antibody development in plasma of mice administered a dose-sparing regimen of 15 μg of either WT ASNase or ERY1-ASNase every 3 weeks for a total of four doses (end-point IgG titers, Mann-Whitney U test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). ( C ) Immunogenicity incidence rates of the dosing regimens of ERY1-ASNase and WT ASNase (Mantel-Cox test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). ( D to G ) End-point plasma anti-ASNase antibody titers of subclass (D) IgG1, (E) IgG2a, (F) IgG2b, and (G) IgG3 (Mann-Whitney U test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). Initial dilutions were 100-fold for all samples; dotted lines indicate mean values for the naïve population ( n = 149); data are presented as means ± SEM ( n = 4 to 9 per group until 80 days; n = 3 to 9 from 80 days onward). ( H ) End-point IgG subclass profile, as calculated by normalization of the sum of all titers across subclasses ( n = 3 to 9).

    Techniques Used: Binding Assay, Mouse Assay, MANN-WHITNEY

    ERY1-ASNase treatment induces antigen-specific tolerance in a nontoxic and age-independent manner. ( A ) Mice were assessed for their capacity to respond to the irrelevant (nontolerized) antigen OVA after a single tolerogenic dose of ERY1-ASNase. ( B ) Time course of the development of anti-OVA total IgG in mice tolerized with 15 μg of ERY1-ASNase or WT ASNase and challenged with five weekly intravenous doses of OVA. ( C ) Mice were assessed for hematological parameters and for capacity to respond to the irrelevant antigen OVA after chronic intravenous administration with 15 μg of ERY1-ASNase or WT ASNase every 3 weeks for a total of four injections. ( D ) Hematological parameters at day 87 (data presented as ERY1-ASNase fold increase over WT ASNase; Student’s two-tailed t test, P > 0.05 for all parameters, n = 3 to 5). ( E ) Quantification of end-point antigen-specific total IgG responses to a challenge with the irrelevant T cell–dependent antigen OVA ( n = 3 to 5, Mann-Whitney U test). ( F and G ) Time course of anti-ASNase IgG development in plasma of aged (27-week-old) mice receiving 15 μg of ERY1-ASNase or WT ASNase, represented as (F) log 10 titer ( n = 3 to 4, Mann-Whitney U test, * P
    Figure Legend Snippet: ERY1-ASNase treatment induces antigen-specific tolerance in a nontoxic and age-independent manner. ( A ) Mice were assessed for their capacity to respond to the irrelevant (nontolerized) antigen OVA after a single tolerogenic dose of ERY1-ASNase. ( B ) Time course of the development of anti-OVA total IgG in mice tolerized with 15 μg of ERY1-ASNase or WT ASNase and challenged with five weekly intravenous doses of OVA. ( C ) Mice were assessed for hematological parameters and for capacity to respond to the irrelevant antigen OVA after chronic intravenous administration with 15 μg of ERY1-ASNase or WT ASNase every 3 weeks for a total of four injections. ( D ) Hematological parameters at day 87 (data presented as ERY1-ASNase fold increase over WT ASNase; Student’s two-tailed t test, P > 0.05 for all parameters, n = 3 to 5). ( E ) Quantification of end-point antigen-specific total IgG responses to a challenge with the irrelevant T cell–dependent antigen OVA ( n = 3 to 5, Mann-Whitney U test). ( F and G ) Time course of anti-ASNase IgG development in plasma of aged (27-week-old) mice receiving 15 μg of ERY1-ASNase or WT ASNase, represented as (F) log 10 titer ( n = 3 to 4, Mann-Whitney U test, * P

    Techniques Used: Mouse Assay, Two Tailed Test, MANN-WHITNEY

    26) Product Images from "The Costimulatory Molecule ICOS Regulates Host Th1 and Follicular Th Cell Differentiation in Response to Plasmodium chabaudi chabaudi AS Infection"

    Article Title: The Costimulatory Molecule ICOS Regulates Host Th1 and Follicular Th Cell Differentiation in Response to Plasmodium chabaudi chabaudi AS Infection

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.1403206

    Inhibition of ICOS signaling between days 0 and 6 p.i. does not impact T FH differentiation. Two hundred micrograms of anti-ICOSL or rat IgG isotype control Ab was i.p. injected into WT mice every other day beginning at day −1 p.i. ( A ) Cumulative data showing representative PD-1 and CXCR5 expression, and frequency of PD-1 + CXCR5 + CD4 + T cells at day 6 p.i. FACS plots gated on live, CD4 + T cells. ( B ) Bcl6 expression of gated area in (A). ( C ) Total number of CD4 + PD-1 + Bcl6 + CXCR5 + cells per spleen. Data are representative of three independent experiments; n = 3–5 mice/group. Error bars represent SEM. Symbols represent individual mice. FMO, fluorescence minus one control.
    Figure Legend Snippet: Inhibition of ICOS signaling between days 0 and 6 p.i. does not impact T FH differentiation. Two hundred micrograms of anti-ICOSL or rat IgG isotype control Ab was i.p. injected into WT mice every other day beginning at day −1 p.i. ( A ) Cumulative data showing representative PD-1 and CXCR5 expression, and frequency of PD-1 + CXCR5 + CD4 + T cells at day 6 p.i. FACS plots gated on live, CD4 + T cells. ( B ) Bcl6 expression of gated area in (A). ( C ) Total number of CD4 + PD-1 + Bcl6 + CXCR5 + cells per spleen. Data are representative of three independent experiments; n = 3–5 mice/group. Error bars represent SEM. Symbols represent individual mice. FMO, fluorescence minus one control.

    Techniques Used: Inhibition, Injection, Mouse Assay, Expressing, FACS, Fluorescence

    Inhibition of ICOS signaling between days 6 and 21 p.i. inhibits T FH differentiation. Two hundred micrograms anti-ICOSL or rat IgG isotype control Ab was i.p. injected into WT mice every other day beginning at day 6 p.i. ( A ) Cumulative data showing representative PD-1 and CXCR5 expression, and frequency of PD-1 + CXCR5 + CD4 + T cells. FACS plots gated on live, CD4 + T cells (D11: p = 0.008, D21: p = 0.008). ( B ) Bcl6 expression of gated area in (A). ( C ) Total number of CD4 + PD-1 + Bcl6 + CXCR5 + cells per spleen (D11: p = 0.016, D21: p = 0.008). Data are representative of three independent experiments; n = 3–5 mice/group. Symbols (A and C) represent individual mice. Error bars represent SEM. * p
    Figure Legend Snippet: Inhibition of ICOS signaling between days 6 and 21 p.i. inhibits T FH differentiation. Two hundred micrograms anti-ICOSL or rat IgG isotype control Ab was i.p. injected into WT mice every other day beginning at day 6 p.i. ( A ) Cumulative data showing representative PD-1 and CXCR5 expression, and frequency of PD-1 + CXCR5 + CD4 + T cells. FACS plots gated on live, CD4 + T cells (D11: p = 0.008, D21: p = 0.008). ( B ) Bcl6 expression of gated area in (A). ( C ) Total number of CD4 + PD-1 + Bcl6 + CXCR5 + cells per spleen (D11: p = 0.016, D21: p = 0.008). Data are representative of three independent experiments; n = 3–5 mice/group. Symbols (A and C) represent individual mice. Error bars represent SEM. * p

    Techniques Used: Inhibition, Injection, Mouse Assay, Expressing, FACS

    GC B cell differentiation, GC organization, and affinity maturation are dysregulated in Icos −/− mice. ( A ) Cumulative data showing representative GL-7 and Fas expression at day 21 p.i. FACS staining gated on live, CD19 + B220 + IgD − B cells. ( B ) Total number of CD19 + B220 + IgD − GL-7 + Fas + cells per spleen (D21: p = 0.001). ( C ) Splenic immunofluorescence staining and total number of PNA + GCs per mm 2 of splenic tissue at day 21 p.i. Green represents PNA, red represents CD3e, blue represents IgD (D21: p = 0.008). Original magnification ×10. ( D ) Relative MSP-1 42 –specific Ab at day 21 p.i. as measured by ELISA. ( E ) Normalized absorbance of MSP-1 42 -specific IgG Ab avidity at day 21 p.i. as determined by elution ELISA. The x -axis (log2) displays increasing molar concentrations of NH 4 SCN. A nonlinear regression curve fit is shown; significance was assessed by unpaired, two-tailed Student t test (0.25M p = 0.003, 0.5M p = 0.0001, 1.0M p = 0.001). ( F ) Frequency of T FH s (CD4 + PD-1 + CXCR5 + ) interacting with total B cells (CD19 + B220 + ) of total T FH s (naive: p = 0.0006, D6: p = 0.010, D11: p = 0.003, D21: p = 0.0006). ( G ) Frequency of T FH s (CD4 + PD-1 + CXCR5 + ) interacting with GC B cells (CD19 + B220 + GL-7 + Fas + ) of total T FH s (D21: p = 0.0006). Data are representative of three independent experiments (A–E); n = 4–5 mice/group. Data are pooled from two independent experiments (F and G); n = 4–8 mice/group. Symbols (B–G) represent individual mice. Error bars (A–G) represent SEM. * p
    Figure Legend Snippet: GC B cell differentiation, GC organization, and affinity maturation are dysregulated in Icos −/− mice. ( A ) Cumulative data showing representative GL-7 and Fas expression at day 21 p.i. FACS staining gated on live, CD19 + B220 + IgD − B cells. ( B ) Total number of CD19 + B220 + IgD − GL-7 + Fas + cells per spleen (D21: p = 0.001). ( C ) Splenic immunofluorescence staining and total number of PNA + GCs per mm 2 of splenic tissue at day 21 p.i. Green represents PNA, red represents CD3e, blue represents IgD (D21: p = 0.008). Original magnification ×10. ( D ) Relative MSP-1 42 –specific Ab at day 21 p.i. as measured by ELISA. ( E ) Normalized absorbance of MSP-1 42 -specific IgG Ab avidity at day 21 p.i. as determined by elution ELISA. The x -axis (log2) displays increasing molar concentrations of NH 4 SCN. A nonlinear regression curve fit is shown; significance was assessed by unpaired, two-tailed Student t test (0.25M p = 0.003, 0.5M p = 0.0001, 1.0M p = 0.001). ( F ) Frequency of T FH s (CD4 + PD-1 + CXCR5 + ) interacting with total B cells (CD19 + B220 + ) of total T FH s (naive: p = 0.0006, D6: p = 0.010, D11: p = 0.003, D21: p = 0.0006). ( G ) Frequency of T FH s (CD4 + PD-1 + CXCR5 + ) interacting with GC B cells (CD19 + B220 + GL-7 + Fas + ) of total T FH s (D21: p = 0.0006). Data are representative of three independent experiments (A–E); n = 4–5 mice/group. Data are pooled from two independent experiments (F and G); n = 4–8 mice/group. Symbols (B–G) represent individual mice. Error bars (A–G) represent SEM. * p

    Techniques Used: Cell Differentiation, Mouse Assay, Expressing, FACS, Staining, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    Disruption of ICOS signaling between days 6 and 21 p.i. curtails GC B cell differentiation and isotype-switched Ab production, but not affinity maturation. Two hundred micrograms of anti-ICOSL or rat IgG isotype control Ab was i.p. injected into WT mice every other day beginning at day 6 p.i. ( A ) Cumulative data showing representative GL-7 and Fas expression at day 21 p.i. FACS staining gated on live, CD19 + B220 + IgD − B cells (D21: p = 0.008). ( B ) Total number of CD19 + B220 + IgD − GL-7 + Fas + cells per spleen (D21: p = 0.016). ( C ) Relative MSP-1 42 –specific Ab at day 21 p.i. as measured by ELISA. ( D ) Normalized absorbance of MSP-1 42 –specific IgG Ab affinity at day 21 p.i. as determined by elution ELISA. The x -axis (log2) displays increasing molar concentrations of NH 4 SCN. A nonlinear regression curve fit is shown; significance was assessed by unpaired, two-tailed Student t test. Data are representative of three independent experiments; n = 3–5 mice/group. Symbols (A and B) represent individual mice. Error bars represent SEM. * p
    Figure Legend Snippet: Disruption of ICOS signaling between days 6 and 21 p.i. curtails GC B cell differentiation and isotype-switched Ab production, but not affinity maturation. Two hundred micrograms of anti-ICOSL or rat IgG isotype control Ab was i.p. injected into WT mice every other day beginning at day 6 p.i. ( A ) Cumulative data showing representative GL-7 and Fas expression at day 21 p.i. FACS staining gated on live, CD19 + B220 + IgD − B cells (D21: p = 0.008). ( B ) Total number of CD19 + B220 + IgD − GL-7 + Fas + cells per spleen (D21: p = 0.016). ( C ) Relative MSP-1 42 –specific Ab at day 21 p.i. as measured by ELISA. ( D ) Normalized absorbance of MSP-1 42 –specific IgG Ab affinity at day 21 p.i. as determined by elution ELISA. The x -axis (log2) displays increasing molar concentrations of NH 4 SCN. A nonlinear regression curve fit is shown; significance was assessed by unpaired, two-tailed Student t test. Data are representative of three independent experiments; n = 3–5 mice/group. Symbols (A and B) represent individual mice. Error bars represent SEM. * p

    Techniques Used: Cell Differentiation, Injection, Mouse Assay, Expressing, FACS, Staining, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    27) Product Images from "B cell responses to apoptotic cells in MFG-E8-/- mice"

