igg1  (SouthernBiotech)

 
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  • 99
    Name:
    Goat Anti Mouse IgG Fab HRP
    Description:

    Catalog Number:
    1015-05
    Price:
    None
    Applications:
    Quality tested applications for relevant formats include -ELISA 1-4FLISA
    Isotype:
    Goat IgG
    Source:
    Pooled antisera from goat hyperimmunized with mouse IgG
    Format:
    HRP (Horseradish Peroxidase)
    Buy from Supplier


    Structured Review

    SouthernBiotech igg1
    Goat Anti Mouse IgG Fab HRP

    https://www.bioz.com/result/igg1/product/SouthernBiotech
    Average 99 stars, based on 899 article reviews
    Price from $9.99 to $1999.99
    igg1 - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "A Strong Humoral Immune Response Induced by a Vaccine Formulation Containing rSm29 Adsorbed to Alum Is Associated With Protection Against Schistosoma mansoni Reinfection in Mice"

    Article Title: A Strong Humoral Immune Response Induced by a Vaccine Formulation Containing rSm29 Adsorbed to Alum Is Associated With Protection Against Schistosoma mansoni Reinfection in Mice

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02488

    Production of Sm29-specific antibodies in immunized mice. Sera from mice were obtained 15 days after each immunization dose and were assessed to determine the levels of IgG (A) , IgG1 (B) , IgG2a (C) , and IgE (D) antibodies against rSm29 in the animals inoculated with alum (open bars), MPLA (bright-gray bars), Sm29Alum (black bars) or Sm29/MPLA (dark-gray bar). Significant differences between adjuvant control and experimental groups are indicated by an asterisk ( p
    Figure Legend Snippet: Production of Sm29-specific antibodies in immunized mice. Sera from mice were obtained 15 days after each immunization dose and were assessed to determine the levels of IgG (A) , IgG1 (B) , IgG2a (C) , and IgE (D) antibodies against rSm29 in the animals inoculated with alum (open bars), MPLA (bright-gray bars), Sm29Alum (black bars) or Sm29/MPLA (dark-gray bar). Significant differences between adjuvant control and experimental groups are indicated by an asterisk ( p

    Techniques Used: Mouse Assay

    2) Product Images from "Uncoupling splicing from transcription using antisense oligonucleotides reveals a dual role for I exon donor splice sites in antibody class switching"

    Article Title: Uncoupling splicing from transcription using antisense oligonucleotides reveals a dual role for I exon donor splice sites in antibody class switching

    Journal: bioRxiv

    doi: 10.1101/850867

    Defective IgG1 class switching in B cells treated by Iμ exon dss ASO (A) Antisense oligonucleotide targeting the donor splice site of Iμ exon (Iμ dss ASO) was designed and synthetized as “vivo-morpholino ASO” permitting passive administration of ASO in the cells (Gene Tools, LLC). Targeted μ GLT (uppercase: exon sequence; lowercase: intron sequence) and Iμ dss ASO sequences are indicated. Iμ, μ I exon; Sμ, μ switch region; CH1μ, μ constant exon 1; dss, donor splice site. (B-E) Splenic B cells were isolated from C57BL/6 mice, stimulated with LPS + IL4 and treated with 2 μM Iμ dss ASO or an irrelevant control ASO for 2 days (B, E) or 3 days (C, D). (B) Constitutively and alternatively spliced μ transcripts analysed as described in figure 2 using Iμ-for and Cμ-rev primers, described in schema A. (C) Quantification of IgG1 in culture supernatants by ELISA. (D) Percentage of IgG1 positive cells determined by flow cytometry as described in figure 2 . (E) Unspliced γ1 GLTs expression monitored as described in figure 2 . (C-E) Data are means ± SEM, n=3 for each group. Unpaired two-tailed Student’s t test was used to determine significance. ns: non significant, * P
    Figure Legend Snippet: Defective IgG1 class switching in B cells treated by Iμ exon dss ASO (A) Antisense oligonucleotide targeting the donor splice site of Iμ exon (Iμ dss ASO) was designed and synthetized as “vivo-morpholino ASO” permitting passive administration of ASO in the cells (Gene Tools, LLC). Targeted μ GLT (uppercase: exon sequence; lowercase: intron sequence) and Iμ dss ASO sequences are indicated. Iμ, μ I exon; Sμ, μ switch region; CH1μ, μ constant exon 1; dss, donor splice site. (B-E) Splenic B cells were isolated from C57BL/6 mice, stimulated with LPS + IL4 and treated with 2 μM Iμ dss ASO or an irrelevant control ASO for 2 days (B, E) or 3 days (C, D). (B) Constitutively and alternatively spliced μ transcripts analysed as described in figure 2 using Iμ-for and Cμ-rev primers, described in schema A. (C) Quantification of IgG1 in culture supernatants by ELISA. (D) Percentage of IgG1 positive cells determined by flow cytometry as described in figure 2 . (E) Unspliced γ1 GLTs expression monitored as described in figure 2 . (C-E) Data are means ± SEM, n=3 for each group. Unpaired two-tailed Student’s t test was used to determine significance. ns: non significant, * P

    Techniques Used: Allele-specific Oligonucleotide, Sequencing, Isolation, Mouse Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Two Tailed Test

    Specific IgG1 class switching inhibition in B cells treated by Iγ1 exon dss ASO Splenic B cells were isolated from C57BL/6 mice, stimulated with anti-CD40 + IL4 and treated with 2 μM Iγ1 dss ASO (ASO) or an irrelevant ASO (control) during 2 days (B, E) or 4 days (C, D, F, G). (A) Schematic representation of unspliced ε GLT. Iε, ε I exon; Sε, ε switch region; CH1ε, ε constant exon 1; dss, donor splice site. (B, E) Unspliced γ1 (B) and ε (E) GLTs expression monitored as described in figure 2 . Iε-for-Q and SεU-rev-Q primers, described in schema A, were used for ε GLTs expression determination. (C, F) Post-switch Iμ-Cγ1 and Iμ-Cε mRNA expression monitored as described in figure 2 . (D, G) Quantification of IgG1 and IgE in culture supernatants by ELISA. (B-G) Data are means ± SEM of two independent experiments, n=3 to 4 for each group. Unpaired two-tailed Student’s t test was used to determine significance. ns: non significant, ** P
    Figure Legend Snippet: Specific IgG1 class switching inhibition in B cells treated by Iγ1 exon dss ASO Splenic B cells were isolated from C57BL/6 mice, stimulated with anti-CD40 + IL4 and treated with 2 μM Iγ1 dss ASO (ASO) or an irrelevant ASO (control) during 2 days (B, E) or 4 days (C, D, F, G). (A) Schematic representation of unspliced ε GLT. Iε, ε I exon; Sε, ε switch region; CH1ε, ε constant exon 1; dss, donor splice site. (B, E) Unspliced γ1 (B) and ε (E) GLTs expression monitored as described in figure 2 . Iε-for-Q and SεU-rev-Q primers, described in schema A, were used for ε GLTs expression determination. (C, F) Post-switch Iμ-Cγ1 and Iμ-Cε mRNA expression monitored as described in figure 2 . (D, G) Quantification of IgG1 and IgE in culture supernatants by ELISA. (B-G) Data are means ± SEM of two independent experiments, n=3 to 4 for each group. Unpaired two-tailed Student’s t test was used to determine significance. ns: non significant, ** P

    Techniques Used: Inhibition, Allele-specific Oligonucleotide, Isolation, Mouse Assay, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    Treatment with Iγ1 exon dss ASO inhibited γ1 GLTs constitutive splicing and IgG1 class switching in B cells (A) Antisense oligonucleotide targeting the donor splice site of Iγ1 exon (Iγ1 dss ASO) was designed and synthetized as “vivo-morpholino ASO” permitting passive administration of ASO in cells (Gene Tools, LLC). Targeted γ1 GLT (uppercase: exon sequence; lowercase: intron sequence) and Iγ1 dss ASO sequences are indicated. Iγ1, γ1 I exon; Sγ1, γ1 switch region; CH1γ1, γ1 constant exon 1; dss, donor splice site. (B-G) Splenic B cells were isolated from C57BL/6 mice, stimulated with LPS + IL4 and treated with 2 μM Iγ1 dss ASO (ASO) or an irrelevant ASO (control) during 2 days (B-C) or 3 days (D-G). (B) Unspliced γ1 GLTs expression relative to GAPDH mRNA expression was monitored by quantitative RT-PCR using Iγ1-for-Q and Sγ1U-rev-Q primers described in schema A. Expression of γ1 GLTs in control B cells was normalized to 1. (C-up) RT-PCR was performed using Iγ1-for and Cγ1-rev primers (position described in schema C-middle) to identify constitutively and alternatively spliced transcripts. PCR products were analysed on agarose gels. Expression of actin mRNA is also shown. Molecular markers in base pairs are indicated. Schematic representation of the different γ1 spliced transcripts is indicated on the right and transcript sequences are given in Supplementary figure 3. One experiment out of three is shown. Quantification of amplification products was done using an Agilent Bioanalyzer. Data are expressed as percentage of each isoforms among the detected transcripts. ND: not detected. (C-middle) Schematic representation of γ1 spliced transcripts detected in B cells from C57BL/6 mice after treatment with an irrelevant ASO (control) or Iγ1 dss ASO (ASO). Grey hatched lines represent splicing events involving constitutive and alternative splice sites. Donor and acceptor splice sites are indicated in red and green respectively. (C-down) Consensus value (ranging from 0 to 100) of each predicted splice site determined using HSF 3.1 tool. (D) Flow cytometry analysis of purified B cell populations using the indicated cell surface markers. Plots are gated on live cells. The percentage of B220 + IgG1 + cells is indicated. One experiment out of five is shown. (E) Percentage of IgG1 positive cells determined by flow cytometry. (F) Quantification of IgG1 in culture supernatants by ELISA. (G) Post-switch Iμ-Cγ1 mRNA expression relative to GAPDH expression was monitored by quantitative RT-PCR. Expression of post-switch mRNAs in control B cells was normalized to 1. Sequence of primers used for RT-PCR and quantitative RT-PCR are indicated in Supplementary table 1. (B and E-G) Data are means ± SEM of two independent experiments, n=5 to 8 for each group. Unpaired two-tailed Student’s t test was used to determine significance. *** P
    Figure Legend Snippet: Treatment with Iγ1 exon dss ASO inhibited γ1 GLTs constitutive splicing and IgG1 class switching in B cells (A) Antisense oligonucleotide targeting the donor splice site of Iγ1 exon (Iγ1 dss ASO) was designed and synthetized as “vivo-morpholino ASO” permitting passive administration of ASO in cells (Gene Tools, LLC). Targeted γ1 GLT (uppercase: exon sequence; lowercase: intron sequence) and Iγ1 dss ASO sequences are indicated. Iγ1, γ1 I exon; Sγ1, γ1 switch region; CH1γ1, γ1 constant exon 1; dss, donor splice site. (B-G) Splenic B cells were isolated from C57BL/6 mice, stimulated with LPS + IL4 and treated with 2 μM Iγ1 dss ASO (ASO) or an irrelevant ASO (control) during 2 days (B-C) or 3 days (D-G). (B) Unspliced γ1 GLTs expression relative to GAPDH mRNA expression was monitored by quantitative RT-PCR using Iγ1-for-Q and Sγ1U-rev-Q primers described in schema A. Expression of γ1 GLTs in control B cells was normalized to 1. (C-up) RT-PCR was performed using Iγ1-for and Cγ1-rev primers (position described in schema C-middle) to identify constitutively and alternatively spliced transcripts. PCR products were analysed on agarose gels. Expression of actin mRNA is also shown. Molecular markers in base pairs are indicated. Schematic representation of the different γ1 spliced transcripts is indicated on the right and transcript sequences are given in Supplementary figure 3. One experiment out of three is shown. Quantification of amplification products was done using an Agilent Bioanalyzer. Data are expressed as percentage of each isoforms among the detected transcripts. ND: not detected. (C-middle) Schematic representation of γ1 spliced transcripts detected in B cells from C57BL/6 mice after treatment with an irrelevant ASO (control) or Iγ1 dss ASO (ASO). Grey hatched lines represent splicing events involving constitutive and alternative splice sites. Donor and acceptor splice sites are indicated in red and green respectively. (C-down) Consensus value (ranging from 0 to 100) of each predicted splice site determined using HSF 3.1 tool. (D) Flow cytometry analysis of purified B cell populations using the indicated cell surface markers. Plots are gated on live cells. The percentage of B220 + IgG1 + cells is indicated. One experiment out of five is shown. (E) Percentage of IgG1 positive cells determined by flow cytometry. (F) Quantification of IgG1 in culture supernatants by ELISA. (G) Post-switch Iμ-Cγ1 mRNA expression relative to GAPDH expression was monitored by quantitative RT-PCR. Expression of post-switch mRNAs in control B cells was normalized to 1. Sequence of primers used for RT-PCR and quantitative RT-PCR are indicated in Supplementary table 1. (B and E-G) Data are means ± SEM of two independent experiments, n=5 to 8 for each group. Unpaired two-tailed Student’s t test was used to determine significance. *** P

    Techniques Used: Allele-specific Oligonucleotide, Sequencing, Isolation, Mouse Assay, Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Flow Cytometry, Purification, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    3) Product Images from "Modified vaccinia virus Ankara as a vaccine against feline coronavirus: immunogenicity and efficacy"

    Article Title: Modified vaccinia virus Ankara as a vaccine against feline coronavirus: immunogenicity and efficacy

    Journal: Journal of Feline Medicine and Surgery

    doi: 10.1016/j.jfms.2003.12.011

    FIPV-specific IgG response in cats immunized once (MVA-M ×1, at week 0) or twice (MVA-M ×2, at weeks 0 and 3) with 10 8 pfu of MVA-M and challenged with FIPV 79-1146 (black arrow, at week 6). Titers correspond to the mean of each group. Control cats were unvaccinated.
    Figure Legend Snippet: FIPV-specific IgG response in cats immunized once (MVA-M ×1, at week 0) or twice (MVA-M ×2, at weeks 0 and 3) with 10 8 pfu of MVA-M and challenged with FIPV 79-1146 (black arrow, at week 6). Titers correspond to the mean of each group. Control cats were unvaccinated.