    Article Title: B cell responses to apoptotic cells in MFG-E8-/- mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0205172

    MFG-E8 deficiency selectively promotes MZ B cell differentiation of anti-dsDNA specific 56R/Vκ38c B cells. A: MFG-E8-/- mice were crossed with 56R anti-dsDNA BCR transgenic mice. The percentages of non-autoreactive 56R/Vκ21D and autoreactive 56R/Vκ38c B cells in the spleens of 3 month old female mice were determined by idiotype specific antibodies using flow cytometry (left) and Immunofluorescence staining (right). The images are representative of at least 5 mice in each strain. B : Mertk-/- mice were crossed with 56R mice. The percentages of non-autoreactive 56R/Vκ21D and autoreactive 56R/Vκ38c in the spleens were determined by flow cytometry. C : Bone marrow from CD45.1+ w.t. or MFG-E8-/- mice was mixed with genotype matched bone marrow from CD45.2+56R mice at a 1:1 ratio. The mixed bone marrow cells were used to reconstitute irradiated CD45.1+ w.t. or MFG-E8-/- mice. At 3–4 month after the reconstitution, spleen (SP) and bone marrow (BM) cells derived from 56R mice were distinguished by the CD45.2 congenic marker. The ratios of autoreactive 56R/Vκ38c to non-autoreactive 56R/Vκ21D (left) and their surface phenotypes (right) were determined by flow cytometry. D : Serum titers of anti-dsDNA IgM and IgG from the bone marrow chimeras constructed in C were determined by ELISA. In C and D, the results are representative of two independent experiments. In A-D, each data point represents an individual animal.
    Figure Legend Snippet: MFG-E8 deficiency selectively promotes MZ B cell differentiation of anti-dsDNA specific 56R/Vκ38c B cells. A: MFG-E8-/- mice were crossed with 56R anti-dsDNA BCR transgenic mice. The percentages of non-autoreactive 56R/Vκ21D and autoreactive 56R/Vκ38c B cells in the spleens of 3 month old female mice were determined by idiotype specific antibodies using flow cytometry (left) and Immunofluorescence staining (right). The images are representative of at least 5 mice in each strain. B : Mertk-/- mice were crossed with 56R mice. The percentages of non-autoreactive 56R/Vκ21D and autoreactive 56R/Vκ38c in the spleens were determined by flow cytometry. C : Bone marrow from CD45.1+ w.t. or MFG-E8-/- mice was mixed with genotype matched bone marrow from CD45.2+56R mice at a 1:1 ratio. The mixed bone marrow cells were used to reconstitute irradiated CD45.1+ w.t. or MFG-E8-/- mice. At 3–4 month after the reconstitution, spleen (SP) and bone marrow (BM) cells derived from 56R mice were distinguished by the CD45.2 congenic marker. The ratios of autoreactive 56R/Vκ38c to non-autoreactive 56R/Vκ21D (left) and their surface phenotypes (right) were determined by flow cytometry. D : Serum titers of anti-dsDNA IgM and IgG from the bone marrow chimeras constructed in C were determined by ELISA. In C and D, the results are representative of two independent experiments. In A-D, each data point represents an individual animal.

    Techniques Used: Cell Differentiation, Mouse Assay, Transgenic Assay, Flow Cytometry, Cytometry, Immunofluorescence, Staining, Irradiation, Derivative Assay, Marker, Construct, Enzyme-linked Immunosorbent Assay

    Activated 56R and H564 B cells show different sensitivities to apoptotic cell accumulation in MFG-E8-/- mice. A: CD45.2+ 56R and H564 B cells were activated by LPS (1μg/ml) for 3 days. Activated B cells were extensively washed and transferred into w.t. and MFG-E8-/- hosts (CD45.1+). At day 5 after the transfer, id+ autoreactive B cells in the circulation were identified by idiotype specific antibodies. The surface levels of BCR on transferred cells were determined by flow cytometry. The expression levels of various BCRs in w.t. and MFG-E8-/- recipients were summarized below. The results are representative of two experiments. B: Activated 56R B cells were transferred as in A. At day 1 and day 7 after the transfer, sera were collected from the recipients. The levels of autoantibody produced by transferred 56R/Vk38c B cells in w.t. and MFG-E8-/- recipients were determined by ELISA using an idiotype specific antibody. Allotype specific anti-IgMa and anti-IgG2a were used to distinguish them from the endogenous IgMb and IgG2c in the recipients. The results are representative of three experiments. Supernatant from in vitro LPS stimulated 56R B cells was used as a positive control. The control contained approximately 1μg/ml antigen specific antibody. C: Activated H564 B cells were transferred as in B. The levels of autoantibody produced by transferred H564 B cells in w.t. and MFG-E8-/-recipients were determined by ELISA using idiotype specific antibody. Allotype specific anti-IgMa and anti-IgG2a were used to distinguish them from the endogenous IgMb and IgG2c in the recipients. The results are representative of two experiments. Supernatant from in vitro LPS stimulated H564 B cells was used as a positive control. The control contained approximately 1μg/ml antigen specific antibody. In B and C, note that neither 56R nor H564 B cells underwent class switching after 3 day LPS treatment in vitro .
    Figure Legend Snippet: Activated 56R and H564 B cells show different sensitivities to apoptotic cell accumulation in MFG-E8-/- mice. A: CD45.2+ 56R and H564 B cells were activated by LPS (1μg/ml) for 3 days. Activated B cells were extensively washed and transferred into w.t. and MFG-E8-/- hosts (CD45.1+). At day 5 after the transfer, id+ autoreactive B cells in the circulation were identified by idiotype specific antibodies. The surface levels of BCR on transferred cells were determined by flow cytometry. The expression levels of various BCRs in w.t. and MFG-E8-/- recipients were summarized below. The results are representative of two experiments. B: Activated 56R B cells were transferred as in A. At day 1 and day 7 after the transfer, sera were collected from the recipients. The levels of autoantibody produced by transferred 56R/Vk38c B cells in w.t. and MFG-E8-/- recipients were determined by ELISA using an idiotype specific antibody. Allotype specific anti-IgMa and anti-IgG2a were used to distinguish them from the endogenous IgMb and IgG2c in the recipients. The results are representative of three experiments. Supernatant from in vitro LPS stimulated 56R B cells was used as a positive control. The control contained approximately 1μg/ml antigen specific antibody. C: Activated H564 B cells were transferred as in B. The levels of autoantibody produced by transferred H564 B cells in w.t. and MFG-E8-/-recipients were determined by ELISA using idiotype specific antibody. Allotype specific anti-IgMa and anti-IgG2a were used to distinguish them from the endogenous IgMb and IgG2c in the recipients. The results are representative of two experiments. Supernatant from in vitro LPS stimulated H564 B cells was used as a positive control. The control contained approximately 1μg/ml antigen specific antibody. In B and C, note that neither 56R nor H564 B cells underwent class switching after 3 day LPS treatment in vitro .

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Expressing, Produced, Enzyme-linked Immunosorbent Assay, In Vitro, Positive Control

    Enhanced antibody responses to apoptotic cells in MFG-E8-/- B6 mice. A: Total IgM, IgG, IgG1, IgG3, IgG2b, and IgG2c levels in the sera collected from 4 month old w.t. and MFG-E8-/- female mice were determined by ELISA. B: Localization of IgG2c+ (red) plasma cells in the spleens of 4 month old w.t. and MFG-E8-/- mice was examined by immunofluorescence staining. Red pulp macrophages and Marginal zone macrophages were visualized by anti-F4/80(green, upper) and anti-CD169 (green, lower), respectively. The images are representative of at least 5 mice in each strain. C: Sera from 2 and 4 month old w.t. and MFG-E8-/- female mice were incubated with apoptotic cells. Binding intensities of IgG2c (MFI) to apoptotic cells were determined by flow cytometry. To investigate the effect of injecting exogenous apoptotic cells, 4 month old w.t. mice were injected with 20x 10 6 apoptotic thymocytes every three days for 5 times and their serum samples were collected one week after the final injection. D: Levels of IgG2c antibody against MDA-LDL in the sera of 4 month old w.t. and MFG-E8-/- female mice were determined by ELISA. In A-D, each data point represents an individual animal.
    Figure Legend Snippet: Enhanced antibody responses to apoptotic cells in MFG-E8-/- B6 mice. A: Total IgM, IgG, IgG1, IgG3, IgG2b, and IgG2c levels in the sera collected from 4 month old w.t. and MFG-E8-/- female mice were determined by ELISA. B: Localization of IgG2c+ (red) plasma cells in the spleens of 4 month old w.t. and MFG-E8-/- mice was examined by immunofluorescence staining. Red pulp macrophages and Marginal zone macrophages were visualized by anti-F4/80(green, upper) and anti-CD169 (green, lower), respectively. The images are representative of at least 5 mice in each strain. C: Sera from 2 and 4 month old w.t. and MFG-E8-/- female mice were incubated with apoptotic cells. Binding intensities of IgG2c (MFI) to apoptotic cells were determined by flow cytometry. To investigate the effect of injecting exogenous apoptotic cells, 4 month old w.t. mice were injected with 20x 10 6 apoptotic thymocytes every three days for 5 times and their serum samples were collected one week after the final injection. D: Levels of IgG2c antibody against MDA-LDL in the sera of 4 month old w.t. and MFG-E8-/- female mice were determined by ELISA. In A-D, each data point represents an individual animal.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Incubation, Binding Assay, Flow Cytometry, Cytometry, Injection, Multiple Displacement Amplification

    Enlarged MZ B cell compartment and enhanced antibody response to NP-Ficoll in MFG-E8-/- mice. A: The percentages of (CD19+/CD21high/CD23low) MZ B cells in the spleens of 4 month old w.t. and MFG-E8-/- female mice were determined by flow cytometry. Each data point represents an individual animal. B : Four month old w.t. and MFG-E8-/- female mice were challenged with NP-Ficoll. Serum titers of IgM and IgG (1–3) against NP were determined by ELISA at day 7 after the challenge. Each data point represents an individual animal. Similar results were obtained from male mice.
    Figure Legend Snippet: Enlarged MZ B cell compartment and enhanced antibody response to NP-Ficoll in MFG-E8-/- mice. A: The percentages of (CD19+/CD21high/CD23low) MZ B cells in the spleens of 4 month old w.t. and MFG-E8-/- female mice were determined by flow cytometry. Each data point represents an individual animal. B : Four month old w.t. and MFG-E8-/- female mice were challenged with NP-Ficoll. Serum titers of IgM and IgG (1–3) against NP were determined by ELISA at day 7 after the challenge. Each data point represents an individual animal. Similar results were obtained from male mice.

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    MFG-E8 deficiency promotes plasma B cell differentiation of anti-ssRNA specific H564 B cells. Plasma cell differentiation of id+ B cells was examined in the spleens of 3 month old w.t. and MFG-E8-/- H564 BCR transgenic mice. A: The numbers of id+/B220low plasma cells per 10,000 id+ cells in the spleens were enumerated by flow cytometry. Surface expression of CD138 on id+/B220low cells was significantly higher than id+/B220+ cells. (Histogram: blue—id+/B220+high, red—id+/B220low). B: To locate id+ plasma cells in the spleens, frozen spleen sections from w.t. and MFG-E8-/- H564 mice were stained with anti-CD169(green), anti-B220(blue), and idiotype specific antibody (red). Right: The number of id+ plasma cells in each spleen section was enumerated using Image J. Each data point represents the average of three sections from an individual animal. C: The numbers of id specific IgG producing H564 B cells in the spleens of w.t. and MFG-E8-/-BCR transgenic mice were determined by ELISPOT. D : The titers of id specific IgG, IgG2a, and IgG2b antibodies in the sera of 4 month old w.t. and MFG-E8-/-H564 mice were determined by ELISA. In A-D, each data point represents an individual animal.
    Figure Legend Snippet: MFG-E8 deficiency promotes plasma B cell differentiation of anti-ssRNA specific H564 B cells. Plasma cell differentiation of id+ B cells was examined in the spleens of 3 month old w.t. and MFG-E8-/- H564 BCR transgenic mice. A: The numbers of id+/B220low plasma cells per 10,000 id+ cells in the spleens were enumerated by flow cytometry. Surface expression of CD138 on id+/B220low cells was significantly higher than id+/B220+ cells. (Histogram: blue—id+/B220+high, red—id+/B220low). B: To locate id+ plasma cells in the spleens, frozen spleen sections from w.t. and MFG-E8-/- H564 mice were stained with anti-CD169(green), anti-B220(blue), and idiotype specific antibody (red). Right: The number of id+ plasma cells in each spleen section was enumerated using Image J. Each data point represents the average of three sections from an individual animal. C: The numbers of id specific IgG producing H564 B cells in the spleens of w.t. and MFG-E8-/-BCR transgenic mice were determined by ELISPOT. D : The titers of id specific IgG, IgG2a, and IgG2b antibodies in the sera of 4 month old w.t. and MFG-E8-/-H564 mice were determined by ELISA. In A-D, each data point represents an individual animal.