    Techniques Used:

    Humoral responses after MVA-M inoculation into naive or MVA-immunized mice. Animals received10 8 pfu of MVA or MVA-M or were non-inoculated. Two weeks later (A) or 4 weeks later (B), they all received 10 8 pfu of MVA-M. Injections were administered subcutaneously. Sera were collected two weeks after the last immunization. IgG1 and IgG2a titers correspond to the mean of the five mice of each group, total IgG titers correspond to the values of pooled sera.
    Figure Legend Snippet: Humoral responses after MVA-M inoculation into naive or MVA-immunized mice. Animals received10 8 pfu of MVA or MVA-M or were non-inoculated. Two weeks later (A) or 4 weeks later (B), they all received 10 8 pfu of MVA-M. Injections were administered subcutaneously. Sera were collected two weeks after the last immunization. IgG1 and IgG2a titers correspond to the mean of the five mice of each group, total IgG titers correspond to the values of pooled sera.

    Techniques Used: Mouse Assay

    4) Product Images from "In vivo and T Cell Cross-Reactivity between Walnut, Cashew and Peanut"

    Article Title: In vivo and T Cell Cross-Reactivity between Walnut, Cashew and Peanut

    Journal: International Archives of Allergy and Immunology

    doi: 10.1159/000155741

    Nut-specific antibodies in the serum of mice following sensitization. Values shown are averages with standard deviation. a IgE in response to cashew, walnut and peanut in the sensitized mice. IgE levels in naïve mice for each nut were subtracted from those in the sensitized mice and are thus set to zero (not shown). b IgG1 in response to cashew, walnut and peanut in the sensitized mice. IgG1 levels in naïve mice were less than 0.3 µg/ml for each nut and are not shown. * p
    Figure Legend Snippet: Nut-specific antibodies in the serum of mice following sensitization. Values shown are averages with standard deviation. a IgE in response to cashew, walnut and peanut in the sensitized mice. IgE levels in naïve mice for each nut were subtracted from those in the sensitized mice and are thus set to zero (not shown). b IgG1 in response to cashew, walnut and peanut in the sensitized mice. IgG1 levels in naïve mice were less than 0.3 µg/ml for each nut and are not shown. * p

    Techniques Used: Mouse Assay, Standard Deviation

    Western blot analysis showing IgG1-binding proteins in the CSH mice and CSH + WN mice. a Coomassie blue-stained SDS-PAGE gel showing the protein profile of peanut (P), cashew (C) and walnut (W) extracts. b Proteins recognized by IgG1 in pooled CSH mice sera for peanut (P), cashew (C) and walnut (W). Inhibition studies were performed with no inhibitor (Ø), walnut or peanut (PN), and demonstrated that the cross-reacting proteins can be inhibited with the appropriate nut extract. The cross-reactive peanut protein was identified as Ara h 1, and the cross-reactive walnut protein was found to be Jug r 2. c Western blot showing the cashew (C) and walnut (W) allergens in the CSH + WN mice. Notice that additional bands are recognized in walnut compared to the CSH mice.
    Figure Legend Snippet: Western blot analysis showing IgG1-binding proteins in the CSH mice and CSH + WN mice. a Coomassie blue-stained SDS-PAGE gel showing the protein profile of peanut (P), cashew (C) and walnut (W) extracts. b Proteins recognized by IgG1 in pooled CSH mice sera for peanut (P), cashew (C) and walnut (W). Inhibition studies were performed with no inhibitor (Ø), walnut or peanut (PN), and demonstrated that the cross-reacting proteins can be inhibited with the appropriate nut extract. The cross-reactive peanut protein was identified as Ara h 1, and the cross-reactive walnut protein was found to be Jug r 2. c Western blot showing the cashew (C) and walnut (W) allergens in the CSH + WN mice. Notice that additional bands are recognized in walnut compared to the CSH mice.

    Techniques Used: Western Blot, Binding Assay, Cell Surface Hydrophobicity, Mouse Assay, Staining, SDS Page, Inhibition, Acetylene Reduction Assay

    5) Product Images from "Synthesis and Evaluation of QS-7-Based Vaccine Adjuvants"

    Article Title: Synthesis and Evaluation of QS-7-Based Vaccine Adjuvants

    Journal: ACS infectious diseases

    doi: 10.1021/acsinfecdis.9b00039

    Serum IgG anti-Dnak activity at weeks 2, 4, and 6. Data are expressed as the mean with the range. Statistical significance compared to no-adjuvant control at * P
    Figure Legend Snippet: Serum IgG anti-Dnak activity at weeks 2, 4, and 6. Data are expressed as the mean with the range. Statistical significance compared to no-adjuvant control at * P

    Techniques Used: Activity Assay

    Serum IgG anti-rHagB activity at weeks 2, 4, and 6. Data are expressed as the mean with the range. Statistical significance compared to no-adjuvant control group at * P
    Figure Legend Snippet: Serum IgG anti-rHagB activity at weeks 2, 4, and 6. Data are expressed as the mean with the range. Statistical significance compared to no-adjuvant control group at * P

    Techniques Used: Activity Assay

    6) Product Images from "From the Cover: PNAS Plus: MZB1 promotes the secretion of J-chain–containing dimeric IgA and is critical for the suppression of gut inflammation"

    Article Title: From the Cover: PNAS Plus: MZB1 promotes the secretion of J-chain–containing dimeric IgA and is critical for the suppression of gut inflammation

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

    doi: 10.1073/pnas.1904204116

    MZB1 is required for efficient production of IgM and IgA, but not IgG 1 , in vivo. ( A , Left ) Five pairs of Mzb1 +/+ and Mzb1 −/− mice were immunized with 25 μg of NP-LPS and analyzed for the production of NP-specific IgM in the serum at 1–4 wk after immunization. ( A , Right ) ELISPOT assay for IgM AFC in the spleen at 1 wk postimmunization. ( B and C ) Eight pairs of Mzb1 +/+ and Mzb1 −/− mice were immunized with 25 μg of NP-CGG in alum at week 0 and boosted with the same amount of NP-CGG in PBS 9 wk later. Serum levels of NP-specific IgM ( B ) and low-affinity ( C , Upper ) and high-affinity ( C , Lower ) NP-specific IgG 1 were measured at the indicated time points by ELISA. ( D , Upper ) FACS profiles showing GC B cells (B220 + CD38 − FAS + ) in the spleen at 12 d after immunization with 25 μg of NP-CGG in alum. ( D , Lower ) Mean ± SD of six pairs of Mzb1 +/+ and Mzb1 −/− mice. ( E ) Mzb1 −/− mice are impaired in secreting IgA into the gut in response to acute inflammation. Four pairs of Mzb1 +/+ and Mzb1 −/− ( E , Left ) or five Mzb1 +/+ and six Mzb1 −/− mice ( E , Right ) were injected i.p. with 20 μg of LPS (arrowheads), and the fecal IgA was analyzed at the indicated time points by ELISA. Means ± SD are shown. ns, not significant. * P
    Figure Legend Snippet: MZB1 is required for efficient production of IgM and IgA, but not IgG 1 , in vivo. ( A , Left ) Five pairs of Mzb1 +/+ and Mzb1 −/− mice were immunized with 25 μg of NP-LPS and analyzed for the production of NP-specific IgM in the serum at 1–4 wk after immunization. ( A , Right ) ELISPOT assay for IgM AFC in the spleen at 1 wk postimmunization. ( B and C ) Eight pairs of Mzb1 +/+ and Mzb1 −/− mice were immunized with 25 μg of NP-CGG in alum at week 0 and boosted with the same amount of NP-CGG in PBS 9 wk later. Serum levels of NP-specific IgM ( B ) and low-affinity ( C , Upper ) and high-affinity ( C , Lower ) NP-specific IgG 1 were measured at the indicated time points by ELISA. ( D , Upper ) FACS profiles showing GC B cells (B220 + CD38 − FAS + ) in the spleen at 12 d after immunization with 25 μg of NP-CGG in alum. ( D , Lower ) Mean ± SD of six pairs of Mzb1 +/+ and Mzb1 −/− mice. ( E ) Mzb1 −/− mice are impaired in secreting IgA into the gut in response to acute inflammation. Four pairs of Mzb1 +/+ and Mzb1 −/− ( E , Left ) or five Mzb1 +/+ and six Mzb1 −/− mice ( E , Right ) were injected i.p. with 20 μg of LPS (arrowheads), and the fecal IgA was analyzed at the indicated time points by ELISA. Means ± SD are shown. ns, not significant. * P

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

    MZB1 is required for efficient secretion of IgA and IgM, but not IgG, in vitro. ( A ) Reduced serum IgA and IgM levels in MZB1-deficient mice. Twenty pairs of age-matched Mzb1 +/+ and Mzb1 −/− mice were bled, and serum Ig levels were measured by ELISA. ( B and C ) MZB1 deficiency does not affect CSR to IgA or IgG 1 . Purified spleen B cells were cultured for 72 h with CD40 ligand–CD8a (1/2 volume), 2 μg/mL α-hCD8a, 10 ng/mL IL-4, 3 ng/mL dextran-conjugated α-IgD, and 2 ng/mL TGFβ (CIDT) to induce CSR to IgA, or CD40 ligand + IL-4 (CI) to induce CSR to IgG 1 , and analyzed by flow cytometry. Fresh purified spleen B cells were used as a control (Ctrl). SSC, side scatter. ( B ) CSR to IgA. ( C ) CSR to IgG 1 . Representative results of three independent experiments are shown. Means ± SD of three experiments are indicated in B and C , Right . ( D – H ) MZB1 promotes the secretion of IgA and IgM, but not IgG. Spleen B cells were purified and cultured for 4 d with LPS ( D – H , Top ; for production of IgM), CIDT ( D – H , Middle ; for production of IgA), and CI ( D – H , Bottom ; for production of IgG 1 ). ( D ) Representative profiles showing B220 low CD138 + plasma cells. ( E ) Summary of three independent experiments shown in D . ( F ) ELISPOT assay for AFC ( Top , IgM-AFC; Middle , IgA-AFC; Bottom , IgG 1 -AFC). ( G ) The amount of Ab in the culture supernatants was analyzed by ELISA ( Top , IgM; Middle , IgA; Bottom , IgG 1 ). ( H ) The average amount of Ab secreted by each AFC, calculated based on the results of F and G . Means ± SD of three experiments are shown. ns, not significant. * P
    Figure Legend Snippet: MZB1 is required for efficient secretion of IgA and IgM, but not IgG, in vitro. ( A ) Reduced serum IgA and IgM levels in MZB1-deficient mice. Twenty pairs of age-matched Mzb1 +/+ and Mzb1 −/− mice were bled, and serum Ig levels were measured by ELISA. ( B and C ) MZB1 deficiency does not affect CSR to IgA or IgG 1 . Purified spleen B cells were cultured for 72 h with CD40 ligand–CD8a (1/2 volume), 2 μg/mL α-hCD8a, 10 ng/mL IL-4, 3 ng/mL dextran-conjugated α-IgD, and 2 ng/mL TGFβ (CIDT) to induce CSR to IgA, or CD40 ligand + IL-4 (CI) to induce CSR to IgG 1 , and analyzed by flow cytometry. Fresh purified spleen B cells were used as a control (Ctrl). SSC, side scatter. ( B ) CSR to IgA. ( C ) CSR to IgG 1 . Representative results of three independent experiments are shown. Means ± SD of three experiments are indicated in B and C , Right . ( D – H ) MZB1 promotes the secretion of IgA and IgM, but not IgG. Spleen B cells were purified and cultured for 4 d with LPS ( D – H , Top ; for production of IgM), CIDT ( D – H , Middle ; for production of IgA), and CI ( D – H , Bottom ; for production of IgG 1 ). ( D ) Representative profiles showing B220 low CD138 + plasma cells. ( E ) Summary of three independent experiments shown in D . ( F ) ELISPOT assay for AFC ( Top , IgM-AFC; Middle , IgA-AFC; Bottom , IgG 1 -AFC). ( G ) The amount of Ab in the culture supernatants was analyzed by ELISA ( Top , IgM; Middle , IgA; Bottom , IgG 1 ). ( H ) The average amount of Ab secreted by each AFC, calculated based on the results of F and G . Means ± SD of three experiments are shown. ns, not significant. * P

    Techniques Used: In Vitro, Mouse Assay, Enzyme-linked Immunosorbent Assay, Purification, Cell Culture, Flow Cytometry, Cytometry, Enzyme-linked Immunospot