    Techniques Used: Cell Differentiation, Transgenic Assay, Mouse Assay, Flow Cytometry, Cytometry, Expressing, Staining, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay

    28) Product Images from "Critical role for granulocyte colony-stimulating factor in inflammatory arthritis"

    Article Title: Critical role for granulocyte colony-stimulating factor in inflammatory arthritis

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0404328101

    Defective anti-CII Ab isotype switching but normal T cell responses and antigen presentation by DCs in immunized G-CSF –/– mice. Shown are T cell proliferation ( a ) and IFN-γ and IL-2 production ( b ) from inguinal LN cells of CIA-immunized B6 (▪ and •) and G-CSF –/– (□ and ○) mice stimulated with denatured CII in vitro .( c ) Serum anti-CII IgG, IgM, IgG2b, IgG2c, IgG1, and IgG3 levels in CIA-immunized B6 (▪) and G-CSF –/– (□) mice at day 62 of CIA; n ≥ 30 mice per group. ( d ) Proliferation of OVA-specific OT-II CD4 + T cells in response to B6 (•) and G-CSF –/– (□) OVA-pulsed DC. Proliferation data are the mean [ 3 H]thymidine uptake cpm × 10 –4 ± SD, and ELISA data are the mean ± SEM; n = 3 experiments. * , P
    Figure Legend Snippet: Defective anti-CII Ab isotype switching but normal T cell responses and antigen presentation by DCs in immunized G-CSF –/– mice. Shown are T cell proliferation ( a ) and IFN-γ and IL-2 production ( b ) from inguinal LN cells of CIA-immunized B6 (▪ and •) and G-CSF –/– (□ and ○) mice stimulated with denatured CII in vitro .( c ) Serum anti-CII IgG, IgM, IgG2b, IgG2c, IgG1, and IgG3 levels in CIA-immunized B6 (▪) and G-CSF –/– (□) mice at day 62 of CIA; n ≥ 30 mice per group. ( d ) Proliferation of OVA-specific OT-II CD4 + T cells in response to B6 (•) and G-CSF –/– (□) OVA-pulsed DC. Proliferation data are the mean [ 3 H]thymidine uptake cpm × 10 –4 ± SD, and ELISA data are the mean ± SEM; n = 3 experiments. * , P

    Techniques Used: Mouse Assay, In Vitro, Enzyme-linked Immunosorbent Assay

    29) Product Images from "Immunogenicity and efficacy following sequential parenterally-administered doses of Salmonella Enteritidis COPS:FliC glycoconjugates in infant and adult mice"

    Article Title: Immunogenicity and efficacy following sequential parenterally-administered doses of Salmonella Enteritidis COPS:FliC glycoconjugates in infant and adult mice

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0006522

    Anti-FliC IgG responses in adult and infant mice after immunization with S . Enteritidis COPS:FliC alone or formulated with different adjuvants. Infant and adult mice ( n = 16–20/group) were immunized with PBS or S . Enteritidis COPS:FliC formulated alone (ɸ), adsorbed to alum, or admixed with MPL. (A) Serum anti-FliC IgG titers taken 12–14 days after each dose were determined by ELISA. Each point represents an individual mouse. Red squares indicate mice that succumbed to subsequent challenge. Bars represent the GMT for adults (grey) and infants (white), and were compared using a two-tailed Mann-Whitney U test. Adjustments for multiple comparisons were not made. ns, not significant. * P ≤ 0.05; ** P ≤ 0.005; *** P ≤ 0.0005 for indicated comparisons. (B) Reverse cumulative distribution curves for post 3 rd immunization anti-FliC IgG titers for adults (grey circles) and infants (white circles) are depicted.
    Figure Legend Snippet: Anti-FliC IgG responses in adult and infant mice after immunization with S . Enteritidis COPS:FliC alone or formulated with different adjuvants. Infant and adult mice ( n = 16–20/group) were immunized with PBS or S . Enteritidis COPS:FliC formulated alone (ɸ), adsorbed to alum, or admixed with MPL. (A) Serum anti-FliC IgG titers taken 12–14 days after each dose were determined by ELISA. Each point represents an individual mouse. Red squares indicate mice that succumbed to subsequent challenge. Bars represent the GMT for adults (grey) and infants (white), and were compared using a two-tailed Mann-Whitney U test. Adjustments for multiple comparisons were not made. ns, not significant. * P ≤ 0.05; ** P ≤ 0.005; *** P ≤ 0.0005 for indicated comparisons. (B) Reverse cumulative distribution curves for post 3 rd immunization anti-FliC IgG titers for adults (grey circles) and infants (white circles) are depicted.

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

    Isotype and avidity of anti-COPS IgG in adult mice after immunization with S . Enteritidis COPS:FliC formulated with MPL. (A) The avidity of anti-COPS IgG after the 3 rd immunization ( n = 6) was determined by ELISA. (B) Post 3 rd immunization serum COPS-specific IgG1 and IgG2b titers were determined by ELISA. The ratio of the two titers is plotted. Each point represents an individual mouse. The bar represents the median.
    Figure Legend Snippet: Isotype and avidity of anti-COPS IgG in adult mice after immunization with S . Enteritidis COPS:FliC formulated with MPL. (A) The avidity of anti-COPS IgG after the 3 rd immunization ( n = 6) was determined by ELISA. (B) Post 3 rd immunization serum COPS-specific IgG1 and IgG2b titers were determined by ELISA. The ratio of the two titers is plotted. Each point represents an individual mouse. The bar represents the median.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Anti-COPS IgG responses in adult and infant mice after immunization with S . Enteritidis COPS:FliC alone or formulated with different adjuvants. (A) Infant and adult mice ( n = 16–20/group) were immunized as described in Fig 1 . Serum anti-COPS IgG titers taken 12–14 days after each dose were determined by ELISA. Each point represents an individual mouse. Red squares indicate mice that succumbed to subsequent challenge. Bars represent the GMT for adults (grey) and infants (white), and were compared using a two-tailed Mann-Whitney U test. Adjustments for multiple comparisons were not made. ns, not significant. * P ≤ 0.05; ** P ≤ 0.005; *** P ≤ 0.0005 for indicated comparisons. (B) Reverse cumulative distribution curves for post 3 rd immunization anti-COPS IgG titers for adults (grey circles) and infants (white circles) are depicted. Dotted lines indicate the cut-off for seroconversion (50 EU/mL), which represents a 4-fold rise over the anti-COPS IgG GMT for PBS controls.
    Figure Legend Snippet: Anti-COPS IgG responses in adult and infant mice after immunization with S . Enteritidis COPS:FliC alone or formulated with different adjuvants. (A) Infant and adult mice ( n = 16–20/group) were immunized as described in Fig 1 . Serum anti-COPS IgG titers taken 12–14 days after each dose were determined by ELISA. Each point represents an individual mouse. Red squares indicate mice that succumbed to subsequent challenge. Bars represent the GMT for adults (grey) and infants (white), and were compared using a two-tailed Mann-Whitney U test. Adjustments for multiple comparisons were not made. ns, not significant. * P ≤ 0.05; ** P ≤ 0.005; *** P ≤ 0.0005 for indicated comparisons. (B) Reverse cumulative distribution curves for post 3 rd immunization anti-COPS IgG titers for adults (grey circles) and infants (white circles) are depicted. Dotted lines indicate the cut-off for seroconversion (50 EU/mL), which represents a 4-fold rise over the anti-COPS IgG GMT for PBS controls.

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

    Isotype and avidity of anti-FliC IgG in adult and infant mice after immunization with S . Enteritidis COPS:FliC alone or formulated with different adjuvants. (A) The avidity of anti-FliC IgG in sera taken after each immunization ( n = 8–10) was determined by sensitivity to urea treatment in an ELISA format. (B) Post 3 rd immunization FliC-specific serum IgG1 and IgG2b titers were determined by ELISA ( n = 9–10). The ratio of the two titers is plotted. Each point represents an individual mouse. Bars represent the median for adults (grey) and infants (white) that was compared using a two-tailed Mann-Whitney U test. Adjustments for multiple comparisons were not made. P-values ≤ 0.05 were considered to be statistically significant; ns, not significant. * P ≤ 0.05; ** P ≤ 0.005; *** P ≤ 0.0005 for indicated comparisons.
    Figure Legend Snippet: Isotype and avidity of anti-FliC IgG in adult and infant mice after immunization with S . Enteritidis COPS:FliC alone or formulated with different adjuvants. (A) The avidity of anti-FliC IgG in sera taken after each immunization ( n = 8–10) was determined by sensitivity to urea treatment in an ELISA format. (B) Post 3 rd immunization FliC-specific serum IgG1 and IgG2b titers were determined by ELISA ( n = 9–10). The ratio of the two titers is plotted. Each point represents an individual mouse. Bars represent the median for adults (grey) and infants (white) that was compared using a two-tailed Mann-Whitney U test. Adjustments for multiple comparisons were not made. P-values ≤ 0.05 were considered to be statistically significant; ns, not significant. * P ≤ 0.05; ** P ≤ 0.005; *** P ≤ 0.0005 for indicated comparisons.

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

    30) Product Images from "Nasal delivery of H5N1 avian influenza vaccine formulated with GenJet™ or in vivo-jetPEI® induces enhanced serological, cellular and protective immune responses"

    Article Title: Nasal delivery of H5N1 avian influenza vaccine formulated with GenJet™ or in vivo-jetPEI® induces enhanced serological, cellular and protective immune responses

    Journal: Drug Delivery

    doi: 10.1080/10717544.2018.1450909

    GenJet™ and in vivo -jetPEI ® enhanced the H5N1-specific T-cell responses. Balb/c mice (3–5 mice/group) were intranasally administered with A/IN/05 vaccine with or without GenJet™ or in vivo -jetPEI ® using prime-boost regimen as described in Figure 1 . (A) One week after booster immunization, the lung tissues were harvested and single cell suspensions were prepared. About 10 6 cells from the lung were stimulated in vitro with HA peptide for 6 h to examine the antigen-specific CD8 T-cell response; or with RG A/IN/05 virus at an MOI of 1 for 16 h to examine the antigen-specific CD4 T-cell response. GolgiPlug™ was added during the last 5 h of incubation. Cells were surface stained with anti-CD44, anti-CD4 or anti-CD8 antibody (BD Bioscience), followed by intracellular staining with anti-IFNγ antibody (BD Bioscience). The frequency of IFN-γ producing T cells in total activated T cells was presented. (B) One-week post-booster immunization, the draining lymph nodes, lungs and spleen tissues were harvested and the frequency of HA518-specific CD8 T cells in total activated CD8 T cells was stained using H-2K d /IYSTVASSL tetramer. (C) Three weeks after booster immunization, sera were collected and IgG2a, IgG2b IgG3 and IgG1 antibodies against A/IN/05 were assessed by ELISA. The data are representative of two independent experiments (3–5 mice each group) and error bars represent SEM. One-way ANOVA with Bonferroni post-analysis was used to analyze differences among treatments. p
    Figure Legend Snippet: GenJet™ and in vivo -jetPEI ® enhanced the H5N1-specific T-cell responses. Balb/c mice (3–5 mice/group) were intranasally administered with A/IN/05 vaccine with or without GenJet™ or in vivo -jetPEI ® using prime-boost regimen as described in Figure 1 . (A) One week after booster immunization, the lung tissues were harvested and single cell suspensions were prepared. About 10 6 cells from the lung were stimulated in vitro with HA peptide for 6 h to examine the antigen-specific CD8 T-cell response; or with RG A/IN/05 virus at an MOI of 1 for 16 h to examine the antigen-specific CD4 T-cell response. GolgiPlug™ was added during the last 5 h of incubation. Cells were surface stained with anti-CD44, anti-CD4 or anti-CD8 antibody (BD Bioscience), followed by intracellular staining with anti-IFNγ antibody (BD Bioscience). The frequency of IFN-γ producing T cells in total activated T cells was presented. (B) One-week post-booster immunization, the draining lymph nodes, lungs and spleen tissues were harvested and the frequency of HA518-specific CD8 T cells in total activated CD8 T cells was stained using H-2K d /IYSTVASSL tetramer. (C) Three weeks after booster immunization, sera were collected and IgG2a, IgG2b IgG3 and IgG1 antibodies against A/IN/05 were assessed by ELISA. The data are representative of two independent experiments (3–5 mice each group) and error bars represent SEM. One-way ANOVA with Bonferroni post-analysis was used to analyze differences among treatments. p

    Techniques Used: In Vivo, Mouse Assay, In Vitro, Incubation, Staining, Enzyme-linked Immunosorbent Assay

    GenJet™ and in vivo -jetPEI enhances the H5N1 vaccine-induced systemic antibody responses and memory B-cell responses. Balb/c mice (5 mice/group) were intranasally administered with 3 µg of A/IN/05 vaccine with or without GenJet™ (10 µl/each mouse as described in previous study (Kulkarni et al., 2014 )) or in vivo -jetPEI ® (0.6 μl/mouse according to manufacturer’s protocol). One month later, mice were boosted with the same vaccine formulations. The control mouse group received GenJet™, in vivo -jetPEI ® or PBS at both time points. (A) Three weeks after booster immunization, sera were collected and IgG, IgA and IgM antibodies against A/IN/05 were assessed by ELISA. (B) One week after booster immunization, the spleen were harvested and the frequency of A/IN/05-specific IgG + ASCs in the spleen were measured by ELISPOT assay. The number of A/IN/05-specific IgG + ASCs were normalized against the number of total IgG + secreting ASCs and presented as % Ag-specific IgG + B cells. The data are representative of two independent experiments (3–5 mice each group) and error bars represent SEM. One-way ANOVA with Bonferroni post-analysis was used to analyze differences among different groups. p
    Figure Legend Snippet: GenJet™ and in vivo -jetPEI enhances the H5N1 vaccine-induced systemic antibody responses and memory B-cell responses. Balb/c mice (5 mice/group) were intranasally administered with 3 µg of A/IN/05 vaccine with or without GenJet™ (10 µl/each mouse as described in previous study (Kulkarni et al., 2014 )) or in vivo -jetPEI ® (0.6 μl/mouse according to manufacturer’s protocol). One month later, mice were boosted with the same vaccine formulations. The control mouse group received GenJet™, in vivo -jetPEI ® or PBS at both time points. (A) Three weeks after booster immunization, sera were collected and IgG, IgA and IgM antibodies against A/IN/05 were assessed by ELISA. (B) One week after booster immunization, the spleen were harvested and the frequency of A/IN/05-specific IgG + ASCs in the spleen were measured by ELISPOT assay. The number of A/IN/05-specific IgG + ASCs were normalized against the number of total IgG + secreting ASCs and presented as % Ag-specific IgG + B cells. The data are representative of two independent experiments (3–5 mice each group) and error bars represent SEM. One-way ANOVA with Bonferroni post-analysis was used to analyze differences among different groups. p