    MZB1 binds to IgA via the αtp dependent on the penultimate Cys (C482). ( A and B ) Generation of MZB1-deficient Ag8 cells. ( A ) Positions and sequences of two guide RNAs. ( B ) Immunoblot for MZB1 expression in MZB1-deficient Ag8 clones, #5 and #9, derived from #5 and #9 gRNA, respectively. ( C and D ) Ag8, #5, and #9 cells were transduced with retrovirus expressing α+λ 1 or γ 1 +λ 1 , and the levels of IgA ( C ) or IgG 1 ( D ) in the culture supernatants were measured at the indicated time points by ELISA. ( E and F ) Reexpression of MZB1 in MZB1-deficient #9(α+λ 1 ) cells restores normal IgA secretion. The #9(α+λ 1 ). Ag8(α+λ 1 ) cells were used as a control. ( E ) Immunoblot analysis for MZB1, GFP, αHC, λ 1 LC, and β-actin protein expression. ( F ) The levels of IgA in the culture supernatants. ( G – I ) MZB1 interacts with IgA via αtp dependent on the penultimate Cys (C482). ( G ) MZB1 physically interacted with IgA. Ag8(α+λ 1 ) cells were lysed and immunoprecipitated with α-IgA Ab. Normal goat IgG was used as a negative control (Ctrl), and whole-cell lysate was used as input. Immunoblot was probed with α-IgA, -MZB1, -BiP, or -GRP94 Ab. ( H ) MZB1 did not interact with IgG 1 . Ag8 and Ag8(γ 1 +λ 1 ) cells were lysed, and IgG 1 was immunoprecipitated. Whole-cell lysate was used as input. Immunoblot was performed as in G . ( I ) MZB1 did not interact with IgA lacking the αtp (Δαtp) or IgA with mutated αtp (penultimate Cys mutated to Ser, C482S). Ag8 cells expressing Δαtp or C482S were lysed and immunoprecipitated with α-IgA Ab. Immunoblot was performed as in G . Means ± SD are shown. ** P
    Figure Legend Snippet: MZB1 binds to IgA via the αtp dependent on the penultimate Cys (C482). ( A and B ) Generation of MZB1-deficient Ag8 cells. ( A ) Positions and sequences of two guide RNAs. ( B ) Immunoblot for MZB1 expression in MZB1-deficient Ag8 clones, #5 and #9, derived from #5 and #9 gRNA, respectively. ( C and D ) Ag8, #5, and #9 cells were transduced with retrovirus expressing α+λ 1 or γ 1 +λ 1 , and the levels of IgA ( C ) or IgG 1 ( D ) in the culture supernatants were measured at the indicated time points by ELISA. ( E and F ) Reexpression of MZB1 in MZB1-deficient #9(α+λ 1 ) cells restores normal IgA secretion. The #9(α+λ 1 ). Ag8(α+λ 1 ) cells were used as a control. ( E ) Immunoblot analysis for MZB1, GFP, αHC, λ 1 LC, and β-actin protein expression. ( F ) The levels of IgA in the culture supernatants. ( G – I ) MZB1 interacts with IgA via αtp dependent on the penultimate Cys (C482). ( G ) MZB1 physically interacted with IgA. Ag8(α+λ 1 ) cells were lysed and immunoprecipitated with α-IgA Ab. Normal goat IgG was used as a negative control (Ctrl), and whole-cell lysate was used as input. Immunoblot was probed with α-IgA, -MZB1, -BiP, or -GRP94 Ab. ( H ) MZB1 did not interact with IgG 1 . Ag8 and Ag8(γ 1 +λ 1 ) cells were lysed, and IgG 1 was immunoprecipitated. Whole-cell lysate was used as input. Immunoblot was performed as in G . ( I ) MZB1 did not interact with IgA lacking the αtp (Δαtp) or IgA with mutated αtp (penultimate Cys mutated to Ser, C482S). Ag8 cells expressing Δαtp or C482S were lysed and immunoprecipitated with α-IgA Ab. Immunoblot was performed as in G . Means ± SD are shown. ** P

    Techniques Used: Expressing, Clone Assay, Derivative Assay, Transduction, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Negative Control

    7) Product Images from "Rapid Reconstitution of Antibody Responses Following Transplantation of Purified Allogeneic Hematopoietic Stem Cells"

    Article Title: Rapid Reconstitution of Antibody Responses Following Transplantation of Purified Allogeneic Hematopoietic Stem Cells

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

    doi: 10.4049/jimmunol.1003674

    Donor- and host-derived TNP-OVA specific Abs 6 wk post-HSCT. Allotype-specific Abs were used to detect the origin, donor or host, of IgG 2a/c . Donor-derived IgG 2a/c were completely absent from fully MHC-disparate full and mixed chimeras ( A ). All IgG 2a/c
    Figure Legend Snippet: Donor- and host-derived TNP-OVA specific Abs 6 wk post-HSCT. Allotype-specific Abs were used to detect the origin, donor or host, of IgG 2a/c . Donor-derived IgG 2a/c were completely absent from fully MHC-disparate full and mixed chimeras ( A ). All IgG 2a/c

    Techniques Used: Derivative Assay

    Donor- and host-derived TNP-OVA specific Abs in fully MHC-disparate chimeras at 15 wk posttransplant. Allotype-specific Abs were used to detect the origin, donor or host, of TNP-OVA specific IgG 2a/c . Donor-derived ( A ) but not host-derived ( B ) IgG 2a/c
    Figure Legend Snippet: Donor- and host-derived TNP-OVA specific Abs in fully MHC-disparate chimeras at 15 wk posttransplant. Allotype-specific Abs were used to detect the origin, donor or host, of TNP-OVA specific IgG 2a/c . Donor-derived ( A ) but not host-derived ( B ) IgG 2a/c

    Techniques Used: Derivative Assay

    8) Product Images from "Combined proinflammatory cytokine and cognate activation of invariant natural killer T cells enhances anti-DNA antibody responses"

    Article Title: Combined proinflammatory cytokine and cognate activation of invariant natural killer T cells enhances anti-DNA antibody responses

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

    doi: 10.1073/pnas.1920463117

    αGalCer promotes germinal center formation during chronic inflammation. ( A and B ) Percent of ( A ) plasma cells (CD138+, B220−) and ( B ) germinal center B cells (IgD−, B220+, GL-7+, CD95+) in the spleen 12 d after in vivo stimulation with the indicated treatments. ( C ) Immunofluorescence microscopy of the spleen (day 12; mice are treated as indicated) stained with B220 (blue), GL-7 (green), and CD138 (red). (Scale bars, 100 μm.) ( D ) Representative plots of intracellular IgE and IgG1 staining of splenic plasma cells on day 12. ( E and F ) Percent of ( E ) IgE+ and ( F ) IgG1+ CD138+ B220− plasma cells in the spleen of mice on day 12 of treatment as identified in the key. Representative plots are from two to four experiments, n = 3 to 5 mice per group. One-way ANOVA was employed to assess statistical differences. * P
    Figure Legend Snippet: αGalCer promotes germinal center formation during chronic inflammation. ( A and B ) Percent of ( A ) plasma cells (CD138+, B220−) and ( B ) germinal center B cells (IgD−, B220+, GL-7+, CD95+) in the spleen 12 d after in vivo stimulation with the indicated treatments. ( C ) Immunofluorescence microscopy of the spleen (day 12; mice are treated as indicated) stained with B220 (blue), GL-7 (green), and CD138 (red). (Scale bars, 100 μm.) ( D ) Representative plots of intracellular IgE and IgG1 staining of splenic plasma cells on day 12. ( E and F ) Percent of ( E ) IgE+ and ( F ) IgG1+ CD138+ B220− plasma cells in the spleen of mice on day 12 of treatment as identified in the key. Representative plots are from two to four experiments, n = 3 to 5 mice per group. One-way ANOVA was employed to assess statistical differences. * P

    Techniques Used: In Vivo, Immunofluorescence, Microscopy, Mouse Assay, Staining

    αGalCer-mediated i NKT cell activation enhances self-reactive innate B cell responses in chronic inflammation. ( A ) Experimental scheme. Mice were immunized i.p. with 5 μg α-galactosylceramide (αGC) on day 1 and i.p. 2 μg IL-18 for days 1 to 10. To follow NP-specific B cell responses, 0.5 μg of NP-αGC was injected intravenously. ( B ) ELISA was used to measure total Ig concentrations from serum at the indicated time points (the statistical difference between IL-18 and IL-18 + αGalCer is shown). ( C ) OD of anti-DNA IgE, IgM, and IgG on day 12. ( D ) Anti-DNA IgG1, IgG2b, and IgG3 and the ratio of IgG2b vs. IgG1 on day 12. Two-way ( B ) or one-way ANOVA ( C and D ) was employed to assess statistical differences. * P
    Figure Legend Snippet: αGalCer-mediated i NKT cell activation enhances self-reactive innate B cell responses in chronic inflammation. ( A ) Experimental scheme. Mice were immunized i.p. with 5 μg α-galactosylceramide (αGC) on day 1 and i.p. 2 μg IL-18 for days 1 to 10. To follow NP-specific B cell responses, 0.5 μg of NP-αGC was injected intravenously. ( B ) ELISA was used to measure total Ig concentrations from serum at the indicated time points (the statistical difference between IL-18 and IL-18 + αGalCer is shown). ( C ) OD of anti-DNA IgE, IgM, and IgG on day 12. ( D ) Anti-DNA IgG1, IgG2b, and IgG3 and the ratio of IgG2b vs. IgG1 on day 12. Two-way ( B ) or one-way ANOVA ( C and D ) was employed to assess statistical differences. * P

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

    Cognate activation of i NKT cells during chronic inflammation provides B cell help and IL-18 + αGalCer exacerbates collagen-induced arthritis. ( A and B ) Percent of ( A ) GFP+ plasma cells and ( B ) hCD2+ germinal center B cells from spleens of Aicda Cre GT Rosa Flox mice treated as indicated. Data are representative of three experiments ( n = 3 mice per group). ( C ) OD (450 nm) of NP-reactive IgM, IgG, IgG1, and IgG3 measured by ELISA. Sera were diluted as shown on the x axis. Data are representative of two experiments ( n = 4 or 5 mice per group). ( D ) Percent of intracellular, NP24-reactive IgG3+ cells among plasma cells (CD138+, B220−). ( E ) Representative flow cytometry plots for intracellular staining. ( F ) Representative immunofluorescence microscopy of the spleen (day 12) showing B220 (blue), hCD2 (green), and IgG3 (red) antibody-forming cells. (Scale bars, 100 μm.) Data are representative of two experiments ( n = 4 or 5 mice per group). One-way ( A , B , D ) or two-way ( C ) ANOVA, Student's t test ( H ). *P
    Figure Legend Snippet: Cognate activation of i NKT cells during chronic inflammation provides B cell help and IL-18 + αGalCer exacerbates collagen-induced arthritis. ( A and B ) Percent of ( A ) GFP+ plasma cells and ( B ) hCD2+ germinal center B cells from spleens of Aicda Cre GT Rosa Flox mice treated as indicated. Data are representative of three experiments ( n = 3 mice per group). ( C ) OD (450 nm) of NP-reactive IgM, IgG, IgG1, and IgG3 measured by ELISA. Sera were diluted as shown on the x axis. Data are representative of two experiments ( n = 4 or 5 mice per group). ( D ) Percent of intracellular, NP24-reactive IgG3+ cells among plasma cells (CD138+, B220−). ( E ) Representative flow cytometry plots for intracellular staining. ( F ) Representative immunofluorescence microscopy of the spleen (day 12) showing B220 (blue), hCD2 (green), and IgG3 (red) antibody-forming cells. (Scale bars, 100 μm.) Data are representative of two experiments ( n = 4 or 5 mice per group). One-way ( A , B , D ) or two-way ( C ) ANOVA, Student's t test ( H ). *P

    Techniques Used: Activation Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining, Immunofluorescence, Microscopy

    9) Product Images from "Extended delivery of vaccines to the skin improves immune responses"

    Article Title: Extended delivery of vaccines to the skin improves immune responses

    Journal: Journal of controlled release : official journal of the Controlled Release Society

    doi: 10.1016/j.jconrel.2019.05.006

    Applicability of extended delivery to multiple vaccines. Antibody responses to extended-delivery vaccination were improved relative to bolus vaccination for tetanus toxoid and subunit influenza vaccines, but not for live-attenuated measles vaccine. (a) Anti-tetanus toxoid IgG determined 42 days after initial vaccination using 5 Lf tetanus toxoid vaccine in Balb/c mice (**** p
    Figure Legend Snippet: Applicability of extended delivery to multiple vaccines. Antibody responses to extended-delivery vaccination were improved relative to bolus vaccination for tetanus toxoid and subunit influenza vaccines, but not for live-attenuated measles vaccine. (a) Anti-tetanus toxoid IgG determined 42 days after initial vaccination using 5 Lf tetanus toxoid vaccine in Balb/c mice (**** p

    Techniques Used: Mouse Assay

    10) Product Images from "A forward genetic screen reveals roles for Nfkbid, Zeb1, and Ruvbl2 in humoral immunity"

    Article Title: A forward genetic screen reveals roles for Nfkbid, Zeb1, and Ruvbl2 in humoral immunity

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

    doi: 10.1073/pnas.1209134109

    The primary T-dependent antibody response is impaired in bumble , cellophane , and Worker mice. WT and mutant mice were immunized with alum-precipitated NP-CGG. Serum levels of NP-specific IgM ( A ), total NP-specific IgG1 ( B ), or high-affinity NP-specific
    Figure Legend Snippet: The primary T-dependent antibody response is impaired in bumble , cellophane , and Worker mice. WT and mutant mice were immunized with alum-precipitated NP-CGG. Serum levels of NP-specific IgM ( A ), total NP-specific IgG1 ( B ), or high-affinity NP-specific

    Techniques Used: Mouse Assay, Mutagenesis

    Recovery of the bumble , cellophane , and Worker phenovariants by forward genetic screening. Serum NP-specific IgM ( A , C , and E ) and βGal-specific IgG ( B , D , and F ) measured by ELISA in WT and G3 mice immunized 5 or 14 d before with NP-Ficoll and
    Figure Legend Snippet: Recovery of the bumble , cellophane , and Worker phenovariants by forward genetic screening. Serum NP-specific IgM ( A , C , and E ) and βGal-specific IgG ( B , D , and F ) measured by ELISA in WT and G3 mice immunized 5 or 14 d before with NP-Ficoll and

    Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay

    The antibody response to rSFV-encoded antigen is dependent on T-cell help but not IFN or TLR signaling or MZ B cells. Serum levels of βGal-specific IgG determined by ELISA 14 d after priming with rSFV-βGal in ( A ) WT mice and mice deficient
    Figure Legend Snippet: The antibody response to rSFV-encoded antigen is dependent on T-cell help but not IFN or TLR signaling or MZ B cells. Serum levels of βGal-specific IgG determined by ELISA 14 d after priming with rSFV-βGal in ( A ) WT mice and mice deficient

    Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay

    11) Product Images from "TRAF3 is required for T cell-mediated immunity and T cell receptor/CD28 signaling 1"

    Article Title: TRAF3 is required for T cell-mediated immunity and T cell receptor/CD28 signaling 1

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

    doi: 10.4049/jimmunol.1000290

    Defective T-dependent IgG1 response in T-TRAF3 −/− mice
    Figure Legend Snippet: Defective T-dependent IgG1 response in T-TRAF3 −/− mice

    Techniques Used: Mouse Assay

    12) Product Images from "Macrophage Foam Cell-Targeting Immunization Attenuates Atherosclerosis"

    Article Title: Macrophage Foam Cell-Targeting Immunization Attenuates Atherosclerosis

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.03127

    FCs immunization results in a specific antibody response in initial atherosclerosis. (A) ELISA was performed to measure the specific antibodies to FCs and macrophages as well as immunoglobulin isotypes including IgG1 and IgG2a titers. Data are shown at plasma dilutions of 1: 800 for each group ( n = 8). OD, optical density. (B) Peritoneal macrophages and FCs were fixed and stained with PBS-immune, Macrophages-immune or FCs-immune plasma (dilution 1:50). Scale bars, 50 μm.
    Figure Legend Snippet: FCs immunization results in a specific antibody response in initial atherosclerosis. (A) ELISA was performed to measure the specific antibodies to FCs and macrophages as well as immunoglobulin isotypes including IgG1 and IgG2a titers. Data are shown at plasma dilutions of 1: 800 for each group ( n = 8). OD, optical density. (B) Peritoneal macrophages and FCs were fixed and stained with PBS-immune, Macrophages-immune or FCs-immune plasma (dilution 1:50). Scale bars, 50 μm.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Staining

    13) Product Images from "Ezh2-mediated epigenetic modification is required for allogeneic T cell-induced lupus disease"

    Article Title: Ezh2-mediated epigenetic modification is required for allogeneic T cell-induced lupus disease

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-020-02225-9

    Dependence of cGVHD-associated autoimmunity on CD4 + T cell Ezh2. bm12 mice (5 mice/group) were injected ip with 1 × 10 7 negatively selected CD4 + T cells from C57BL/6 (WT) or B6.CD4.Ezh2-KO (Ezh2) mice. Mouse sera were collected weekly. Total IgG and autoantibodies against dsDNA and chromatin were analyzed by ELISA [ 16 , 19 ]. Data are representative of four repeats. Error bars show standard deviations. Student’s t test, * p
    Figure Legend Snippet: Dependence of cGVHD-associated autoimmunity on CD4 + T cell Ezh2. bm12 mice (5 mice/group) were injected ip with 1 × 10 7 negatively selected CD4 + T cells from C57BL/6 (WT) or B6.CD4.Ezh2-KO (Ezh2) mice. Mouse sera were collected weekly. Total IgG and autoantibodies against dsDNA and chromatin were analyzed by ELISA [ 16 , 19 ]. Data are representative of four repeats. Error bars show standard deviations. Student’s t test, * p

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

    14) Product Images from "Uncoupling Splicing From Transcription Using Antisense Oligonucleotides Reveals a Dual Role for I Exon Donor Splice Sites in Antibody Class Switching"

    Article Title: Uncoupling Splicing From Transcription Using Antisense Oligonucleotides Reveals a Dual Role for I Exon Donor Splice Sites in Antibody Class Switching

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.00780

    Treatment with Iγ1 exon dss ASO inhibited γ1 GLT constitutive splicing and IgG1 class switching in B cells. (A) Antisense oligonucleotide targeting the donor splice site of Iγ1 exon (Iγ1 dss ASO) was designed and synthetized as “ vivo -morpholino ASO” permitting passive administration of ASO in cells (Gene Tools, LLC). Targeted γ1 GLT (uppercase: exon sequence; lowercase: intron sequence) and Iγ1 dss ASO sequences are indicated. Iγ1, γ1 I exon; Sγ1, γ1 switch region; CH1γ1, γ1 constant exon 1; dss, donor splice site. (B–G) Splenic B cells were isolated from C57BL/6 mice, stimulated with LPS + IL4, and treated with 2 μM Iγ1 dss ASO (ASO) or an irrelevant ASO (control) for 2 days (B,C) or 3 days (D–G) . (B) Unspliced γ1 GLT expression relative to GAPDH mRNA expression was monitored by quantitative RT-PCR using Iγ1-for-Q and Sγ1U-rev-Q primers described in schema A . Expression of γ1 GLTs in control B cells was normalized to 1. ( C , top) RT-PCR was performed using Iγ1-for and Cγ1-rev primers (position described in schema C , middle) to identify constitutively and alternatively spliced transcripts. PCR products were analyzed on agarose gels. Expression of actin mRNA is also shown. Molecular markers in base pairs are indicated. Schematic representation of the different γ1 spliced transcripts is indicated on the right, and transcript sequences are given in Supplementary Figure 3 . One experiment out of three is shown. Relative quantification of amplification products was done using ImageJ software and expressed after normalization to actin band intensity. ( C , middle) Schematic representation of γ1 spliced transcripts detected in B cells from C57BL / 6 mice after treatment with an irrelevant ASO (control) or Iγ1 dss ASO (ASO). Gray hatched lines represent splicing events involving constitutive and alternative splice sites. Donor and acceptor splice sites are indicated in red and green, respectively. ( C , bottom) Consensus value (ranging from 0 to 100) of each predicted splice site determined using the HSF 3.1 tool. (D) Flow cytometry analysis of purified B-cell populations using the indicated cell surface markers. Plots are gated on live cells. The percentage of B220 + IgG1 + cells is indicated. One experiment out of five is shown. (E) Percentage of IgG1-positive cells determined by flow cytometry. (F) Quantification of IgG1 in culture supernatants by ELISA. (G) Post-switch Iμ-Cγ1 mRNA expression relative to GAPDH expression was monitored by quantitative RT-PCR. Expression of post-switch mRNAs in control B cells was normalized to 1. Sequences of primers used for RT-PCR and quantitative RT-PCR are indicated in Supplementary Table 1 . (B,C,E – G) Data are means ± SEM of two independent experiments, n = 3–8 for each group. Unpaired two-tailed Student's t test was used to determine significance. ** P
    Figure Legend Snippet: Treatment with Iγ1 exon dss ASO inhibited γ1 GLT constitutive splicing and IgG1 class switching in B cells. (A) Antisense oligonucleotide targeting the donor splice site of Iγ1 exon (Iγ1 dss ASO) was designed and synthetized as “ vivo -morpholino ASO” permitting passive administration of ASO in cells (Gene Tools, LLC). Targeted γ1 GLT (uppercase: exon sequence; lowercase: intron sequence) and Iγ1 dss ASO sequences are indicated. Iγ1, γ1 I exon; Sγ1, γ1 switch region; CH1γ1, γ1 constant exon 1; dss, donor splice site. (B–G) Splenic B cells were isolated from C57BL/6 mice, stimulated with LPS + IL4, and treated with 2 μM Iγ1 dss ASO (ASO) or an irrelevant ASO (control) for 2 days (B,C) or 3 days (D–G) . (B) Unspliced γ1 GLT expression relative to GAPDH mRNA expression was monitored by quantitative RT-PCR using Iγ1-for-Q and Sγ1U-rev-Q primers described in schema A . Expression of γ1 GLTs in control B cells was normalized to 1. ( C , top) RT-PCR was performed using Iγ1-for and Cγ1-rev primers (position described in schema C , middle) to identify constitutively and alternatively spliced transcripts. PCR products were analyzed on agarose gels. Expression of actin mRNA is also shown. Molecular markers in base pairs are indicated. Schematic representation of the different γ1 spliced transcripts is indicated on the right, and transcript sequences are given in Supplementary Figure 3 . One experiment out of three is shown. Relative quantification of amplification products was done using ImageJ software and expressed after normalization to actin band intensity. ( C , middle) Schematic representation of γ1 spliced transcripts detected in B cells from C57BL / 6 mice after treatment with an irrelevant ASO (control) or Iγ1 dss ASO (ASO). Gray hatched lines represent splicing events involving constitutive and alternative splice sites. Donor and acceptor splice sites are indicated in red and green, respectively. ( C , bottom) Consensus value (ranging from 0 to 100) of each predicted splice site determined using the HSF 3.1 tool. (D) Flow cytometry analysis of purified B-cell populations using the indicated cell surface markers. Plots are gated on live cells. The percentage of B220 + IgG1 + cells is indicated. One experiment out of five is shown. (E) Percentage of IgG1-positive cells determined by flow cytometry. (F) Quantification of IgG1 in culture supernatants by ELISA. (G) Post-switch Iμ-Cγ1 mRNA expression relative to GAPDH expression was monitored by quantitative RT-PCR. Expression of post-switch mRNAs in control B cells was normalized to 1. Sequences of primers used for RT-PCR and quantitative RT-PCR are indicated in Supplementary Table 1 . (B,C,E – G) Data are means ± SEM of two independent experiments, n = 3–8 for each group. Unpaired two-tailed Student's t test was used to determine significance. ** P

    Techniques Used: Allele-specific Oligonucleotide, Sequencing, Isolation, Mouse Assay, Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Software, Flow Cytometry, Purification, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    Specific IgG1 class switching inhibition in B cells treated by Iγ1 exon dss ASO. Splenic B cells were isolated from C57BL / 6 mice, stimulated with anti-CD40 + IL4 and treated with 2 μM Iγ1 dss ASO (ASO) or an irrelevant ASO (control) for 2 days (B,E) or 4 days (C,D,F,G) . (A) Schematic representation of unspliced ε GLT. Iε, ε I exon; Sε, ε switch region; CH1ε, ε constant exon 1; dss, donor splice site. (B,E) Unspliced γ1 (B) and ε (E) GLT expression monitored as described in Figure 2 . Iε-for-Q and SεU-rev-Q primers, described in schema A , were used for ε GLT expression determination. (C,F) Post-switch Iμ-Cγ1 and Iμ-Cε mRNA expressions monitored as described in Figure 2 . (D,G) Quantification of IgG1 and IgE in culture supernatants by ELISA. (B–G) Data are means ± SEM of two independent experiments, n = 3–4 for each group. Unpaired two-tailed Student's t test was used to determine significance. ns: non-significant, ** P
    Figure Legend Snippet: Specific IgG1 class switching inhibition in B cells treated by Iγ1 exon dss ASO. Splenic B cells were isolated from C57BL / 6 mice, stimulated with anti-CD40 + IL4 and treated with 2 μM Iγ1 dss ASO (ASO) or an irrelevant ASO (control) for 2 days (B,E) or 4 days (C,D,F,G) . (A) Schematic representation of unspliced ε GLT. Iε, ε I exon; Sε, ε switch region; CH1ε, ε constant exon 1; dss, donor splice site. (B,E) Unspliced γ1 (B) and ε (E) GLT expression monitored as described in Figure 2 . Iε-for-Q and SεU-rev-Q primers, described in schema A , were used for ε GLT expression determination. (C,F) Post-switch Iμ-Cγ1 and Iμ-Cε mRNA expressions monitored as described in Figure 2 . (D,G) Quantification of IgG1 and IgE in culture supernatants by ELISA. (B–G) Data are means ± SEM of two independent experiments, n = 3–4 for each group. Unpaired two-tailed Student's t test was used to determine significance. ns: non-significant, ** P

    Techniques Used: Inhibition, Allele-specific Oligonucleotide, Isolation, Mouse Assay, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    Impaired antibody class switching in B cells treated by Iμ exon dss ASO. (A) Antisense oligonucleotide targeting the donor splice site of Iμ exon (Iμ dss ASO) was designed and synthetized as “ vivo -morpholino ASO” permitting passive administration of ASO in the cells (Gene Tools, LLC). Targeted μ GLT (uppercase: exon sequence; lowercase: intron sequence) and Iμ dss ASO sequences are indicated. Iμ, μ I exon; Sμ, μ switch region; CH1μ, μ constant exon 1; dss, donor splice site. (B–K) Splenic B cells were isolated from C57BL / 6 mice, stimulated with LPS + IL4 (B–E) or LPS (F–K) and treated with 2 μM (B–E) , 3 μM (F,G,J) or 4 μM (H,I,K) Iμ dss ASO or an irrelevant control ASO for 2 days (B,E) or 3 days (C,D,F–K) . (B) Constitutively and alternatively spliced μ transcripts analyzed as described in Figure 2 using Iμ-for and Cμ-rev primers, described in schema A . One experiment out of three is shown. Relative quantification of amplification products was done using ImageJ software and expressed after normalization to actin band intensity. (C) Quantification of IgG1 in culture supernatants by ELISA. (D) Percentage of IgG1-positive cells determined by flow cytometry as described in Figure 2 . (E) Unspliced γ1 GLT expression monitored as described in Figure 2 . (F,I) Flow cytometry analysis of purified B-cell populations using the indicated cell surface markers. Plots are gated on live cells. The percentage of B220 + IgG3 + (F) or B220 + IgG2b + (I) cells is indicated. One experiment out of three is shown. (G,H,J,K) Percentage of IgG3-positive (G,H) or IgG2b-positive (J,K) cells determined by flow cytometry. (B–K) Data are means ± SEM, n = 3 for each group. Unpaired two-tailed Student's t test was used to determine significance. ns: non-significant, * P
    Figure Legend Snippet: Impaired antibody class switching in B cells treated by Iμ exon dss ASO. (A) Antisense oligonucleotide targeting the donor splice site of Iμ exon (Iμ dss ASO) was designed and synthetized as “ vivo -morpholino ASO” permitting passive administration of ASO in the cells (Gene Tools, LLC). Targeted μ GLT (uppercase: exon sequence; lowercase: intron sequence) and Iμ dss ASO sequences are indicated. Iμ, μ I exon; Sμ, μ switch region; CH1μ, μ constant exon 1; dss, donor splice site. (B–K) Splenic B cells were isolated from C57BL / 6 mice, stimulated with LPS + IL4 (B–E) or LPS (F–K) and treated with 2 μM (B–E) , 3 μM (F,G,J) or 4 μM (H,I,K) Iμ dss ASO or an irrelevant control ASO for 2 days (B,E) or 3 days (C,D,F–K) . (B) Constitutively and alternatively spliced μ transcripts analyzed as described in Figure 2 using Iμ-for and Cμ-rev primers, described in schema A . One experiment out of three is shown. Relative quantification of amplification products was done using ImageJ software and expressed after normalization to actin band intensity. (C) Quantification of IgG1 in culture supernatants by ELISA. (D) Percentage of IgG1-positive cells determined by flow cytometry as described in Figure 2 . (E) Unspliced γ1 GLT expression monitored as described in Figure 2 . (F,I) Flow cytometry analysis of purified B-cell populations using the indicated cell surface markers. Plots are gated on live cells. The percentage of B220 + IgG3 + (F) or B220 + IgG2b + (I) cells is indicated. One experiment out of three is shown. (G,H,J,K) Percentage of IgG3-positive (G,H) or IgG2b-positive (J,K) cells determined by flow cytometry. (B–K) Data are means ± SEM, n = 3 for each group. Unpaired two-tailed Student's t test was used to determine significance. ns: non-significant, * P