    Techniques Used: In Vivo, Mouse Assay, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

    31) Product Images from "Nasal delivery of H5N1 avian influenza vaccine formulated with GenJet™ or in vivo-jetPEI® induces enhanced serological, cellular and protective immune responses"

    Article Title: Nasal delivery of H5N1 avian influenza vaccine formulated with GenJet™ or in vivo-jetPEI® induces enhanced serological, cellular and protective immune responses

    Journal: Drug Delivery

    doi: 10.1080/10717544.2018.1450909

    GenJet™ and in vivo -jetPEI ® enhanced the H5N1-specific T-cell responses. Balb/c mice (3–5 mice/group) were intranasally administered with A/IN/05 vaccine with or without GenJet™ or in vivo -jetPEI ® using prime-boost regimen as described in Figure 1 . (A) One week after booster immunization, the lung tissues were harvested and single cell suspensions were prepared. About 10 6 cells from the lung were stimulated in vitro with HA peptide for 6 h to examine the antigen-specific CD8 T-cell response; or with RG A/IN/05 virus at an MOI of 1 for 16 h to examine the antigen-specific CD4 T-cell response. GolgiPlug™ was added during the last 5 h of incubation. Cells were surface stained with anti-CD44, anti-CD4 or anti-CD8 antibody (BD Bioscience), followed by intracellular staining with anti-IFNγ antibody (BD Bioscience). The frequency of IFN-γ producing T cells in total activated T cells was presented. (B) One-week post-booster immunization, the draining lymph nodes, lungs and spleen tissues were harvested and the frequency of HA518-specific CD8 T cells in total activated CD8 T cells was stained using H-2K d /IYSTVASSL tetramer. (C) Three weeks after booster immunization, sera were collected and IgG2a, IgG2b IgG3 and IgG1 antibodies against A/IN/05 were assessed by ELISA. The data are representative of two independent experiments (3–5 mice each group) and error bars represent SEM. One-way ANOVA with Bonferroni post-analysis was used to analyze differences among treatments. p
    Figure Legend Snippet: GenJet™ and in vivo -jetPEI ® enhanced the H5N1-specific T-cell responses. Balb/c mice (3–5 mice/group) were intranasally administered with A/IN/05 vaccine with or without GenJet™ or in vivo -jetPEI ® using prime-boost regimen as described in Figure 1 . (A) One week after booster immunization, the lung tissues were harvested and single cell suspensions were prepared. About 10 6 cells from the lung were stimulated in vitro with HA peptide for 6 h to examine the antigen-specific CD8 T-cell response; or with RG A/IN/05 virus at an MOI of 1 for 16 h to examine the antigen-specific CD4 T-cell response. GolgiPlug™ was added during the last 5 h of incubation. Cells were surface stained with anti-CD44, anti-CD4 or anti-CD8 antibody (BD Bioscience), followed by intracellular staining with anti-IFNγ antibody (BD Bioscience). The frequency of IFN-γ producing T cells in total activated T cells was presented. (B) One-week post-booster immunization, the draining lymph nodes, lungs and spleen tissues were harvested and the frequency of HA518-specific CD8 T cells in total activated CD8 T cells was stained using H-2K d /IYSTVASSL tetramer. (C) Three weeks after booster immunization, sera were collected and IgG2a, IgG2b IgG3 and IgG1 antibodies against A/IN/05 were assessed by ELISA. The data are representative of two independent experiments (3–5 mice each group) and error bars represent SEM. One-way ANOVA with Bonferroni post-analysis was used to analyze differences among treatments. p

    Techniques Used: In Vivo, Mouse Assay, In Vitro, Incubation, Staining, Enzyme-linked Immunosorbent Assay

    GenJet™ and in vivo -jetPEI enhances the H5N1 vaccine-induced systemic antibody responses and memory B-cell responses. Balb/c mice (5 mice/group) were intranasally administered with 3 µg of A/IN/05 vaccine with or without GenJet™ (10 µl/each mouse as described in previous study (Kulkarni et al., 2014 )) or in vivo -jetPEI ® (0.6 μl/mouse according to manufacturer’s protocol). One month later, mice were boosted with the same vaccine formulations. The control mouse group received GenJet™, in vivo -jetPEI ® or PBS at both time points. (A) Three weeks after booster immunization, sera were collected and IgG, IgA and IgM antibodies against A/IN/05 were assessed by ELISA. (B) One week after booster immunization, the spleen were harvested and the frequency of A/IN/05-specific IgG + ASCs in the spleen were measured by ELISPOT assay. The number of A/IN/05-specific IgG + ASCs were normalized against the number of total IgG + secreting ASCs and presented as % Ag-specific IgG + B cells. The data are representative of two independent experiments (3–5 mice each group) and error bars represent SEM. One-way ANOVA with Bonferroni post-analysis was used to analyze differences among different groups. p
    Figure Legend Snippet: GenJet™ and in vivo -jetPEI enhances the H5N1 vaccine-induced systemic antibody responses and memory B-cell responses. Balb/c mice (5 mice/group) were intranasally administered with 3 µg of A/IN/05 vaccine with or without GenJet™ (10 µl/each mouse as described in previous study (Kulkarni et al., 2014 )) or in vivo -jetPEI ® (0.6 μl/mouse according to manufacturer’s protocol). One month later, mice were boosted with the same vaccine formulations. The control mouse group received GenJet™, in vivo -jetPEI ® or PBS at both time points. (A) Three weeks after booster immunization, sera were collected and IgG, IgA and IgM antibodies against A/IN/05 were assessed by ELISA. (B) One week after booster immunization, the spleen were harvested and the frequency of A/IN/05-specific IgG + ASCs in the spleen were measured by ELISPOT assay. The number of A/IN/05-specific IgG + ASCs were normalized against the number of total IgG + secreting ASCs and presented as % Ag-specific IgG + B cells. The data are representative of two independent experiments (3–5 mice each group) and error bars represent SEM. One-way ANOVA with Bonferroni post-analysis was used to analyze differences among different groups. p

    Techniques Used: In Vivo, Mouse Assay, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

    32) Product Images from "Linkage of Exogenous T-cell Epitopes to the 19-Kilodalton Region of Plasmodium yoelii Merozoite Surface Protein 1 (MSP119) Can Enhance Protective Immunity against Malaria and Modulate the Immunoglobulin Subclass Response to MSP119"

    Article Title: Linkage of Exogenous T-cell Epitopes to the 19-Kilodalton Region of Plasmodium yoelii Merozoite Surface Protein 1 (MSP119) Can Enhance Protective Immunity against Malaria and Modulate the Immunoglobulin Subclass Response to MSP119

    Journal: Infection and Immunity

    doi:

    GST-specific IgG subclass responses in B10.BR mice immunized with GST fusion proteins, as determined by ELISA. Groups of five mice were immunized with GST/MAP-P2, GST-MSP1 19 , GST-P2-MSP1 19 , or GST-P8-MSP1 19 . The bars display reactivities of pooled sera analyzed for reactivity with GST at dilutions of 1:2 15 (IgG1 and IgG2b) or 1:2 10 (IgG2a and IgG3). Sera from naive mice did not react significantly at these dilutions (not shown). Abs 492 , absorbance at 492 nm.
    Figure Legend Snippet: GST-specific IgG subclass responses in B10.BR mice immunized with GST fusion proteins, as determined by ELISA. Groups of five mice were immunized with GST/MAP-P2, GST-MSP1 19 , GST-P2-MSP1 19 , or GST-P8-MSP1 19 . The bars display reactivities of pooled sera analyzed for reactivity with GST at dilutions of 1:2 15 (IgG1 and IgG2b) or 1:2 10 (IgG2a and IgG3). Sera from naive mice did not react significantly at these dilutions (not shown). Abs 492 , absorbance at 492 nm.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Comparison of MSP1 19 -specific IgG subclass responses in B10.A(4R) mice immunized with GST-MSP1 19 or MSP1 19 as measured by ELISA. Groups of five mice were immunized s.c. with either GST-MSP1 19 or GST-free MSP1 19 . The bars display mean reactivities of sera analyzed for reactivity with MSP1 19 at dilutions of 1:2 19 (IgG1), 1:2 14 (IgG2a), 1:2 18 (IgG2b), or 1:2 13 (IgG3). Abs 492 , absorbance at 492 nm.
    Figure Legend Snippet: Comparison of MSP1 19 -specific IgG subclass responses in B10.A(4R) mice immunized with GST-MSP1 19 or MSP1 19 as measured by ELISA. Groups of five mice were immunized s.c. with either GST-MSP1 19 or GST-free MSP1 19 . The bars display mean reactivities of sera analyzed for reactivity with MSP1 19 at dilutions of 1:2 19 (IgG1), 1:2 14 (IgG2a), 1:2 18 (IgG2b), or 1:2 13 (IgG3). Abs 492 , absorbance at 492 nm.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Relationship between the MSP1 19 -specific IgG titers of antisera to GST-MSP1 19 variants and the capacity to inhibit the binding of MAb F5 or MAb B10 to MSP1 19 . The MSP1 19 -specific titers of sera from B10.A(4R), B10.BR, C57BL/6, and B10.D2 mice immunized with GST-MSP1 19 , GST-P2-MSP1 19 , or GST-P8-MSP1 19 [B10.A(4R) and B10.BR mice]) were determined by ELISA. To establish the capacity of the sera to inhibit the binding of MAb to MSP1 19 , ELISA plates were incubated first with serum diluted 1:600 and subsequently with biotinylated MAb F5 (A) or MAb B10 (B). The IgG titers of individual sera are plotted against the level of competition obtained with the corresponding sera. Open circles, mice that died following challenge infection. The lines display the association between IgG titer and inhibition of MAb F5 ( r = 0.52; n = 50; P ≤ 0.0001) (A) and MAb B10 ( r = 0.66; n = 50; P ≤ 0.0001) (B). The binding of biotinylated MAb could be inhibited by adding an excess of nonlabeled homologous but not heterologous MAb (not shown).
    Figure Legend Snippet: Relationship between the MSP1 19 -specific IgG titers of antisera to GST-MSP1 19 variants and the capacity to inhibit the binding of MAb F5 or MAb B10 to MSP1 19 . The MSP1 19 -specific titers of sera from B10.A(4R), B10.BR, C57BL/6, and B10.D2 mice immunized with GST-MSP1 19 , GST-P2-MSP1 19 , or GST-P8-MSP1 19 [B10.A(4R) and B10.BR mice]) were determined by ELISA. To establish the capacity of the sera to inhibit the binding of MAb to MSP1 19 , ELISA plates were incubated first with serum diluted 1:600 and subsequently with biotinylated MAb F5 (A) or MAb B10 (B). The IgG titers of individual sera are plotted against the level of competition obtained with the corresponding sera. Open circles, mice that died following challenge infection. The lines display the association between IgG titer and inhibition of MAb F5 ( r = 0.52; n = 50; P ≤ 0.0001) (A) and MAb B10 ( r = 0.66; n = 50; P ≤ 0.0001) (B). The binding of biotinylated MAb could be inhibited by adding an excess of nonlabeled homologous but not heterologous MAb (not shown).

    Techniques Used: Binding Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, Incubation, Infection, Inhibition

    33) Product Images from "Distinct B-cell populations contribute to vaccine antigen-specific antibody production in a transgenic mouse model"

    Article Title: Distinct B-cell populations contribute to vaccine antigen-specific antibody production in a transgenic mouse model

    Journal: Immunology

    doi: 10.1111/imm.12287

    Immunization of ROSA yellow fluorescent protein transgenic (YFP Tg) mice induces vaccine-specific IgG antibodies (a) Total IgG and (b) IgM antibodies specific for inactivated influenza (A/PR8) virus antigen day 9 post prime and boost immunization ( n =
    Figure Legend Snippet: Immunization of ROSA yellow fluorescent protein transgenic (YFP Tg) mice induces vaccine-specific IgG antibodies (a) Total IgG and (b) IgM antibodies specific for inactivated influenza (A/PR8) virus antigen day 9 post prime and boost immunization ( n =

    Techniques Used: Transgenic Assay, Mouse Assay

    34) Product Images from "Interferon alpha treatment of NZW/BXSB F1 females mimics some but not all features associated with the Yaa mutation"

    Article Title: Interferon alpha treatment of NZW/BXSB F1 females mimics some but not all features associated with the Yaa mutation

    Journal: Arthritis and rheumatism

    doi: 10.1002/art.24414

    IFNα treated mice had higher titers of autoantibodies than age matched untreated controls (2A, 2B). ELISpot analysis of spleens showed an increase in the frequency (2C) and total number (2D) of IgG and IgG anti-cardiolipin producing B cells in
    Figure Legend Snippet: IFNα treated mice had higher titers of autoantibodies than age matched untreated controls (2A, 2B). ELISpot analysis of spleens showed an increase in the frequency (2C) and total number (2D) of IgG and IgG anti-cardiolipin producing B cells in

    Techniques Used: Mouse Assay, Enzyme-linked Immunospot

    35) Product Images from "Apremilast Ameliorates Experimental Arthritis via Suppression of Th1 and Th17 Cells and Enhancement of CD4+Foxp3+ Regulatory T Cells Differentiation"

    Article Title: Apremilast Ameliorates Experimental Arthritis via Suppression of Th1 and Th17 Cells and Enhancement of CD4+Foxp3+ Regulatory T Cells Differentiation

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01662

    Apremilast delayed arthritis onset and reduced arthritis scores in the collagen-induced arthritis (CIA) model. DBA/1J-FoxP3 gfp mice were immunized with type II collagen emulsified with Freund’s complete adjuvant. On day 14 after immunization, Apremilast (5 or 25 mg/kg) was given orally once daily for 10 days. Vehicle alone (0.5% carboxymethyl cellulose, 0.25% Tween 80, CMC) was administered by oral gavage as a negative control. (A) The incidence of arthritis and (B) arthritis severity scores were determined at various time points after immunization. (C) Total serum IgG, IgG1, IgG2a, and IgG2b anti-mouse type II collagen antibody levels were measured by enzyme-linked immunosorbent assay. The data indicate the mean ± SEM of five mice per group from two independent experiments. Data were analyzed using the one-way ANOVA for comparison among multiple groups, followed by Turkey’s test (* p
    Figure Legend Snippet: Apremilast delayed arthritis onset and reduced arthritis scores in the collagen-induced arthritis (CIA) model. DBA/1J-FoxP3 gfp mice were immunized with type II collagen emulsified with Freund’s complete adjuvant. On day 14 after immunization, Apremilast (5 or 25 mg/kg) was given orally once daily for 10 days. Vehicle alone (0.5% carboxymethyl cellulose, 0.25% Tween 80, CMC) was administered by oral gavage as a negative control. (A) The incidence of arthritis and (B) arthritis severity scores were determined at various time points after immunization. (C) Total serum IgG, IgG1, IgG2a, and IgG2b anti-mouse type II collagen antibody levels were measured by enzyme-linked immunosorbent assay. The data indicate the mean ± SEM of five mice per group from two independent experiments. Data were analyzed using the one-way ANOVA for comparison among multiple groups, followed by Turkey’s test (* p

    Techniques Used: Mouse Assay, Negative Control, Enzyme-linked Immunosorbent Assay

    36) Product Images from "Role for DNA repair factor XRCC4 in immunoglobulin class switch recombination"

    Article Title: Role for DNA repair factor XRCC4 in immunoglobulin class switch recombination

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20070255

    Ig production is impaired in conditional X4 KO mice. Sera from ΔX4T and control X4T mice were collected and total IgM, IgG1, IgG2b, IgG3, and IgA were determined by ELISA (microgram/milliliter). The results of statistical tests are indicated; * indicates a statistically significant difference (P
    Figure Legend Snippet: Ig production is impaired in conditional X4 KO mice. Sera from ΔX4T and control X4T mice were collected and total IgM, IgG1, IgG2b, IgG3, and IgA were determined by ELISA (microgram/milliliter). The results of statistical tests are indicated; * indicates a statistically significant difference (P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    37) Product Images from "Repression of the B cell identity factor Pax5 is not required for plasma cell development"

    Article Title: Repression of the B cell identity factor Pax5 is not required for plasma cell development

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20200147

    Normal CSR and plasma cell development under T cell–independent stimulation conditions. (A) Flow-cytometric analysis of plasma cells (TACI + CD138 + ; upper row) in the spleen of Igh Pax5/+ and Igh +/+ littermates 14 d after immunization with TNP-Ficoll. The frequency of IgG2b-expressing plasma cells was determined by intracellular IgG2b staining (lower row). The results shown are representative of two experiments. (B) Normal generation of GC B cells in the Peyer’s patches of Igh Pax5/+ and Igh +/+ littermates at steady state. The frequencies of total GC B cells (CD19 + GL7 + Fas + ) and IgA + GC cells were determined by flow cytometry (left) and quantified (right). (C) Normal generation of IgA-expressing plasma cells in the Peyer’s patches at steady state. The frequencies of total (TACI + CD138 + ) and IgA-expressing plasma cells were determined by flow cytometry (left) and quantified (right). One experiment (B and C) was performed. Statistical data (A–C) are shown as mean values with SD and were analyzed by the two-tailed unpaired Student’s t test; NS, not significant (P > 0.05). Each dot corresponds to one mouse. TACI, transmembrane activator CAML interactor.
    Figure Legend Snippet: Normal CSR and plasma cell development under T cell–independent stimulation conditions. (A) Flow-cytometric analysis of plasma cells (TACI + CD138 + ; upper row) in the spleen of Igh Pax5/+ and Igh +/+ littermates 14 d after immunization with TNP-Ficoll. The frequency of IgG2b-expressing plasma cells was determined by intracellular IgG2b staining (lower row). The results shown are representative of two experiments. (B) Normal generation of GC B cells in the Peyer’s patches of Igh Pax5/+ and Igh +/+ littermates at steady state. The frequencies of total GC B cells (CD19 + GL7 + Fas + ) and IgA + GC cells were determined by flow cytometry (left) and quantified (right). (C) Normal generation of IgA-expressing plasma cells in the Peyer’s patches at steady state. The frequencies of total (TACI + CD138 + ) and IgA-expressing plasma cells were determined by flow cytometry (left) and quantified (right). One experiment (B and C) was performed. Statistical data (A–C) are shown as mean values with SD and were analyzed by the two-tailed unpaired Student’s t test; NS, not significant (P > 0.05). Each dot corresponds to one mouse. TACI, transmembrane activator CAML interactor.

    Techniques Used: Expressing, Staining, Flow Cytometry, Two Tailed Test

    Immune responses to SRBC immunization in Igh Pax5/+ mice. (A) GC B cell response in the spleen of Igh +/+ and Igh Pax5/+ mice 14 d after immunization with SRBCs. Total GC B cells (CD19 + GL7 + Fas + ) and IgG1 + GC B cells were analyzed by flow cytometry (left), and their frequency was quantified (right) together with the frequency of splenic plasma cells (Lin – B220 int CD138 + Blimp1-GFP + ). The data are pooled from two independent experiments. (B) Schematic diagram describing the generation of mixed bone marrow chimeras. Lineage-depleted bone marrow cells from Igh Pax5/+ or control Igh +/+ mice were mixed at a 1:1 ratio with lineage-depleted bone marrow cells from J H T mice before injection into lethally irradiated J H T recipient mice. 3 mo after reconstitution, the bone marrow chimeric mice were immunized with SRBCs and analyzed 2 wk later. (C) Flow-cytometric analysis of intracellular Pax5 protein levels in T FH cells (CD4 + CXCR5 + PD1 + Bcl6 + ) from the spleen of chimeric mice reconstituted with J H T and Igh Pax5/+ (black line) or Igh +/+ (gray filled) bone marrow cells. (D) Flow-cytometric analysis of the GC B cell response (left) and frequency of the indicated cell types (right) in the spleen of chimeric mice 2 wk after immunization with SRBCs. The data shown are representative of two independent experiments. Statistical data (A and D) are shown as mean values with SD and were analyzed by the two-tailed unpaired Student’s t test; *, P
    Figure Legend Snippet: Immune responses to SRBC immunization in Igh Pax5/+ mice. (A) GC B cell response in the spleen of Igh +/+ and Igh Pax5/+ mice 14 d after immunization with SRBCs. Total GC B cells (CD19 + GL7 + Fas + ) and IgG1 + GC B cells were analyzed by flow cytometry (left), and their frequency was quantified (right) together with the frequency of splenic plasma cells (Lin – B220 int CD138 + Blimp1-GFP + ). The data are pooled from two independent experiments. (B) Schematic diagram describing the generation of mixed bone marrow chimeras. Lineage-depleted bone marrow cells from Igh Pax5/+ or control Igh +/+ mice were mixed at a 1:1 ratio with lineage-depleted bone marrow cells from J H T mice before injection into lethally irradiated J H T recipient mice. 3 mo after reconstitution, the bone marrow chimeric mice were immunized with SRBCs and analyzed 2 wk later. (C) Flow-cytometric analysis of intracellular Pax5 protein levels in T FH cells (CD4 + CXCR5 + PD1 + Bcl6 + ) from the spleen of chimeric mice reconstituted with J H T and Igh Pax5/+ (black line) or Igh +/+ (gray filled) bone marrow cells. (D) Flow-cytometric analysis of the GC B cell response (left) and frequency of the indicated cell types (right) in the spleen of chimeric mice 2 wk after immunization with SRBCs. The data shown are representative of two independent experiments. Statistical data (A and D) are shown as mean values with SD and were analyzed by the two-tailed unpaired Student’s t test; *, P

    Techniques Used: Mouse Assay, Flow Cytometry, Injection, Irradiation, Two Tailed Test

    Rescue of IgG1 CSR in the presence of functional T cells. (A) Experimental strategy to interrogate B-lineage–intrinsic effects of ectopic Pax5 expression in response to immunization. CD43 – B cells isolated from the spleens of Igh B1-8hi/Pax5 Prdm1 Gfp/+ or Igh B1-8hi/+ Prdm1 Gfp/+ mice (CD45.1 + CD45.2 + ) were cotransferred with splenic T cells from OT-II TCR-tg Rag2 −/− mice (CD45.2 + ) into C57BL/6 recipients (CD45.1 + ). 1 d after cell transfer, the recipients were immunized with NP-OVA (in alum) and were analyzed 6 d after immunization. (B) Flow-cytometric analysis of splenic GC B cells of Igh B1-8hi donor origin. The frequencies of IgG1 + GC B cells of the Igh B1-8hi/+ Prdm1 Gfp/+ and Igh B1-8hi/Pax5 Prdm1 Gfp/+ genotypes were determined by sequential gating on donor cells (CD45.1 + CD45.2 + ), B cells (B220 + ), and GC B cells (GL7 + Fas + ) by flow cytometry (above). The quantification of the frequencies of GC B cells and IgG1 + GC B cells is shown below. The data are pooled from three independent experiments. (C) Analysis of splenic GFP + plasma cells derived from the transferred Igh B1-8hi/+ Prdm1 Gfp/+ or Igh B1-8hi/Pax5 Prdm1 Gfp/+ B cells. The flow-cytometric analysis is shown on the left, and the frequency of the GFP + plasma cells is quantified on the right. The data are pooled from two independent experiments. (D) ELISPOT assay to determine the frequency of anti–NP-IgM and anti–NP-IgG1 ASCs derived from the transferred Igh B1-8hi/+ Prdm1 Gfp/+ or Igh B1-8hi/Pax5 Prdm1 Gfp/+ cells. Six days after immunization with NP-OVA, 500 GFP + CD138 + plasma cells of donor origin were directly sorted onto NP 20 -BSA–coated plates. Cells were incubated for 6 h, before the number and size of anti–NP-IgM and anti–NP-IgG1 ELISPOTs were determined. Images of representative ELISPOT wells (left), the mean cell number (middle), and the median ELISPOT size (right) are shown for anti–NP-IgM and anti–NP-IgG1 ASCs. Statistical data (B–D) are shown as mean values with SD and were analyzed by the two-tailed unpaired Student’s t test; **, P
    Figure Legend Snippet: Rescue of IgG1 CSR in the presence of functional T cells. (A) Experimental strategy to interrogate B-lineage–intrinsic effects of ectopic Pax5 expression in response to immunization. CD43 – B cells isolated from the spleens of Igh B1-8hi/Pax5 Prdm1 Gfp/+ or Igh B1-8hi/+ Prdm1 Gfp/+ mice (CD45.1 + CD45.2 + ) were cotransferred with splenic T cells from OT-II TCR-tg Rag2 −/− mice (CD45.2 + ) into C57BL/6 recipients (CD45.1 + ). 1 d after cell transfer, the recipients were immunized with NP-OVA (in alum) and were analyzed 6 d after immunization. (B) Flow-cytometric analysis of splenic GC B cells of Igh B1-8hi donor origin. The frequencies of IgG1 + GC B cells of the Igh B1-8hi/+ Prdm1 Gfp/+ and Igh B1-8hi/Pax5 Prdm1 Gfp/+ genotypes were determined by sequential gating on donor cells (CD45.1 + CD45.2 + ), B cells (B220 + ), and GC B cells (GL7 + Fas + ) by flow cytometry (above). The quantification of the frequencies of GC B cells and IgG1 + GC B cells is shown below. The data are pooled from three independent experiments. (C) Analysis of splenic GFP + plasma cells derived from the transferred Igh B1-8hi/+ Prdm1 Gfp/+ or Igh B1-8hi/Pax5 Prdm1 Gfp/+ B cells. The flow-cytometric analysis is shown on the left, and the frequency of the GFP + plasma cells is quantified on the right. The data are pooled from two independent experiments. (D) ELISPOT assay to determine the frequency of anti–NP-IgM and anti–NP-IgG1 ASCs derived from the transferred Igh B1-8hi/+ Prdm1 Gfp/+ or Igh B1-8hi/Pax5 Prdm1 Gfp/+ cells. Six days after immunization with NP-OVA, 500 GFP + CD138 + plasma cells of donor origin were directly sorted onto NP 20 -BSA–coated plates. Cells were incubated for 6 h, before the number and size of anti–NP-IgM and anti–NP-IgG1 ELISPOTs were determined. Images of representative ELISPOT wells (left), the mean cell number (middle), and the median ELISPOT size (right) are shown for anti–NP-IgM and anti–NP-IgG1 ASCs. Statistical data (B–D) are shown as mean values with SD and were analyzed by the two-tailed unpaired Student’s t test; **, P