    Techniques Used: Allele-specific Oligonucleotide, Sequencing, Isolation, Mouse Assay, Amplification, Software, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Purification, Two Tailed Test

    15) Product Images from "Modulation of Vaccine-Induced HIV-1-Specific Immune Responses by Co-Electroporation of PD-L1 Encoding DNA"

    Article Title: Modulation of Vaccine-Induced HIV-1-Specific Immune Responses by Co-Electroporation of PD-L1 Encoding DNA

    Journal: Vaccines

    doi: 10.3390/vaccines8010027

    Soluble PD-1 and PD-L1 ectodomain co-expression is not inducing an autologous antibody response. 293T cells were transfected with plasmids encoding for Env, Gag, sPD-1, or sPD-L1. Twenty-four hours after transfection, cells were treated with Brefeldin A for 6 h in order to inhibit protein transport. Subsequently, cells were fixed, permeabilized, and incubated with sera from mice immunized with Env- and Gag-DNA either with empty vector (mock) or with corresponding checkpoint ectodomain DNA (sPD-1, sPD-L1). Murine antibodies were detected using an APC-conjugated anti-mouse IgG1 antibody. Shown are the histograms of transfected cells ( A ) as well as the Geometric Mean Fluorescence Intensity of Env- ( B ), Gag- ( C ), sPD-1 ( D ), and sPD-L1 transfected cells ( E ) after incubation with immunized mouse sera and the respective secondary antibody. ( A ) Data are representative of three independent experiments. ( B – E ) Data represent the mean with SEM of one out of three representative experiments with three sera samples from each group.
    Figure Legend Snippet: Soluble PD-1 and PD-L1 ectodomain co-expression is not inducing an autologous antibody response. 293T cells were transfected with plasmids encoding for Env, Gag, sPD-1, or sPD-L1. Twenty-four hours after transfection, cells were treated with Brefeldin A for 6 h in order to inhibit protein transport. Subsequently, cells were fixed, permeabilized, and incubated with sera from mice immunized with Env- and Gag-DNA either with empty vector (mock) or with corresponding checkpoint ectodomain DNA (sPD-1, sPD-L1). Murine antibodies were detected using an APC-conjugated anti-mouse IgG1 antibody. Shown are the histograms of transfected cells ( A ) as well as the Geometric Mean Fluorescence Intensity of Env- ( B ), Gag- ( C ), sPD-1 ( D ), and sPD-L1 transfected cells ( E ) after incubation with immunized mouse sera and the respective secondary antibody. ( A ) Data are representative of three independent experiments. ( B – E ) Data represent the mean with SEM of one out of three representative experiments with three sera samples from each group.

    Techniques Used: Expressing, Transfection, Incubation, Mouse Assay, Plasmid Preparation, Fluorescence

    Checkpoint inhibition by monoclonal antibody administration after virus-like particle (VLP) immunization. ( A ) Six-week old BALB/c mice were intramuscularly immunized with VLPs containing Env and Gag. Two days after immunization, 200 µg of checkpoint inhibitors (CPIs) or isotype control were administered intraperitoneally in three-day intervals over a total time-period of two weeks. Animals received a booster immunization with a follow-up CPI administration that was identical to the priming regimen. Blood was drawn at weeks 3 and 6 and antibody responses analyzed. Env-specific IgG1 ( B ) and IgG2a ( C ) antibody responses and IgG2a:IgG1 ratios ( D ) in the sera of BALB/c mice six weeks after priming. Shown are mean values with standard errors of the means (SEM) ( n = 4 for experimental groups, n = 2 for controls (naïve, PBS)).
    Figure Legend Snippet: Checkpoint inhibition by monoclonal antibody administration after virus-like particle (VLP) immunization. ( A ) Six-week old BALB/c mice were intramuscularly immunized with VLPs containing Env and Gag. Two days after immunization, 200 µg of checkpoint inhibitors (CPIs) or isotype control were administered intraperitoneally in three-day intervals over a total time-period of two weeks. Animals received a booster immunization with a follow-up CPI administration that was identical to the priming regimen. Blood was drawn at weeks 3 and 6 and antibody responses analyzed. Env-specific IgG1 ( B ) and IgG2a ( C ) antibody responses and IgG2a:IgG1 ratios ( D ) in the sera of BALB/c mice six weeks after priming. Shown are mean values with standard errors of the means (SEM) ( n = 4 for experimental groups, n = 2 for controls (naïve, PBS)).

    Techniques Used: Inhibition, Mouse Assay

    Checkpoint inhibition by monoclonal antibodies after DNA electroporation. ( A ) Six-week old BALB/c mice were electroporated intramuscularly with expression plasmids encoding for Env and Gag. Two days after electroporation, 200 µg of CPIs or isotype control were administered via the intraperitoneal route in three-day intervals over a total time-period of two weeks. After two weeks, half of the animals were sacrificed, and T cell responses analyzed. The other animals received a booster immunization with a follow-up CPI administration identical to the priming regimen. Blood was drawn at weeks 3 and 6 and antibody responses analyzed. ( B ) Percentage of CD4+ T cells producing IFNγ after in vitro stimulation with HIV Env T helper peptide (measured by intracellular cytokine staining). Shown are mean values with SEM ( n = 4). Env-specific IgG1 ( C ) and IgG2a ( D ) antibody responses in the sera of BALB/c mice six weeks after priming. Shown are mean values with SEM ( n = 4–6) and significant differences between the groups (one-way ANOVA analyses followed by Tukey’s multiple comparison test; **** p
    Figure Legend Snippet: Checkpoint inhibition by monoclonal antibodies after DNA electroporation. ( A ) Six-week old BALB/c mice were electroporated intramuscularly with expression plasmids encoding for Env and Gag. Two days after electroporation, 200 µg of CPIs or isotype control were administered via the intraperitoneal route in three-day intervals over a total time-period of two weeks. After two weeks, half of the animals were sacrificed, and T cell responses analyzed. The other animals received a booster immunization with a follow-up CPI administration identical to the priming regimen. Blood was drawn at weeks 3 and 6 and antibody responses analyzed. ( B ) Percentage of CD4+ T cells producing IFNγ after in vitro stimulation with HIV Env T helper peptide (measured by intracellular cytokine staining). Shown are mean values with SEM ( n = 4). Env-specific IgG1 ( C ) and IgG2a ( D ) antibody responses in the sera of BALB/c mice six weeks after priming. Shown are mean values with SEM ( n = 4–6) and significant differences between the groups (one-way ANOVA analyses followed by Tukey’s multiple comparison test; **** p

    Techniques Used: Inhibition, Electroporation, Mouse Assay, Expressing, In Vitro, Staining

    16) Product Images from "Intranasal Vaccination with Chitosan-DNA Nanoparticles Expressing Pneumococcal Surface Antigen A Protects Mice against Nasopharyngeal Colonization by Streptococcus pneumoniae ▿"

    Article Title: Intranasal Vaccination with Chitosan-DNA Nanoparticles Expressing Pneumococcal Surface Antigen A Protects Mice against Nasopharyngeal Colonization by Streptococcus pneumoniae ▿

    Journal: Clinical and Vaccine Immunology : CVI

    doi: 10.1128/CVI.00263-10

    ELISA analysis of total anti-PsaA IgG in serum (A), anti-PsaA IgG isotypes in serum (B), and IgA antibody in nasal washes, BALF, and MEL (C). Mice were intranasally immunized with four doses of chitosan- psaA , naked psaA , or chitosan- pVAX1 at 2-week intervals,
    Figure Legend Snippet: ELISA analysis of total anti-PsaA IgG in serum (A), anti-PsaA IgG isotypes in serum (B), and IgA antibody in nasal washes, BALF, and MEL (C). Mice were intranasally immunized with four doses of chitosan- psaA , naked psaA , or chitosan- pVAX1 at 2-week intervals,

    Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay

    17) 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

    18) Product Images from "BRCT-domain protein BRIT1 influences class switch recombination"

    Article Title: BRCT-domain protein BRIT1 influences class switch recombination

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

    doi: 10.1073/pnas.1708211114

    CSR is compromised in BRIT1-deficient splenic B cells. ( A ) Immunoblot analysis of BRIT1 and AID in whole-cell lysates of splenic B-cells from Mb1-Cre control (Ctrl) and BRIT1 KO mice (0 h, naive; 48 h and 96 h, LPS + IL-4 stimulation). ( B ) Representative flow cytometry of CSR to IgG1, IgG3, and IgA in splenic B cells from Mb1-Cre Ctrl and BRIT1 KO mice. ( C ) Quantification of CSR to IgG1, IgG3, and IgA in splenic B cells from Mb1-Cre Ctrl and BRIT1 KO mice. ( n = 7; ** P
    Figure Legend Snippet: CSR is compromised in BRIT1-deficient splenic B cells. ( A ) Immunoblot analysis of BRIT1 and AID in whole-cell lysates of splenic B-cells from Mb1-Cre control (Ctrl) and BRIT1 KO mice (0 h, naive; 48 h and 96 h, LPS + IL-4 stimulation). ( B ) Representative flow cytometry of CSR to IgG1, IgG3, and IgA in splenic B cells from Mb1-Cre Ctrl and BRIT1 KO mice. ( C ) Quantification of CSR to IgG1, IgG3, and IgA in splenic B cells from Mb1-Cre Ctrl and BRIT1 KO mice. ( n = 7; ** P

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry

    Depletion of MDC1 exacerbates CSR defect in BRIT1 KO B cells. ( A ) Strategy to deplete MDC1 in ex vivo stimulated splenic B cells. B cells were harvested from control and BRIT1 KO mice, activated with LPS + IL-4, and retrovirally infected with shRNA and a GFP marker (sh3 and sh4). WB, Western blot. ( B ) Representative flow cytometry of CSR to IgG1 from Mb1-Cre control and BRIT1 KO mice with MDC1 knockdown constructs sh3 and sh4. ( C ) Quantification of CSR to IgG1 ( n = 3; ** P
    Figure Legend Snippet: Depletion of MDC1 exacerbates CSR defect in BRIT1 KO B cells. ( A ) Strategy to deplete MDC1 in ex vivo stimulated splenic B cells. B cells were harvested from control and BRIT1 KO mice, activated with LPS + IL-4, and retrovirally infected with shRNA and a GFP marker (sh3 and sh4). WB, Western blot. ( B ) Representative flow cytometry of CSR to IgG1 from Mb1-Cre control and BRIT1 KO mice with MDC1 knockdown constructs sh3 and sh4. ( C ) Quantification of CSR to IgG1 ( n = 3; ** P

    Techniques Used: Ex Vivo, Mouse Assay, Infection, shRNA, Marker, Western Blot, Flow Cytometry, Cytometry, Construct

    BRIT1 and MDC1 could both contribute to the ATM–γ-H2AX axis of DSB resolution during CSR. ( A ) Representative flow cytometry of CSR to IgG1 in splenic B cells derived from Mb1-Cre control, BRIT1 KO, and AID KO mice treated with ATMi or with DMSO vehicle control. ( B ) Quantification of CSR to IgG1 ( n = 3; ** P
    Figure Legend Snippet: BRIT1 and MDC1 could both contribute to the ATM–γ-H2AX axis of DSB resolution during CSR. ( A ) Representative flow cytometry of CSR to IgG1 in splenic B cells derived from Mb1-Cre control, BRIT1 KO, and AID KO mice treated with ATMi or with DMSO vehicle control. ( B ) Quantification of CSR to IgG1 ( n = 3; ** P

    Techniques Used: Flow Cytometry, Cytometry, Derivative Assay, Mouse Assay

    BRIT1 is dispensable for immune response in vivo. ( A ) Schematic of NP-CGG immunization protocol. GC, germinal center. ( B ) IgM, IgG1, IgG3, and IgA concentrations in serum, determined by ELISA on day 0. Ctrl, control. ( C ) Flow cytometric analysis of GC B cells (B220 + DAPI − GL7 + Fas + ) in control, BRIT1 KO, and AID KO mice following NP-CGG immunization on day 14 and quantification of GC B cells in immunized mice on day 14 ( n = 3). ( D ) Quantification of IgG1 + GC B cells in immunized mice on day 14 ( n = 3). ( E ) Quantification of NP + GC B cells in immunized mice on day 14 ( n = 3). ( F ) Quantification of IgG1 + NP + GC B cells in immunized mice on day 14 ( n = 3). ( G ) IgM and IgG1 concentrations in serum from Ctrl, BRIT1 KO, and AID KO mice following NP-CGG immunization on day 28, determined by ELISA. ( H ) Detection of low-affinity and high-affinity NP-specific IgM and IgG1 serum antibodies in Ctrl ( n = 4), BRIT1 KO ( n = 4), and AID KO ( n = 4) mice following NP-CGG immunization on day 28 by ELISA. Conc., concentration.
    Figure Legend Snippet: BRIT1 is dispensable for immune response in vivo. ( A ) Schematic of NP-CGG immunization protocol. GC, germinal center. ( B ) IgM, IgG1, IgG3, and IgA concentrations in serum, determined by ELISA on day 0. Ctrl, control. ( C ) Flow cytometric analysis of GC B cells (B220 + DAPI − GL7 + Fas + ) in control, BRIT1 KO, and AID KO mice following NP-CGG immunization on day 14 and quantification of GC B cells in immunized mice on day 14 ( n = 3). ( D ) Quantification of IgG1 + GC B cells in immunized mice on day 14 ( n = 3). ( E ) Quantification of NP + GC B cells in immunized mice on day 14 ( n = 3). ( F ) Quantification of IgG1 + NP + GC B cells in immunized mice on day 14 ( n = 3). ( G ) IgM and IgG1 concentrations in serum from Ctrl, BRIT1 KO, and AID KO mice following NP-CGG immunization on day 28, determined by ELISA. ( H ) Detection of low-affinity and high-affinity NP-specific IgM and IgG1 serum antibodies in Ctrl ( n = 4), BRIT1 KO ( n = 4), and AID KO ( n = 4) mice following NP-CGG immunization on day 28 by ELISA. Conc., concentration.

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

    BRCT domains of BRIT1 influence CSR. ( A ) Schematic of BRIT1 protein with N-terminal and C-terminal BRCT domains and various deletions. ΔC, C-terminal BRCT domain deletion; ΔN, N-terminal BRCT domain deletion. ( B ) Schematic of retroviral reconstitution of BRIT1 in ex vivo activated splenic B cells. Splenic B cells were harvested from control and BRIT1 KO mice, activated with LPS + IL-4, and retrovirally reconstituted with BRIT1 variants from pMIG vector, which also expresses GFP as a marker. WB, Western blot. ( C ) Representative flow cytometry of CSR to IgG1 in activated splenic BRIT1 KO B cells retrovirally transduced with vector alone (pMIG), BRIT1-FL, BRIT1-ΔN, or BRIT1-ΔC. The vectors express GFP (numbers in parentheses indicate IgG1 + cell in the GFP + gate). Uninfected activated splenic B cells from Mb1-Cre control and BRIT1 KO mice served as negative controls for GFP gating ( n = 3). ( D ) Quantification of the percentage of CSR to IgG1 in splenic B cells from Mb1-Cre control or BRIT1 KO mice retrovirally reconstituted with BRIT1 variants or left uninfected ( n = 3; ** P
    Figure Legend Snippet: BRCT domains of BRIT1 influence CSR. ( A ) Schematic of BRIT1 protein with N-terminal and C-terminal BRCT domains and various deletions. ΔC, C-terminal BRCT domain deletion; ΔN, N-terminal BRCT domain deletion. ( B ) Schematic of retroviral reconstitution of BRIT1 in ex vivo activated splenic B cells. Splenic B cells were harvested from control and BRIT1 KO mice, activated with LPS + IL-4, and retrovirally reconstituted with BRIT1 variants from pMIG vector, which also expresses GFP as a marker. WB, Western blot. ( C ) Representative flow cytometry of CSR to IgG1 in activated splenic BRIT1 KO B cells retrovirally transduced with vector alone (pMIG), BRIT1-FL, BRIT1-ΔN, or BRIT1-ΔC. The vectors express GFP (numbers in parentheses indicate IgG1 + cell in the GFP + gate). Uninfected activated splenic B cells from Mb1-Cre control and BRIT1 KO mice served as negative controls for GFP gating ( n = 3). ( D ) Quantification of the percentage of CSR to IgG1 in splenic B cells from Mb1-Cre control or BRIT1 KO mice retrovirally reconstituted with BRIT1 variants or left uninfected ( n = 3; ** P

    Techniques Used: Ex Vivo, Mouse Assay, Plasmid Preparation, Marker, Western Blot, Flow Cytometry, Cytometry, Transduction

    19) Product Images from "Whole Pichia pastoris Yeast Expressing Measles Virus Nucleoprotein as a Production and Delivery System to Multimerize Plasmodium Antigens"

    Article Title: Whole Pichia pastoris Yeast Expressing Measles Virus Nucleoprotein as a Production and Delivery System to Multimerize Plasmodium Antigens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0086658

    Humoral responses elicited in mice after immunization. ( A ) Kinetics of anti-PbCS IgG responses in mice immunized with N-PbCS yeasts. OD 450 nm are expressed in log 10 scale. Black arrows indicate immunization schedule. ( B ) Isotyping of humoral IgG responses at day 42 in mice immunized with N-PbCS. The bars correspond to median values per group. Asterisks (*) indicate significant median differences (p
    Figure Legend Snippet: Humoral responses elicited in mice after immunization. ( A ) Kinetics of anti-PbCS IgG responses in mice immunized with N-PbCS yeasts. OD 450 nm are expressed in log 10 scale. Black arrows indicate immunization schedule. ( B ) Isotyping of humoral IgG responses at day 42 in mice immunized with N-PbCS. The bars correspond to median values per group. Asterisks (*) indicate significant median differences (p

    Techniques Used: Mouse Assay

    20) Product Images from "Cosmc controls B cell homing"

    Article Title: Cosmc controls B cell homing

    Journal: Nature Communications

    doi: 10.1038/s41467-020-17765-6

    Sialylation differentially regulates T and B cell trafficking. O-Glycans ( a , b ) were extracted from splenic B cells that were purified from WT ( a ) and BC- Cosmc KO ( b ) mice. The released glycans were subjected to mass spectrometric analysis. The peaks with annotated glycan structures are listed. Other peaks are background noise, not matching to any glycan mass. c IgG2b hinge-region O-glycosylation characterization in WT and BC- Cosmc KO serum. The O-glycan distribution of the IgG2b hinge-region tryptic peptide (K)LEPSGPISTINPCPPCK. Thr104 (UniProt annotation) was identified to be O-glycosylated. Glycan compositions are indicated with H—hexose; N—N-acetylhexosamine; G— N-glycolylneuraminic acid. d – f Single cell suspension of splenocytes from WT mice were labeled with CellTrace Violet or CFSE and treated with neuraminidase. The preparation of the splenocytes was co-injected into recipient mice ( n = 8, except for n = 5 for liver). Lymphoid tissues were harvested at 1 h after transfer. d Representative flow cytometric dot plots show input cells ratio before injection and after transfer. Neuraminidase in orange rectangle, and numbers in orange (percentage of total input) indicated donor cells treated with Neuraminidase. PBS in purple numbers (percentage of total input) indicated donor cells treated with PBS. Blue rectangle and numbers indicated neuraminidase-treated and transferred B cell population that recovered from the indicated tissue of recipients. e , f Homing indices were calculated based on the percentage of homed T and B cells. The homing index was calculated as the [percentage of dye + CD19 + B cells or Thy1.2 + T cells] tissue / [percentage of internal control CFSE + B cells or T cells] tissue ratio to the input ratio. Each symbol (black square and open circle for WT and BC- Cosmc KO, respectively) represents an individual mouse. Data are presented as average ±SD of each genotype with each individual value plotted. Source data are provided as a Source Data file.
    Figure Legend Snippet: Sialylation differentially regulates T and B cell trafficking. O-Glycans ( a , b ) were extracted from splenic B cells that were purified from WT ( a ) and BC- Cosmc KO ( b ) mice. The released glycans were subjected to mass spectrometric analysis. The peaks with annotated glycan structures are listed. Other peaks are background noise, not matching to any glycan mass. c IgG2b hinge-region O-glycosylation characterization in WT and BC- Cosmc KO serum. The O-glycan distribution of the IgG2b hinge-region tryptic peptide (K)LEPSGPISTINPCPPCK. Thr104 (UniProt annotation) was identified to be O-glycosylated. Glycan compositions are indicated with H—hexose; N—N-acetylhexosamine; G— N-glycolylneuraminic acid. d – f Single cell suspension of splenocytes from WT mice were labeled with CellTrace Violet or CFSE and treated with neuraminidase. The preparation of the splenocytes was co-injected into recipient mice ( n = 8, except for n = 5 for liver). Lymphoid tissues were harvested at 1 h after transfer. d Representative flow cytometric dot plots show input cells ratio before injection and after transfer. Neuraminidase in orange rectangle, and numbers in orange (percentage of total input) indicated donor cells treated with Neuraminidase. PBS in purple numbers (percentage of total input) indicated donor cells treated with PBS. Blue rectangle and numbers indicated neuraminidase-treated and transferred B cell population that recovered from the indicated tissue of recipients. e , f Homing indices were calculated based on the percentage of homed T and B cells. The homing index was calculated as the [percentage of dye + CD19 + B cells or Thy1.2 + T cells] tissue / [percentage of internal control CFSE + B cells or T cells] tissue ratio to the input ratio. Each symbol (black square and open circle for WT and BC- Cosmc KO, respectively) represents an individual mouse. Data are presented as average ±SD of each genotype with each individual value plotted. Source data are provided as a Source Data file.

    Techniques Used: Purification, Mouse Assay, Labeling, Injection

    21) Product Images from "A polysaccharide isolated from Agaricus blazei Murill (ABP-AW1) as a potential Th1 immunity-stimulating adjuvant"

    Article Title: A polysaccharide isolated from Agaricus blazei Murill (ABP-AW1) as a potential Th1 immunity-stimulating adjuvant

    Journal: Oncology Letters

    doi: 10.3892/ol.2013.1484

    Effect of ABP-AW1 extracted from Agaricus blazei on ovalbumin (OVA)-specific IgG, IgG1 and IgG2b antibody levels in OVA-immunized mice. Data are plotted as the mean ± SD (n=5) of triplicate wells. Significant differences with from the OVA and OVA/alum groups were designated as * P
    Figure Legend Snippet: Effect of ABP-AW1 extracted from Agaricus blazei on ovalbumin (OVA)-specific IgG, IgG1 and IgG2b antibody levels in OVA-immunized mice. Data are plotted as the mean ± SD (n=5) of triplicate wells. Significant differences with from the OVA and OVA/alum groups were designated as * P

    Techniques Used: Mouse Assay

    22) Product Images from "Chlamydia trachomatis recombinant MOMP encapsulated in PLGA nanoparticles triggers primarily T helper 1 cellular and antibody immune responses in mice: a desirable candidate nanovaccine"

    Article Title: Chlamydia trachomatis recombinant MOMP encapsulated in PLGA nanoparticles triggers primarily T helper 1 cellular and antibody immune responses in mice: a desirable candidate nanovaccine

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S44155

    Production of Th1 and Th2 antibodies in sera from immunized mice. ( A and B ) Groups of PLGA-rMOMP and PLGA-PBS mice were immunized as indicated in the Materials and methods section. Pooled sera were collected two weeks following the last immunization and used to determine IgG, IgG2a, and IgG1 responses in PLGA-rMOMP and PLGA-PBS mice at dilutions of 1:1600 and 1:100, respectively ( A ). Also shown are the reciprocal antibody titers for IgG, IgG2a, and IgG1 in PLGA-rMOMP and PLGA-PBS-immunized mice ( B ). ( C and D ) The groups of mice were immunized following the same immunization regimen as in the Materials and methods section, except rMOMP was administered to mice in FIA. Serum IgG, IgG2a and IgG1 responses ( C ) and reciprocal antibody titers ( D ) in FIA-PBS and FIA-rMOMP-immunized mice. Notes: To determine antibody concentrations (titers), two-fold serial dilutions of serum were made and the endpoint titer was considered to be the last serum dilution with readings higher than the mean + 3 standard deviations of negative controls. Anti-rMOMP-specific antibodies were determined by enzyme-linked immunosorbent assay. Asterisk indicates a significant difference in comparison with the corresponding control ( P
    Figure Legend Snippet: Production of Th1 and Th2 antibodies in sera from immunized mice. ( A and B ) Groups of PLGA-rMOMP and PLGA-PBS mice were immunized as indicated in the Materials and methods section. Pooled sera were collected two weeks following the last immunization and used to determine IgG, IgG2a, and IgG1 responses in PLGA-rMOMP and PLGA-PBS mice at dilutions of 1:1600 and 1:100, respectively ( A ). Also shown are the reciprocal antibody titers for IgG, IgG2a, and IgG1 in PLGA-rMOMP and PLGA-PBS-immunized mice ( B ). ( C and D ) The groups of mice were immunized following the same immunization regimen as in the Materials and methods section, except rMOMP was administered to mice in FIA. Serum IgG, IgG2a and IgG1 responses ( C ) and reciprocal antibody titers ( D ) in FIA-PBS and FIA-rMOMP-immunized mice. Notes: To determine antibody concentrations (titers), two-fold serial dilutions of serum were made and the endpoint titer was considered to be the last serum dilution with readings higher than the mean + 3 standard deviations of negative controls. Anti-rMOMP-specific antibodies were determined by enzyme-linked immunosorbent assay. Asterisk indicates a significant difference in comparison with the corresponding control ( P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    23) Product Images from "Correction of Murine SCID-X1 by Lentiviral Gene Therapy Using a Codon-optimized IL2RG Gene and Minimal Pretransplant Conditioning"