    Techniques Used: Functional Assay, Expressing, Isolation, Mouse Assay, Flow Cytometry, Derivative Assay, Enzyme-linked Immunospot, Incubation, Two Tailed Test

    Antibody secretion by Igh Pax5/+ plasma cells. (A and B) Antibody secretion by Igh Pax5/+ and Igh +/+ plasma cells under steady-state conditions. The numbers of IgM and IgG ASCs in the bone marrow of Igh Pax5/+ (black) and Igh +/+ (gray) mice at the age of 2–3 mo (A) and 6–8 mo (B) were determined by ELISPOT assay by incubating RBC-depleted bone marrow cells (2 × 10 5 ) on IgM- or IgG-coated plates for 6 h before determination of the number and size of the ELISPOTs. Images of representative ELISPOT wells (left), the mean cell number (middle), and the median ELISPOT size (right) are shown for IgM and IgG ASCs. (C and D) Antibody secretion by Igh Pax5/+ and Igh +/+ plasma cells 14 d after immunization with NP-KLH (in alum). The numbers of anti–NP-IgM or anti–NP-IgG1 ASCs in the spleen (C) and bone marrow (D) were determined by ELISPOT assay by incubating RBC-depleted cells (2 × 10 6 ) from the spleen or bone marrow on NP 20 -BSA-coated plates before the determination of the number and size of the ELISPOTs. Images of representative ELISPOT wells (left), the mean cell number (middle), and the median ELISPOT size (right) are shown for anti–NP-IgM and anti–NP-IgG1 ASCs. The data (C and D) are pooled from two independent immunization experiments. Statistical data (A–D) are shown as mean values with SD and were analyzed by the two-tailed unpaired Student’s t test; *, P
    Figure Legend Snippet: Antibody secretion by Igh Pax5/+ plasma cells. (A and B) Antibody secretion by Igh Pax5/+ and Igh +/+ plasma cells under steady-state conditions. The numbers of IgM and IgG ASCs in the bone marrow of Igh Pax5/+ (black) and Igh +/+ (gray) mice at the age of 2–3 mo (A) and 6–8 mo (B) were determined by ELISPOT assay by incubating RBC-depleted bone marrow cells (2 × 10 5 ) on IgM- or IgG-coated plates for 6 h before determination of the number and size of the ELISPOTs. Images of representative ELISPOT wells (left), the mean cell number (middle), and the median ELISPOT size (right) are shown for IgM and IgG ASCs. (C and D) Antibody secretion by Igh Pax5/+ and Igh +/+ plasma cells 14 d after immunization with NP-KLH (in alum). The numbers of anti–NP-IgM or anti–NP-IgG1 ASCs in the spleen (C) and bone marrow (D) were determined by ELISPOT assay by incubating RBC-depleted cells (2 × 10 6 ) from the spleen or bone marrow on NP 20 -BSA-coated plates before the determination of the number and size of the ELISPOTs. Images of representative ELISPOT wells (left), the mean cell number (middle), and the median ELISPOT size (right) are shown for anti–NP-IgM and anti–NP-IgG1 ASCs. The data (C and D) are pooled from two independent immunization experiments. Statistical data (A–D) are shown as mean values with SD and were analyzed by the two-tailed unpaired Student’s t test; *, P

    Techniques Used: Mouse Assay, Enzyme-linked Immunospot, Two Tailed Test

    Impaired CSR to IgG1 in Igh Pax5/+ mice. (A and B) Normal CSR to IgG3 and IgG1 under in vitro conditions. Naive B cells from the spleen and lymph nodes of Igh Pax5/+ (black) or Igh +/+ (gray) mice were cultured for 4 d with LPS (A) or anti-CD40, IL-4, and IL-5 (B) before flow-cytometric analysis and quantification of the class-switched B cells. The data are representative of two (A) or three (B) independent experiments. (C) GC B cell response in the spleen of Igh Pax5/+ and Igh +/+ mice 14 d after immunization with NP-KLH (in alum). The frequencies of total GC B cells (CD19 + GL7 + Fas + ), NP-specific GC B cells, and NP-specific IgG1 + GC cells were determined by flow cytometry (above) and quantified (below). The data are pooled from four independent experiments. (D) Frequency of T FH cells (CD4 + CXCR5 + PD1 + Bcl6 + ) in the spleens of Igh Pax5/+ (black) and Igh +/+ (gray) mice 14 d after NP-KLH immunization (left). Flow-cytometric analysis of Pax5 expression in T FH cells from the Igh Pax5/+ (black line) and Igh +/+ (gray filled) spleen is shown on the right. The data are pooled from two independent experiments. Statistical data (A–D) are shown as mean values with SD and were analyzed by the two-tailed unpaired Student’s t test; ****, P
    Figure Legend Snippet: Impaired CSR to IgG1 in Igh Pax5/+ mice. (A and B) Normal CSR to IgG3 and IgG1 under in vitro conditions. Naive B cells from the spleen and lymph nodes of Igh Pax5/+ (black) or Igh +/+ (gray) mice were cultured for 4 d with LPS (A) or anti-CD40, IL-4, and IL-5 (B) before flow-cytometric analysis and quantification of the class-switched B cells. The data are representative of two (A) or three (B) independent experiments. (C) GC B cell response in the spleen of Igh Pax5/+ and Igh +/+ mice 14 d after immunization with NP-KLH (in alum). The frequencies of total GC B cells (CD19 + GL7 + Fas + ), NP-specific GC B cells, and NP-specific IgG1 + GC cells were determined by flow cytometry (above) and quantified (below). The data are pooled from four independent experiments. (D) Frequency of T FH cells (CD4 + CXCR5 + PD1 + Bcl6 + ) in the spleens of Igh Pax5/+ (black) and Igh +/+ (gray) mice 14 d after NP-KLH immunization (left). Flow-cytometric analysis of Pax5 expression in T FH cells from the Igh Pax5/+ (black line) and Igh +/+ (gray filled) spleen is shown on the right. The data are pooled from two independent experiments. Statistical data (A–D) are shown as mean values with SD and were analyzed by the two-tailed unpaired Student’s t test; ****, P

    Techniques Used: Mouse Assay, In Vitro, Cell Culture, Flow Cytometry, Expressing, Two Tailed Test

    38) Product Images from "Deciphering the importance of the palindromic architecture of the immunoglobulin heavy-chain 3' regulatory region"

    Article Title: Deciphering the importance of the palindromic architecture of the immunoglobulin heavy-chain 3' regulatory region

    Journal: Nature Communications

    doi: 10.1038/ncomms10730

    Influence of the 3'RR palindrome on Ig synthesis. ( a ) ELISA analysis of IgG 1 , IgG 2a , IgG 2b , IgG 3 and IgA in supernatants of LPS±IL-4-, INFγ- and TGFβ-stimulated splenocytes of ΔleftPAL, Δ IRIS and wt mice. Data are the mean±s.e.m. of eight experiments with one mouse (8–12 weeks old, male and female). * P
    Figure Legend Snippet: Influence of the 3'RR palindrome on Ig synthesis. ( a ) ELISA analysis of IgG 1 , IgG 2a , IgG 2b , IgG 3 and IgA in supernatants of LPS±IL-4-, INFγ- and TGFβ-stimulated splenocytes of ΔleftPAL, Δ IRIS and wt mice. Data are the mean±s.e.m. of eight experiments with one mouse (8–12 weeks old, male and female). * P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay

    39) Product Images from "Neutrophils are required during immunization with the pneumococcal conjugate vaccine for protective antibody responses and host defense against infection"

    Article Title: Neutrophils are required during immunization with the pneumococcal conjugate vaccine for protective antibody responses and host defense against infection

    Journal: bioRxiv

    doi: 10.1101/2020.02.04.934380

    PMNs are required for optimal antibody function following PCV. (A-C) Sera were collected from naïve, Prevnar-13 immunized and PMN depleted immunized mice four weeks post vaccination following the timeline indicated in Fig 1A . (A) Wild type ( WT ) or a capsule deletion mutant ( Δcps ) S. pneumoniae were incubated with the indicated sera for 30 minutes, washed and stained with fluorescently-labeled anti-mouse IgG. The amount (mean fluorescent intensity or MFI) of bound Abs was determined by flow cytometry. Representative data from one of three separate experiments (n=3 biological replicates) are shown where each condition was tested in triplicate (n=3 technical replicates) per experiment. (B) The ability of PMNs isolated from naïve mice to kill pneumococci pre-opsonized with the indicated sera was determined. Percent bacterial killing was determined with respect to a no PMN control. Data shown are pooled from three separate experiments (n=3 biological replicates) where each condition was tested in triplicate (n=3 technical replicates) per experiment. (A-B) Bar graph represent means+/-SD and asterisks indicate significant differences from vaccinated controls as calculated by One-way ANOVA followed by Dunnet’s test. (C) Naïve C57BL/6 female mice were injected i.p with 200μl of pooled serum from the indicated mice then challenged i.t. 1 hour later with 5×10 5 CFU S. pneumoniae TIGR4. Survival was assessed over time. *, denotes significance by the log-Rank (Mantel-Cox) test. Data were pooled from 8 mice/group from two separate experiments.
    Figure Legend Snippet: PMNs are required for optimal antibody function following PCV. (A-C) Sera were collected from naïve, Prevnar-13 immunized and PMN depleted immunized mice four weeks post vaccination following the timeline indicated in Fig 1A . (A) Wild type ( WT ) or a capsule deletion mutant ( Δcps ) S. pneumoniae were incubated with the indicated sera for 30 minutes, washed and stained with fluorescently-labeled anti-mouse IgG. The amount (mean fluorescent intensity or MFI) of bound Abs was determined by flow cytometry. Representative data from one of three separate experiments (n=3 biological replicates) are shown where each condition was tested in triplicate (n=3 technical replicates) per experiment. (B) The ability of PMNs isolated from naïve mice to kill pneumococci pre-opsonized with the indicated sera was determined. Percent bacterial killing was determined with respect to a no PMN control. Data shown are pooled from three separate experiments (n=3 biological replicates) where each condition was tested in triplicate (n=3 technical replicates) per experiment. (A-B) Bar graph represent means+/-SD and asterisks indicate significant differences from vaccinated controls as calculated by One-way ANOVA followed by Dunnet’s test. (C) Naïve C57BL/6 female mice were injected i.p with 200μl of pooled serum from the indicated mice then challenged i.t. 1 hour later with 5×10 5 CFU S. pneumoniae TIGR4. Survival was assessed over time. *, denotes significance by the log-Rank (Mantel-Cox) test. Data were pooled from 8 mice/group from two separate experiments.

    Techniques Used: Mouse Assay, Mutagenesis, Incubation, Staining, Labeling, Flow Cytometry, Isolation, Injection

    PMNs contribute to IgG2 and IgG3 production following PCV immunization. Sera were collected from naïve (green lines), Prevnar-13 immunized (black lines) and PMN depleted Prevnar-13 immunized mice (blue lines) following the timeline presented in Fig 1A . (A-D) The levels of the indicated antibodies against purified polysaccharide serotype 4 were then measured in the sera by ELISA. Antibody units were calculated based on a hyperimmune standard. p values were determined by student t-test. Asterisks ( p
    Figure Legend Snippet: PMNs contribute to IgG2 and IgG3 production following PCV immunization. Sera were collected from naïve (green lines), Prevnar-13 immunized (black lines) and PMN depleted Prevnar-13 immunized mice (blue lines) following the timeline presented in Fig 1A . (A-D) The levels of the indicated antibodies against purified polysaccharide serotype 4 were then measured in the sera by ELISA. Antibody units were calculated based on a hyperimmune standard. p values were determined by student t-test. Asterisks ( p

    Techniques Used: Mouse Assay, Purification, Enzyme-linked Immunosorbent Assay

    Total levels of anti-pneumococcal IgG and IgM remain unchanged in PMN-depleted PCV immunized mice. Sera were collected from naïve (green lines), Prevnar-13 immunized (black lines) and PMN depleted Prevnar-13 immunized mice (blue lines) over time as indicated in Fig 1A . Circulating levels of IgM (A) and total IgG (B) against purified polysaccharide serotype 4 were then measured by ELISA. Antibody units were calculated based on a hyperimmune standard (see Materials and Methods) included in each ELISA plate. p values were determined by student t-test. Asterisks ( p
    Figure Legend Snippet: Total levels of anti-pneumococcal IgG and IgM remain unchanged in PMN-depleted PCV immunized mice. Sera were collected from naïve (green lines), Prevnar-13 immunized (black lines) and PMN depleted Prevnar-13 immunized mice (blue lines) over time as indicated in Fig 1A . Circulating levels of IgM (A) and total IgG (B) against purified polysaccharide serotype 4 were then measured by ELISA. Antibody units were calculated based on a hyperimmune standard (see Materials and Methods) included in each ELISA plate. p values were determined by student t-test. Asterisks ( p

    Techniques Used: Mouse Assay, Purification, Enzyme-linked Immunosorbent Assay

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    Article Snippet: .. ELISA For assessment of anti-L or anti-D antibodies, sera were collected by cardiac puncture 21 days following hydrogel application of mice (subcutaneous implant or application to wound). for detection of anti-L and anti-D antibodies plates were coated with either L-MMP peptide or D-MMP peptide resepcitvely (GenScript; sequence above) Serum samples were tested at a 1:500 dilution followed by incubation with alkaline phosphatase-labeled goat anti-mouse IgG1 or IgG2a, or IgG3 antibodies (Southern Biotechnology Associates or BD Pharmingen), and development with p -nitrophenylphosphate substrate (Sigma-Aldrich). .. Optical density at 405 nm (OD405) was read using a spectramax i3X microplate reader (Softmax Pro 3.1 software; Molecular Devices).

    Article Title: Immunogenicity and Cross-Protective Efficacy Induced by Outer Membrane Proteins from Salmonella Typhimurium Mutants with Truncated LPS in Mice
    Article Snippet: The plate was washed 3 times with PBST (PBS with 0.1% Tween 20) and then blocked with 2% bovine serum albumin (BSA) (Sigma-Aldrich) solution. .. After incubation of 2 h at room temperature, a 100-μL volume of serially-diluted sample (serum dilution started at 1:50) was added to triplicate individual wells and incubated for 1 h at room temperature and then washing with PBST 3 times; biotinylated goat anti-mouse IgG, IgG1 or IgG2a (Southern Biotechnology Inc., Birmingham, AL, USA) was added in each well, and then, the wells were developed with a streptavidin-alkaline phosphatase conjugate (Southern Biotechnology, Inc., Birmingham, AL, USA) and detected using p-nitrophenylphosphate substrate (Sigma-Aldrich) in diethanolamine buffer (pH 9.8). .. Absorbance was taken at 405 nm using an automated ELISA plate (Bio-Rad iMark Microplate Reader, Hercules, CA, USA) with suitable time.

    Immunoprecipitation:

    Article Title: Evaluation of autoantibodies and immunoglobulin G subclasses in women with suspected macroprolactinemia, et al. Evaluation of autoantibodies and immunoglobulin G subclasses in women with suspected macroprolactinemia
    Article Snippet: .. Lastly, the IgG1 and IgG3 were the predominant subclasses in the PRL‐IgG complex trapped by the immunoprecipitation method, suggesting their pathogenic significance in the development of anti‐PRL autoantibodies. ..

    Produced:

    Article Title: Distinct B-cell populations contribute to vaccine antigen-specific antibody production in a transgenic mouse model
    Article Snippet: IgG2a, IgG2b and IgG2c isotype antibodies were also found at much higher levels in CD43+ (B220− ) cell fractions with low YFP+ cell percentages from both primed and boosted ROSA YFP mice compared with those in CD43− (B220+ ) cell fractions with higher YFP+ cell percentages. .. It is also interesting to note that IgG2b and IgG2c antibodies were produced at detectable levels in the CD43− (B220+ population) cell fraction of boosted splenocytes, although their levels were lower compared with those in CD43+ cell fractions (Fig. c). .. These results indicate that YFP+ ROSA mice can generate vaccine antigen-specific memory B cells capable of differentiating to IgG and isotype antibody-secreting plasma cells after VLP vaccination.

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  • 94
    SouthernBiotech igg2c hrp
    The LION/repRNA-CoV2S vaccine induces Th1-biased and neutralizing antibodies in C57BL/6 mice. Six to eight-week old C57BL/6 mice (n=5/group) received 10, 1, or 0.1 μg LION/repRNA-CoV2S via the intramuscular route on days 0 and 28. ( A ) Anti-S IgG antibody concentrations were determined by enzyme linked immunosorbent assay (ELISA) on days 14, 28, and 40. For day 14 samples, ( B ) 50% inhibitory concentrations (IC50) were determined by pseudovirus (SARS-CoV-2 Wuhan-Hu-1 pseudotype) neutralization assays. For day 14 samples, ( C ) anti-S IgG1 and <t>IgG2c</t> antibody endpoint titers and ( D ) ratios were determined by ELISA. On day 40, 12 days after a booster immunization, ( E ) spleens and ( F ) lungs were harvested and IFN-γ responses were measured by enzyme-linked immune absorbent spot (ELISpot) assay following an 18-hour stimulation with 10 peptide pools encompassing the S protein and consisting of 15 mers overlapping by 11 amino acids (see Fig. S1). Data in A , C , and D are representative of 3 independent experiments; data in B , E , and F are from a single experiment. Dotted lines in A, B, E, and F represent the lower limit of detection. All data are represented as individual values as well as mean ± s.d. *p
    Igg2c Hrp, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    SouthernBiotech peroxidase conjugated anti mouse igg1
    Recognition of Pfs48/45 in fixed P . falciparum parasites in indirect immunofluorescence assays. The representative results for fixed parasites incubated with pooled pre-immune sera, pooled sera from CFA adjuvant alone immunized mice, a representative anti-Pfs48/45 serum, a representative ELISA and WB positive anti-Pvs48/45 serum (CFA group), and a representative ELISA and WB positive anti-Pvs48/45 (Montanide ISA-51 group) at a dilution of 1:100. BF, bright field; DAPI, the nuclei stained by NucBlue reagent; FITC, antibody reactivity to the parasite visualized with FITC-conjugated anti-mouse <t>IgG</t> antibody, and Merge of DAPI and FITC images. All images were visualized and captured at 1000X magnification. Scale bar (white line), 5μm.
    Peroxidase Conjugated Anti Mouse Igg1, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    SouthernBiotech anti igg2b
    Impaired B cell responses in TC10 KO animals. ( A ) Serum was collected from naive WT and TC10 KO animals, and baseline titers of IgM, <t>IgG2b,</t> IgG2c, IgG3, and IgA were measured by sandwich ELISA. ( B – E ) WT and TC10 KO animals were infected with 10 4 PFU VACV (B and C) or 2.10 2 Influenza virus (D and E) by intra footpad (B and C) or intranasal injection (D and E), and the draining popliteal (B and C) or mediastinal (D and E) LNs were isolated 7 (B and C) or 9 (D and E) d later. Germinal center B cells (CD95 + GL-7 + (B and D) and plasma cells [CD138 + IgD lo (C and E)] were analyzed by flow cytometry. Quantifications are shown on the panels on the right and show the percentage of B cells in the indicated gates. ( F and G ) WT and TC10 KO mice were immunized with NP-KLH precipitated in Alum, and serum samples collected weekly for 28 d. NP-specific IgM and IgG3 (F) titers were measured, as well as total IgG titers (G). ( H ) ELISA analysis showing affinity maturation (expressed as the ratio of NP3 to NP23 titers) of total IgG in WT and TC10 KO animals. Data are from one representative of two to three independent experiments with at least three animals in each group. Student t test (ns: p > 0.05, * p
    Anti Igg2b, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    SouthernBiotech biotinylated goat anti human igg1
    Binding of CLL69 Abs to OSE on apoptotic cells and atherosclerotic lesions. ( A ) Flow cytometry analysis of CLL69 rAb binding to apoptotic cells. Representative flow cytometry contour plots of apoptotic murine thymocytes and histogram plots for the binding of rAb to apoptotic cells. Murine thymocytes were induced to undergo apoptosis by incubation with 10 ng/ml PMA for 16 hours. Apoptotic cells were stained with rAbs CLL69 A–D, isotype VH3-21, or no primary Ab, followed by detection with a FITC-conjugated anti-human <t>IgG1</t> and staining with PE-labeled Annexin-V and 7-AAD to identify the viable and apoptotic cells. The contour plot (upper left panel) identifies the viable cells (Q3, PE-Annexin-V −/ 7-AAD − ), early apoptotic (Q4, Annexin-V + /7-AAD − ) and late apoptotic cells (Q2, Annexin-V + /7-AAD + ). Histogram panels Q3 (bottom left), Q4 (bottom right) and Q2 (upper right) represent rAb staining of viable cells, early apoptotic cells, and late apoptotic cells, respectively. Relative cell fluorescence of rAbs CLL69 A–D and IGHV3-21 control rAb are indicted by colored lines, and staining with the secondary Ab alone is shown in grey. ( B ) Immunofluorescence microscopy of CLL69C rAb binding to apoptotic cells. Deconvolution microscopy showing the binding of CLL69C rAb to late apoptotic Jurkat cells (FITC green color, the cell on the left panel) with dense fragmented nucleus detected by Hoechst staining (blue color), and apoptotic cells detected by PE-Annexin-V (red color), but not to a cell with intact nucleus (Panel A). The vector control and secondary Ab only (anti-IgG1-FITC) do not bind the apoptotic cells (panel B and C, respectively). ( C ) Inhibition of MAA-LDL binding to macrophage scavenger receptors by CLL69Ab. Competition assays for the inhibition of <t>biotinylated-MAA-LDL</t> binding to macrophage J774 by CLL69C rAb, UL10 Fab, or an irrelevant control VH3-21 rAb, as indicated. A fixed and limiting amount of biotin-MAA-LDL (5 µg/ml) was added to J774 macrophages in the absence or presence of increasing concentrations of indicated competitors. Extent of binding was determined in RLU/100 msec as described for chemiluminescent ELISA assays. Data shown represent the ratio MAA-LDL binding in the presence or absence of competitors (B/B 0 ) as described in legend to Fig. 1B. Results are representative of three independent experiments, each point determined in triplicate. ( D ) Immunohistochemical staining of human carotid endarterectomy (CEA) specimens. Roughly parallel sections of human carotid endarterectomy tissue were stained with rAbs of CLL69C (panel C), CLL69A (panel B), or a secondary Ab alone (panel A). MAA-epitopes recognized by CLL69C or CLL69A are indicated by red color.
    Biotinylated Goat Anti Human Igg1, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The LION/repRNA-CoV2S vaccine induces Th1-biased and neutralizing antibodies in C57BL/6 mice. Six to eight-week old C57BL/6 mice (n=5/group) received 10, 1, or 0.1 μg LION/repRNA-CoV2S via the intramuscular route on days 0 and 28. ( A ) Anti-S IgG antibody concentrations were determined by enzyme linked immunosorbent assay (ELISA) on days 14, 28, and 40. For day 14 samples, ( B ) 50% inhibitory concentrations (IC50) were determined by pseudovirus (SARS-CoV-2 Wuhan-Hu-1 pseudotype) neutralization assays. For day 14 samples, ( C ) anti-S IgG1 and IgG2c antibody endpoint titers and ( D ) ratios were determined by ELISA. On day 40, 12 days after a booster immunization, ( E ) spleens and ( F ) lungs were harvested and IFN-γ responses were measured by enzyme-linked immune absorbent spot (ELISpot) assay following an 18-hour stimulation with 10 peptide pools encompassing the S protein and consisting of 15 mers overlapping by 11 amino acids (see Fig. S1). Data in A , C , and D are representative of 3 independent experiments; data in B , E , and F are from a single experiment. Dotted lines in A, B, E, and F represent the lower limit of detection. All data are represented as individual values as well as mean ± s.d. *p

    Journal: Science Translational Medicine

    Article Title: An alphavirus-derived replicon RNA vaccine induces SARS-CoV-2 neutralizing antibody and T cell responses in mice and nonhuman primates

    doi: 10.1126/scitranslmed.abc9396

    Figure Lengend Snippet: The LION/repRNA-CoV2S vaccine induces Th1-biased and neutralizing antibodies in C57BL/6 mice. Six to eight-week old C57BL/6 mice (n=5/group) received 10, 1, or 0.1 μg LION/repRNA-CoV2S via the intramuscular route on days 0 and 28. ( A ) Anti-S IgG antibody concentrations were determined by enzyme linked immunosorbent assay (ELISA) on days 14, 28, and 40. For day 14 samples, ( B ) 50% inhibitory concentrations (IC50) were determined by pseudovirus (SARS-CoV-2 Wuhan-Hu-1 pseudotype) neutralization assays. For day 14 samples, ( C ) anti-S IgG1 and IgG2c antibody endpoint titers and ( D ) ratios were determined by ELISA. On day 40, 12 days after a booster immunization, ( E ) spleens and ( F ) lungs were harvested and IFN-γ responses were measured by enzyme-linked immune absorbent spot (ELISpot) assay following an 18-hour stimulation with 10 peptide pools encompassing the S protein and consisting of 15 mers overlapping by 11 amino acids (see Fig. S1). Data in A , C , and D are representative of 3 independent experiments; data in B , E , and F are from a single experiment. Dotted lines in A, B, E, and F represent the lower limit of detection. All data are represented as individual values as well as mean ± s.d. *p

    Article Snippet: Then, in consecutive order, washes in PBS/Tween, serially diluted serum samples, anti-mouse or-monkey IgG, IgG1, IgG2a, or IgG2c-HRP (Southern Biotech) and TMB then HCL were added to the plates.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Neutralization, Enzyme-linked Immunospot

    Recognition of Pfs48/45 in fixed P . falciparum parasites in indirect immunofluorescence assays. The representative results for fixed parasites incubated with pooled pre-immune sera, pooled sera from CFA adjuvant alone immunized mice, a representative anti-Pfs48/45 serum, a representative ELISA and WB positive anti-Pvs48/45 serum (CFA group), and a representative ELISA and WB positive anti-Pvs48/45 (Montanide ISA-51 group) at a dilution of 1:100. BF, bright field; DAPI, the nuclei stained by NucBlue reagent; FITC, antibody reactivity to the parasite visualized with FITC-conjugated anti-mouse IgG antibody, and Merge of DAPI and FITC images. All images were visualized and captured at 1000X magnification. Scale bar (white line), 5μm.