    Article Title: Correction of Murine SCID-X1 by Lentiviral Gene Therapy Using a Codon-optimized IL2RG Gene and Minimal Pretransplant Conditioning

    Journal: Molecular Therapy

    doi: 10.1038/mt.2011.127

    Response of plasma immunoglobulin levels to immunization . ( a ) Plasma immunoglobulin levels quantified for IgM, IgG1 or trinitrophenol keyhole limpet hemocyanin. Il2rg −/− mice were transplanted with wild-type Lin − cells ( n = 3)
    Figure Legend Snippet: Response of plasma immunoglobulin levels to immunization . ( a ) Plasma immunoglobulin levels quantified for IgM, IgG1 or trinitrophenol keyhole limpet hemocyanin. Il2rg −/− mice were transplanted with wild-type Lin − cells ( n = 3)

    Techniques Used: Mouse Assay

    24) Product Images from "Type I IFN Counteracts the Induction of Antigen-Specific Immune Responses by Lipid-Based Delivery of mRNA Vaccines"

    Article Title: Type I IFN Counteracts the Induction of Antigen-Specific Immune Responses by Lipid-Based Delivery of mRNA Vaccines

    Journal: Molecular Therapy

    doi: 10.1038/mt.2012.202

    Prime-boost strategy enhances antigen-specific immune responses. Mice were immunized by 2 s.c. injections of 20 µg of naked or DOTAP gag with a 3-week interval. At week 8, some mice were boosted with 20 µg of the capsid protein p24 either subcutaneously ( a and b ) or intratracheally ( c ). T cell and B cell responses were analyzed 10 days after the p24 protein boost. ( a ) Number of Gag-specific interferon (IFN)-γ and interleukin-2 secreting T cells was determined by enzyme-linked immunosorbent spot (ELISPOT) on isolated spleen and lymph node cells. Means ± SEM are shown of a total of 5 mice per group. ( b ) Anti-p24 immunoglobulin (Ig)M, IgG1, and IgG2c titers were determined by serum enzyme-linked immunosorbent assay. Means ± SEM are shown for 5 mice, immunized with naked gag and 5 with DOTAP gag , each followed by p24 boost. ( c ) Number of Gag-specific IFN-γ secreting CD4 and CD8 T cells was determined by ELISPOT on isolated lung cells. Means ± SEM are shown of a total of 5 mice per group.
    Figure Legend Snippet: Prime-boost strategy enhances antigen-specific immune responses. Mice were immunized by 2 s.c. injections of 20 µg of naked or DOTAP gag with a 3-week interval. At week 8, some mice were boosted with 20 µg of the capsid protein p24 either subcutaneously ( a and b ) or intratracheally ( c ). T cell and B cell responses were analyzed 10 days after the p24 protein boost. ( a ) Number of Gag-specific interferon (IFN)-γ and interleukin-2 secreting T cells was determined by enzyme-linked immunosorbent spot (ELISPOT) on isolated spleen and lymph node cells. Means ± SEM are shown of a total of 5 mice per group. ( b ) Anti-p24 immunoglobulin (Ig)M, IgG1, and IgG2c titers were determined by serum enzyme-linked immunosorbent assay. Means ± SEM are shown for 5 mice, immunized with naked gag and 5 with DOTAP gag , each followed by p24 boost. ( c ) Number of Gag-specific IFN-γ secreting CD4 and CD8 T cells was determined by ELISPOT on isolated lung cells. Means ± SEM are shown of a total of 5 mice per group.

    Techniques Used: Mouse Assay, ELISpot Assay, Enzyme-linked Immunospot, Isolation, Enzyme-linked Immunosorbent Assay

    25) Product Images from "Induction of systemic immunity through nasal-associated lymphoid tissue (NALT) of mice intranasally immunized with Brucella abortus malate dehydrogenase-loaded chitosan nanoparticles"

    Article Title: Induction of systemic immunity through nasal-associated lymphoid tissue (NALT) of mice intranasally immunized with Brucella abortus malate dehydrogenase-loaded chitosan nanoparticles

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0228463

    Antibody measurements at 2 weeks after primary intranasal immunization. (A) Specific IgA antibodies in sera, nasal wash, fecal extract, and genital secretions. Significant production of IgA was detected in the fecal excretions and genital secretions from the CNs-Mdh-immunized group compared to CNs-TF-immunized group. (B) Specific IgG antibodies at 2 wpi. The CNs-Mdh elicited a significantly higher titer of specific IgG than that of the CNs-TF-immunized group which served as the vector control. The main subtype produced after immunization was IgG1, while the titer of IgG2a was not significantly enhanced following immunization. (C) Total IgA antibodies in sera, nasal wash, fecal extract, and genital secretions. Titers of IgA in genital secretions and fecal excretions were significantly increased in CNs-Mdh-immunized group compared with those following the CNs-TF-immunization. In the nasal washes, a high titer of IgA in the CNs-Mdh-immunized group was measured, indicating significance compared to the PBS- and CNs-immunized groups. However, there were no significant differences in sera. Groups were statistically compared using one-way ANOVA with Tukey’s post hoc multiple comparison test. The results for specific antibodies are expressed as the sample to negative control (PBS) ratio (S/N ratio).
    Figure Legend Snippet: Antibody measurements at 2 weeks after primary intranasal immunization. (A) Specific IgA antibodies in sera, nasal wash, fecal extract, and genital secretions. Significant production of IgA was detected in the fecal excretions and genital secretions from the CNs-Mdh-immunized group compared to CNs-TF-immunized group. (B) Specific IgG antibodies at 2 wpi. The CNs-Mdh elicited a significantly higher titer of specific IgG than that of the CNs-TF-immunized group which served as the vector control. The main subtype produced after immunization was IgG1, while the titer of IgG2a was not significantly enhanced following immunization. (C) Total IgA antibodies in sera, nasal wash, fecal extract, and genital secretions. Titers of IgA in genital secretions and fecal excretions were significantly increased in CNs-Mdh-immunized group compared with those following the CNs-TF-immunization. In the nasal washes, a high titer of IgA in the CNs-Mdh-immunized group was measured, indicating significance compared to the PBS- and CNs-immunized groups. However, there were no significant differences in sera. Groups were statistically compared using one-way ANOVA with Tukey’s post hoc multiple comparison test. The results for specific antibodies are expressed as the sample to negative control (PBS) ratio (S/N ratio).

    Techniques Used: Plasmid Preparation, Produced, Negative Control

    26) Product Images from "Genetically Modified Rabies Virus Vector-Based Rift Valley Fever Virus Vaccine is Safe and Induces Efficacious Immune Responses in Mice"

    Article Title: Genetically Modified Rabies Virus Vector-Based Rift Valley Fever Virus Vaccine is Safe and Induces Efficacious Immune Responses in Mice

    Journal: Viruses

    doi: 10.3390/v11100919

    Analysis of the humoral response against RVFV eGn. BALB/c mice were immunized IM in the gastrocnemius muscle with either 10 7 TCID 50 of BPL-inactivated virus supernatant with or without poly (I:C) and ISA 201 VG adjuvants or 10 7 TCID 50 of rSRV9 vector and boosted two times with the same amount on days 14 and 28 ( A ). ELISA analysis of total IgG against RVFV eGn beginning at one week after the first immunization and continuing until two weeks after the last booster immunization ( B ). Serum antibody titres of IgG subtype (IgG1, IgG2a and IgG3) antibodies against RVFV eGn at two weeks after the last booster immunization ( C ). Data are shown as the mean ± SD and were analysed by two-way ANOVA. The ratios of matched IgG1/IgG2a antibody titres are plotted, and one-way ANOVA was applied to check for variance differences ( D ) (* P
    Figure Legend Snippet: Analysis of the humoral response against RVFV eGn. BALB/c mice were immunized IM in the gastrocnemius muscle with either 10 7 TCID 50 of BPL-inactivated virus supernatant with or without poly (I:C) and ISA 201 VG adjuvants or 10 7 TCID 50 of rSRV9 vector and boosted two times with the same amount on days 14 and 28 ( A ). ELISA analysis of total IgG against RVFV eGn beginning at one week after the first immunization and continuing until two weeks after the last booster immunization ( B ). Serum antibody titres of IgG subtype (IgG1, IgG2a and IgG3) antibodies against RVFV eGn at two weeks after the last booster immunization ( C ). Data are shown as the mean ± SD and were analysed by two-way ANOVA. The ratios of matched IgG1/IgG2a antibody titres are plotted, and one-way ANOVA was applied to check for variance differences ( D ) (* P

    Techniques Used: Mouse Assay, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

    27) Product Images from "Dysregulated LIGHT expression on T cells mediates intestinal inflammation and contributes to IgA nephropathy"

    Article Title: Dysregulated LIGHT expression on T cells mediates intestinal inflammation and contributes to IgA nephropathy

    Journal: Journal of Clinical Investigation

    doi: 10.1172/JCI200420096

    Dominant IgA deposition and abnormal UA in Tg mice. (A) Immunofluorescence staining of kidney sections of WT (left) and Tg (right) mice 6–8 months of age. Distinct IgA, C3, IgM, and IgG deposition were observed in aged Tg mice. Representative
    Figure Legend Snippet: Dominant IgA deposition and abnormal UA in Tg mice. (A) Immunofluorescence staining of kidney sections of WT (left) and Tg (right) mice 6–8 months of age. Distinct IgA, C3, IgM, and IgG deposition were observed in aged Tg mice. Representative

    Techniques Used: Mouse Assay, Immunofluorescence, Staining

    28) Product Images from "Deletion of WASp and N-WASp in B cells cripples the germinal center response and results in production of IgM autoantibodies"

    Article Title: Deletion of WASp and N-WASp in B cells cripples the germinal center response and results in production of IgM autoantibodies

    Journal: Journal of autoimmunity

    doi: 10.1016/j.jaut.2015.06.003

    WASp −/− and WASp −/− N-WASp fl/fl CD19 Cre/+ B cells can compete for help from WT T cells. (A) Generation of BM chimeric mice. WASp −/− or WASp −/− -WASp fl/fl CD19 Cre/+ (expressing CD45.2) and WT (CD45.1) BM cells at a 3:1 ratio were intravenous injected into lethally irradiated WT recipient mice. Injections of apoptotic cells started 10 weeks after transplantation. Percentage of cells was analyzed by flow cytometry. (B) Follicular B cells (B220 + CD23 + IgM int CD21 int ), MZB cells (B220 + CD23 − IgM high CD21 high ), total GC cells (B220 + GL7 + CD95 + ), LZ B cells (B220 + GL7 + CD95 + CD83 + CXCR4 low ), DZ B cells (B220 + GL7 + CD95 + CD83 low CXCR4 + ), total plasma cells (B220 − CD138 + ) and switched plasma cells (B220 − CD138 + IgG1 + ) were investigated at d 27. n = 7 per condition. (C) T FH cells (CD4 + CD44 + CD62L − PD1 + CXCR5 + ) were analyzed at d 27. n = 7 per condition. (D) B cell induced T cell activation in vitro . B cells were loaded with ovalbumin (OVA) and thereafter co-cultured with antigen-specific CD4 + T cells from OT-II mice. (E) Synapse formation between OVA-loaded B cells and OT-II CD4 + T cells quantified by immunohistochemistry. The star indicates the T cell and the arrow indicates a polarized synapse as determined by polymerized actin (F-actin) at the synapse interphase. Representative pictures from each strain from two experiments are shown. Bar, 10 μm n = 3. (F) Proliferation of and IL-2 production by CD4 + T cells from OT-II mice was measured by flow cytometry. n = 3. Graphs show three technical replicas and the experiment has been repeated five times with similar results. Abbreviations: WKO; WASp −/− , cDKO; WASp −/− -N-WASp fl/fl CD19 Cre+ .
    Figure Legend Snippet: WASp −/− and WASp −/− N-WASp fl/fl CD19 Cre/+ B cells can compete for help from WT T cells. (A) Generation of BM chimeric mice. WASp −/− or WASp −/− -WASp fl/fl CD19 Cre/+ (expressing CD45.2) and WT (CD45.1) BM cells at a 3:1 ratio were intravenous injected into lethally irradiated WT recipient mice. Injections of apoptotic cells started 10 weeks after transplantation. Percentage of cells was analyzed by flow cytometry. (B) Follicular B cells (B220 + CD23 + IgM int CD21 int ), MZB cells (B220 + CD23 − IgM high CD21 high ), total GC cells (B220 + GL7 + CD95 + ), LZ B cells (B220 + GL7 + CD95 + CD83 + CXCR4 low ), DZ B cells (B220 + GL7 + CD95 + CD83 low CXCR4 + ), total plasma cells (B220 − CD138 + ) and switched plasma cells (B220 − CD138 + IgG1 + ) were investigated at d 27. n = 7 per condition. (C) T FH cells (CD4 + CD44 + CD62L − PD1 + CXCR5 + ) were analyzed at d 27. n = 7 per condition. (D) B cell induced T cell activation in vitro . B cells were loaded with ovalbumin (OVA) and thereafter co-cultured with antigen-specific CD4 + T cells from OT-II mice. (E) Synapse formation between OVA-loaded B cells and OT-II CD4 + T cells quantified by immunohistochemistry. The star indicates the T cell and the arrow indicates a polarized synapse as determined by polymerized actin (F-actin) at the synapse interphase. Representative pictures from each strain from two experiments are shown. Bar, 10 μm n = 3. (F) Proliferation of and IL-2 production by CD4 + T cells from OT-II mice was measured by flow cytometry. n = 3. Graphs show three technical replicas and the experiment has been repeated five times with similar results. Abbreviations: WKO; WASp −/− , cDKO; WASp −/− -N-WASp fl/fl CD19 Cre+ .