    Journal: PLoS ONE

    Article Title: Immunological Cross-Reactivity between Malaria Vaccine Target Antigen P48/45 in Plasmodium vivax and P. falciparum and Cross–Boosting of Immune Responses

    doi: 10.1371/journal.pone.0158212

    Figure Lengend Snippet: Recognition of Pfs48/45 in fixed P . falciparum parasites in indirect immunofluorescence assays. The representative results for fixed parasites incubated with pooled pre-immune sera, pooled sera from CFA adjuvant alone immunized mice, a representative anti-Pfs48/45 serum, a representative ELISA and WB positive anti-Pvs48/45 serum (CFA group), and a representative ELISA and WB positive anti-Pvs48/45 (Montanide ISA-51 group) at a dilution of 1:100. BF, bright field; DAPI, the nuclei stained by NucBlue reagent; FITC, antibody reactivity to the parasite visualized with FITC-conjugated anti-mouse IgG antibody, and Merge of DAPI and FITC images. All images were visualized and captured at 1000X magnification. Scale bar (white line), 5μm.

    Article Snippet: Peroxidase conjugated anti-mouse IgG1, IgG2a, IgG2b and IgG3 (Southern Biotech, 1:2500 dilution) were used as secondary antibodies for antibody isotype analysis.

    Techniques: Immunofluorescence, Incubation, Mouse Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Staining

    Analysis of antibody isotypes. Anti-Pvs48/45 sera showing positive reactivity to Pfs48/45 in ELISA and Western blotting (4 out of 5 from CFA group and 2 out of 5 from Montanide ISA-51 group) were individually tested to compare immunoglobulin isotypes. ELISA plates coated with Pvs48/45 (panel A) or Pfs48/45 (panel B) were incubated with sera (1:10,000 dilution for Fig 3A and 1:100 dilution for Fig 3B). The plates were then incubated with peroxidase-conjugated goat anti-mouse IgG1, IgG2a, IgG2b and IgG3 (1:2,500 dilution) and processed as in standard ELISA. Shown are mean absorbance values for each isotype and the insets in panels A and B show relative proportions of IgG2a, IgG2b and IgG3 isotypes compared to IgG1. The error bars indicate SD.

    Journal: PLoS ONE

    Article Title: Immunological Cross-Reactivity between Malaria Vaccine Target Antigen P48/45 in Plasmodium vivax and P. falciparum and Cross–Boosting of Immune Responses

    doi: 10.1371/journal.pone.0158212

    Figure Lengend Snippet: Analysis of antibody isotypes. Anti-Pvs48/45 sera showing positive reactivity to Pfs48/45 in ELISA and Western blotting (4 out of 5 from CFA group and 2 out of 5 from Montanide ISA-51 group) were individually tested to compare immunoglobulin isotypes. ELISA plates coated with Pvs48/45 (panel A) or Pfs48/45 (panel B) were incubated with sera (1:10,000 dilution for Fig 3A and 1:100 dilution for Fig 3B). The plates were then incubated with peroxidase-conjugated goat anti-mouse IgG1, IgG2a, IgG2b and IgG3 (1:2,500 dilution) and processed as in standard ELISA. Shown are mean absorbance values for each isotype and the insets in panels A and B show relative proportions of IgG2a, IgG2b and IgG3 isotypes compared to IgG1. The error bars indicate SD.

    Article Snippet: Peroxidase conjugated anti-mouse IgG1, IgG2a, IgG2b and IgG3 (Southern Biotech, 1:2500 dilution) were used as secondary antibodies for antibody isotype analysis.

    Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Incubation

    Impaired B cell responses in TC10 KO animals. ( A ) Serum was collected from naive WT and TC10 KO animals, and baseline titers of IgM, IgG2b, IgG2c, IgG3, and IgA were measured by sandwich ELISA. ( B – E ) WT and TC10 KO animals were infected with 10 4 PFU VACV (B and C) or 2.10 2 Influenza virus (D and E) by intra footpad (B and C) or intranasal injection (D and E), and the draining popliteal (B and C) or mediastinal (D and E) LNs were isolated 7 (B and C) or 9 (D and E) d later. Germinal center B cells (CD95 + GL-7 + (B and D) and plasma cells [CD138 + IgD lo (C and E)] were analyzed by flow cytometry. Quantifications are shown on the panels on the right and show the percentage of B cells in the indicated gates. ( F and G ) WT and TC10 KO mice were immunized with NP-KLH precipitated in Alum, and serum samples collected weekly for 28 d. NP-specific IgM and IgG3 (F) titers were measured, as well as total IgG titers (G). ( H ) ELISA analysis showing affinity maturation (expressed as the ratio of NP3 to NP23 titers) of total IgG in WT and TC10 KO animals. Data are from one representative of two to three independent experiments with at least three animals in each group. Student t test (ns: p > 0.05, * p

    Journal: The Journal of Immunology Author Choice

    Article Title: The Small Rho GTPase TC10 Modulates B Cell Immune Responses

    doi: 10.4049/jimmunol.1602167

    Figure Lengend Snippet: Impaired B cell responses in TC10 KO animals. ( A ) Serum was collected from naive WT and TC10 KO animals, and baseline titers of IgM, IgG2b, IgG2c, IgG3, and IgA were measured by sandwich ELISA. ( B – E ) WT and TC10 KO animals were infected with 10 4 PFU VACV (B and C) or 2.10 2 Influenza virus (D and E) by intra footpad (B and C) or intranasal injection (D and E), and the draining popliteal (B and C) or mediastinal (D and E) LNs were isolated 7 (B and C) or 9 (D and E) d later. Germinal center B cells (CD95 + GL-7 + (B and D) and plasma cells [CD138 + IgD lo (C and E)] were analyzed by flow cytometry. Quantifications are shown on the panels on the right and show the percentage of B cells in the indicated gates. ( F and G ) WT and TC10 KO mice were immunized with NP-KLH precipitated in Alum, and serum samples collected weekly for 28 d. NP-specific IgM and IgG3 (F) titers were measured, as well as total IgG titers (G). ( H ) ELISA analysis showing affinity maturation (expressed as the ratio of NP3 to NP23 titers) of total IgG in WT and TC10 KO animals. Data are from one representative of two to three independent experiments with at least three animals in each group. Student t test (ns: p > 0.05, * p

    Article Snippet: Biotinylated anti-IgM (553406; BD Biosciences), anti-IgG2b (Southern Biotech), IgG2c (Southern Biotech), or anti-IL6 (Clone MP5-32C11; BD Biosciences) Abs were used for detection.

    Techniques: Sandwich ELISA, Infection, Injection, Isolation, Flow Cytometry, Cytometry, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Binding of CLL69 Abs to OSE on apoptotic cells and atherosclerotic lesions. ( A ) Flow cytometry analysis of CLL69 rAb binding to apoptotic cells. Representative flow cytometry contour plots of apoptotic murine thymocytes and histogram plots for the binding of rAb to apoptotic cells. Murine thymocytes were induced to undergo apoptosis by incubation with 10 ng/ml PMA for 16 hours. Apoptotic cells were stained with rAbs CLL69 A–D, isotype VH3-21, or no primary Ab, followed by detection with a FITC-conjugated anti-human IgG1 and staining with PE-labeled Annexin-V and 7-AAD to identify the viable and apoptotic cells. The contour plot (upper left panel) identifies the viable cells (Q3, PE-Annexin-V −/ 7-AAD − ), early apoptotic (Q4, Annexin-V + /7-AAD − ) and late apoptotic cells (Q2, Annexin-V + /7-AAD + ). Histogram panels Q3 (bottom left), Q4 (bottom right) and Q2 (upper right) represent rAb staining of viable cells, early apoptotic cells, and late apoptotic cells, respectively. Relative cell fluorescence of rAbs CLL69 A–D and IGHV3-21 control rAb are indicted by colored lines, and staining with the secondary Ab alone is shown in grey. ( B ) Immunofluorescence microscopy of CLL69C rAb binding to apoptotic cells. Deconvolution microscopy showing the binding of CLL69C rAb to late apoptotic Jurkat cells (FITC green color, the cell on the left panel) with dense fragmented nucleus detected by Hoechst staining (blue color), and apoptotic cells detected by PE-Annexin-V (red color), but not to a cell with intact nucleus (Panel A). The vector control and secondary Ab only (anti-IgG1-FITC) do not bind the apoptotic cells (panel B and C, respectively). ( C ) Inhibition of MAA-LDL binding to macrophage scavenger receptors by CLL69Ab. Competition assays for the inhibition of biotinylated-MAA-LDL binding to macrophage J774 by CLL69C rAb, UL10 Fab, or an irrelevant control VH3-21 rAb, as indicated. A fixed and limiting amount of biotin-MAA-LDL (5 µg/ml) was added to J774 macrophages in the absence or presence of increasing concentrations of indicated competitors. Extent of binding was determined in RLU/100 msec as described for chemiluminescent ELISA assays. Data shown represent the ratio MAA-LDL binding in the presence or absence of competitors (B/B 0 ) as described in legend to Fig. 1B. Results are representative of three independent experiments, each point determined in triplicate. ( D ) Immunohistochemical staining of human carotid endarterectomy (CEA) specimens. Roughly parallel sections of human carotid endarterectomy tissue were stained with rAbs of CLL69C (panel C), CLL69A (panel B), or a secondary Ab alone (panel A). MAA-epitopes recognized by CLL69C or CLL69A are indicated by red color.

    Journal: PLoS ONE

    Article Title: IGHV1-69-Encoded Antibodies Expressed in Chronic Lymphocytic Leukemia React with Malondialdehyde-Acetaldehyde Adduct, an Immunodominant Oxidation-Specific Epitope

    doi: 10.1371/journal.pone.0065203

    Figure Lengend Snippet: Binding of CLL69 Abs to OSE on apoptotic cells and atherosclerotic lesions. ( A ) Flow cytometry analysis of CLL69 rAb binding to apoptotic cells. Representative flow cytometry contour plots of apoptotic murine thymocytes and histogram plots for the binding of rAb to apoptotic cells. Murine thymocytes were induced to undergo apoptosis by incubation with 10 ng/ml PMA for 16 hours. Apoptotic cells were stained with rAbs CLL69 A–D, isotype VH3-21, or no primary Ab, followed by detection with a FITC-conjugated anti-human IgG1 and staining with PE-labeled Annexin-V and 7-AAD to identify the viable and apoptotic cells. The contour plot (upper left panel) identifies the viable cells (Q3, PE-Annexin-V −/ 7-AAD − ), early apoptotic (Q4, Annexin-V + /7-AAD − ) and late apoptotic cells (Q2, Annexin-V + /7-AAD + ). Histogram panels Q3 (bottom left), Q4 (bottom right) and Q2 (upper right) represent rAb staining of viable cells, early apoptotic cells, and late apoptotic cells, respectively. Relative cell fluorescence of rAbs CLL69 A–D and IGHV3-21 control rAb are indicted by colored lines, and staining with the secondary Ab alone is shown in grey. ( B ) Immunofluorescence microscopy of CLL69C rAb binding to apoptotic cells. Deconvolution microscopy showing the binding of CLL69C rAb to late apoptotic Jurkat cells (FITC green color, the cell on the left panel) with dense fragmented nucleus detected by Hoechst staining (blue color), and apoptotic cells detected by PE-Annexin-V (red color), but not to a cell with intact nucleus (Panel A). The vector control and secondary Ab only (anti-IgG1-FITC) do not bind the apoptotic cells (panel B and C, respectively). ( C ) Inhibition of MAA-LDL binding to macrophage scavenger receptors by CLL69Ab. Competition assays for the inhibition of biotinylated-MAA-LDL binding to macrophage J774 by CLL69C rAb, UL10 Fab, or an irrelevant control VH3-21 rAb, as indicated. A fixed and limiting amount of biotin-MAA-LDL (5 µg/ml) was added to J774 macrophages in the absence or presence of increasing concentrations of indicated competitors. Extent of binding was determined in RLU/100 msec as described for chemiluminescent ELISA assays. Data shown represent the ratio MAA-LDL binding in the presence or absence of competitors (B/B 0 ) as described in legend to Fig. 1B. Results are representative of three independent experiments, each point determined in triplicate. ( D ) Immunohistochemical staining of human carotid endarterectomy (CEA) specimens. Roughly parallel sections of human carotid endarterectomy tissue were stained with rAbs of CLL69C (panel C), CLL69A (panel B), or a secondary Ab alone (panel A). MAA-epitopes recognized by CLL69C or CLL69A are indicated by red color.

    Article Snippet: Sections of human or rabbit lesions were blocked with PBS containing 5% horse serum and 2% Fc blocker and stained with diluted CLL69 rAbs (1∶200), followed by addition of a biotinylated goat anti-human IgG1 (Southern Biotech) or anti-HA biotin mAb conjugate to detect the bound CLL69 Ab or Fab in the lesions.

    Techniques: Binding Assay, Flow Cytometry, Cytometry, Incubation, Staining, Labeling, Fluorescence, Immunofluorescence, Microscopy, Plasmid Preparation, Inhibition, Chemiluminescent ELISA, Immunohistochemistry