    Techniques Used: Mouse Assay, Expressing, Injection, Irradiation, Transplantation Assay, Flow Cytometry, Cytometry, Activation Assay, In Vitro, Cell Culture, Immunohistochemistry

    Increased autoantibody production in naive WASp −/− and WASp −/− N-WASp fl/fl CD19 Cre+ mice. Serum from naïve mice and mice immunized with apoptotic cells were screened for reactivity to 95 different autoantigens using autoantibody array. Autoantigens are sorted by ANOVA starting with lowest p-value at the top for (A) IgM autoantibodies and for (B) IgG autoantibodies. Serum from 7 to 9 mice were tested, data of individual mice are shown. Black color equals ⩽ 1-fold change as compared with average values in control serum (WT d 0). A yellow square indicates a ⩾10-fold increase of autoantibody titer and a black square no difference as compared with average values in control serum. All yellow nuances in between represent a value larger than 1 and smaller than 10. WT, WASp −/− , WASp −/− N-WASp fl/fl CD19 Cre/+ d 0 n = 7, WT, WASp −/− N-WASp fl/fl CD19 Cre/+ d 27 n = 9. (C–D) Autoantibodies in 8–10 weeks old mice. Serum titers of anti-DNA and anti-chromatin (C) IgM and (D) IgG measured in 8–10 weeks old unchallenged mice. WT n = 3, WASp −/− n = 7, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 7, each dot correspond to one mouse. Significance was assessed with unpaired, two-tailed Student t test. * P
    Figure Legend Snippet: Increased autoantibody production in naive WASp −/− and WASp −/− N-WASp fl/fl CD19 Cre+ mice. Serum from naïve mice and mice immunized with apoptotic cells were screened for reactivity to 95 different autoantigens using autoantibody array. Autoantigens are sorted by ANOVA starting with lowest p-value at the top for (A) IgM autoantibodies and for (B) IgG autoantibodies. Serum from 7 to 9 mice were tested, data of individual mice are shown. Black color equals ⩽ 1-fold change as compared with average values in control serum (WT d 0). A yellow square indicates a ⩾10-fold increase of autoantibody titer and a black square no difference as compared with average values in control serum. All yellow nuances in between represent a value larger than 1 and smaller than 10. WT, WASp −/− , WASp −/− N-WASp fl/fl CD19 Cre/+ d 0 n = 7, WT, WASp −/− N-WASp fl/fl CD19 Cre/+ d 27 n = 9. (C–D) Autoantibodies in 8–10 weeks old mice. Serum titers of anti-DNA and anti-chromatin (C) IgM and (D) IgG measured in 8–10 weeks old unchallenged mice. WT n = 3, WASp −/− n = 7, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 7, each dot correspond to one mouse. Significance was assessed with unpaired, two-tailed Student t test. * P

    Techniques Used: Mouse Assay, Two Tailed Test

    WASp −/− N-WASpn fl/fl CD19 Cre+ mice have increased serum titers of DNA-specific IgM antibodies. (A and B) Confocal imaging analysis of spleen sections from mice 30 min after injection with CFSE-labeled apoptotic cells. Representative images of WT and WASp −/− n = 3, WASp −/− -N-WASpn fl/fl nCD19 Cre+ n = 4, are shown. (A) B cells were visualized with B220 (blue), metallophilic macrophages with CD169 (red), and apoptotic CFSE (green). Bars, 150 μm. (B) DCs were visualized with CD11c (red). White arrows indicate co-localization of CD11c + cells and apoptotic cells. Bars, 300 μm (C and D) Flow cytometry analysis of DC subsets in spleen 30 min after injection with CFSE-labeled apoptotic cells (C) Absolute number of CD11c + DCs, CD11c + DEC2015 + DCs, and CD11c + 33D1 + DCs. (D) DC co-localization of CFSE-labeled apoptotic cells shown for CD11c + DEC205 + and CD11c + 33D1 + DCs. WT n = 5, WASp −/− n = 5, WASp −/− N-WASpn fl/fl nCD19 Cre+ n = 6. (E) Mice were injected with apoptotic cells once a week for four weeks. Serum titers of anti-DNA (F) IgM and (G) IgG antibodies were measured at d 0–27. n = 11 –19 and represent a pool of two experiments, each dot correspond to one mouse. Significance was assessed with unpaired, two-tailed Student t test. * P
    Figure Legend Snippet: WASp −/− N-WASpn fl/fl CD19 Cre+ mice have increased serum titers of DNA-specific IgM antibodies. (A and B) Confocal imaging analysis of spleen sections from mice 30 min after injection with CFSE-labeled apoptotic cells. Representative images of WT and WASp −/− n = 3, WASp −/− -N-WASpn fl/fl nCD19 Cre+ n = 4, are shown. (A) B cells were visualized with B220 (blue), metallophilic macrophages with CD169 (red), and apoptotic CFSE (green). Bars, 150 μm. (B) DCs were visualized with CD11c (red). White arrows indicate co-localization of CD11c + cells and apoptotic cells. Bars, 300 μm (C and D) Flow cytometry analysis of DC subsets in spleen 30 min after injection with CFSE-labeled apoptotic cells (C) Absolute number of CD11c + DCs, CD11c + DEC2015 + DCs, and CD11c + 33D1 + DCs. (D) DC co-localization of CFSE-labeled apoptotic cells shown for CD11c + DEC205 + and CD11c + 33D1 + DCs. WT n = 5, WASp −/− n = 5, WASp −/− N-WASpn fl/fl nCD19 Cre+ n = 6. (E) Mice were injected with apoptotic cells once a week for four weeks. Serum titers of anti-DNA (F) IgM and (G) IgG antibodies were measured at d 0–27. n = 11 –19 and represent a pool of two experiments, each dot correspond to one mouse. Significance was assessed with unpaired, two-tailed Student t test. * P

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

    Repeated apoptotic cell injections induce a GC response in WT, WASp −/− and WASp −/− N-WASp fl/fl CD19 Cre/+ mice. (A) Immunohistochemistry of GC formation in spleen at d 0 and d 27 after apoptotic cell injections. B cells were labeled with B220 (blue), GC cells with PNA (red), and metallophilic macrophages with CD169 (green). Representative pictures from each strain from three experiments are shown. The ratio of GC area of total B220 + area in spleen sections is indicated. WT d 0 n = 8, WT d 27 n = 16, WASp −/− d 0 n = 7, WASp −/− d 27 n = 17, WASp −/− N-WASp fl/fl CD19 Cre/+ d 0 n = 8, WASp −/− N-WASp fl/fl CD19 Cre/+ d 27 n = 16. Bars, 300 μm. (B) Flow cytometry data using B220, GL7 and CD95 to determine quantity of GC B cells. WT n = 14, WASp −/− n = 13, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 11. (C) Absolute number of T FH cells (CD4 + CD44 + CD62L − PD1 + CXCR5 + ) analyzed by flow cytometry. WT n = 8, WASp −/− n = 7, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 5. (D) Flow cytometry data of the GC compartments as determined by LZ B cells (CD83 + CXCR4 − ) and DZ B cells (CD83 − CXCR4 + ). WT n = 14, WASp −/− n = 13, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 11. (E) Immunohistochemistry for GC polarization. FDCs were labeled with CD35 (green) in spleens at d 27 from mice immunized with apoptotic cells. Upper panel shows CD35 alone and lower panel, CD35 together with PNA (red) and B220 (blue). Representative pictures from each strain from two experiments are shown. The percentage of CD35 + area of total PNA + or B220 + area in spleen sections is indicated. n = 3 and three images per mouse were analyzed. Bars, 300 μm. (F) Absolute number of total plasma cells (B220 − CD138 + ) and IgG1 + plasma cells as determined by flow cytometry. WT n = 8, WASp −/− n = 7, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 5. (G) Immunohistochemistry of IgM and IgG1 localization in spleen at d 0 and d 27. WT d 0 n = 3, WT d 27 n = 4, WASp −/− d 0 n = 4, WASp −/− d 27 n = 6, WASp −/− N-WASp fl/fl CD19 Cre/+ d 0 n = 4, WASp −/− N-WASp fl/fl CD19 Cre/+ d 27 n = 6. Bars, 300 μm. Significance was assessed with unpaired, two-tailed Student t test. ** P
    Figure Legend Snippet: Repeated apoptotic cell injections induce a GC response in WT, WASp −/− and WASp −/− N-WASp fl/fl CD19 Cre/+ mice. (A) Immunohistochemistry of GC formation in spleen at d 0 and d 27 after apoptotic cell injections. B cells were labeled with B220 (blue), GC cells with PNA (red), and metallophilic macrophages with CD169 (green). Representative pictures from each strain from three experiments are shown. The ratio of GC area of total B220 + area in spleen sections is indicated. WT d 0 n = 8, WT d 27 n = 16, WASp −/− d 0 n = 7, WASp −/− d 27 n = 17, WASp −/− N-WASp fl/fl CD19 Cre/+ d 0 n = 8, WASp −/− N-WASp fl/fl CD19 Cre/+ d 27 n = 16. Bars, 300 μm. (B) Flow cytometry data using B220, GL7 and CD95 to determine quantity of GC B cells. WT n = 14, WASp −/− n = 13, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 11. (C) Absolute number of T FH cells (CD4 + CD44 + CD62L − PD1 + CXCR5 + ) analyzed by flow cytometry. WT n = 8, WASp −/− n = 7, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 5. (D) Flow cytometry data of the GC compartments as determined by LZ B cells (CD83 + CXCR4 − ) and DZ B cells (CD83 − CXCR4 + ). WT n = 14, WASp −/− n = 13, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 11. (E) Immunohistochemistry for GC polarization. FDCs were labeled with CD35 (green) in spleens at d 27 from mice immunized with apoptotic cells. Upper panel shows CD35 alone and lower panel, CD35 together with PNA (red) and B220 (blue). Representative pictures from each strain from two experiments are shown. The percentage of CD35 + area of total PNA + or B220 + area in spleen sections is indicated. n = 3 and three images per mouse were analyzed. Bars, 300 μm. (F) Absolute number of total plasma cells (B220 − CD138 + ) and IgG1 + plasma cells as determined by flow cytometry. WT n = 8, WASp −/− n = 7, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 5. (G) Immunohistochemistry of IgM and IgG1 localization in spleen at d 0 and d 27. WT d 0 n = 3, WT d 27 n = 4, WASp −/− d 0 n = 4, WASp −/− d 27 n = 6, WASp −/− N-WASp fl/fl CD19 Cre/+ d 0 n = 4, WASp −/− N-WASp fl/fl CD19 Cre/+ d 27 n = 6. Bars, 300 μm. Significance was assessed with unpaired, two-tailed Student t test. ** P

    Techniques Used: Mouse Assay, Immunohistochemistry, Labeling, Flow Cytometry, Cytometry, Two Tailed Test

    Altered B cell affinity maturation in WASp −/− N-WASp fl/fl CD19 Cre/+ mice. (A and B) Characterization of replacement mutations found in the V H 1-186.2 family of (A) IgM and (B) IgG1. Grey background highlights the CDR1, CDR2, and CDR3 regions. Pink arrowhead indicates the high affinity amino acid mutation W33L. Amount of unique clones analyzed; WT Cμ n = 34, WT Cγ1 n = 109, WKO Cμ n = 36, WKO Cγ1 n = 34, cDKO Cμ n = 80, cDKO Cγ1 n = 21. (C) Breakdown of peripheral tolerance inWASp deficiency. N-WASp activity in WASp-deficient B cells supports increased reactivity to self antigens. Abbreviations: WKO; WASp −/− , cDKO; WASp −/− N-WASp fl/fl CD19 Cre/+ .
    Figure Legend Snippet: Altered B cell affinity maturation in WASp −/− N-WASp fl/fl CD19 Cre/+ mice. (A and B) Characterization of replacement mutations found in the V H 1-186.2 family of (A) IgM and (B) IgG1. Grey background highlights the CDR1, CDR2, and CDR3 regions. Pink arrowhead indicates the high affinity amino acid mutation W33L. Amount of unique clones analyzed; WT Cμ n = 34, WT Cγ1 n = 109, WKO Cμ n = 36, WKO Cγ1 n = 34, cDKO Cμ n = 80, cDKO Cγ1 n = 21. (C) Breakdown of peripheral tolerance inWASp deficiency. N-WASp activity in WASp-deficient B cells supports increased reactivity to self antigens. Abbreviations: WKO; WASp −/− , cDKO; WASp −/− N-WASp fl/fl CD19 Cre/+ .

    Techniques Used: Mouse Assay, Mutagenesis, Clone Assay, Activity Assay

    29) Product Images from "Identification of Toxoplasma Gondii Tyrosine Hydroxylase (TH) Activity and Molecular Immunoprotection against Toxoplasmosis"

    Article Title: Identification of Toxoplasma Gondii Tyrosine Hydroxylase (TH) Activity and Molecular Immunoprotection against Toxoplasmosis

    Journal: Vaccines

    doi: 10.3390/vaccines8020158

    Dynamic humoral immunoreaction of BALB/c mice under the induction of recombined TgTH protein. The BALB/c mice were classified into 4 groups at random and each of which contained 5 mice ( n = 5). In the three experimental groups, mice received the immunization of Freund adjuvant accompanied with the recombined protein TgTH (1:1), Freund adjuvant accompanied with pET-32a protein (1:1), and Freund adjuvant alone, and the fourth group was used as a blank control. The titers of IgG and the subclass IgG2a and IgG1 were detected on weeks 0, 2, 4, and 6. As for the absorption at 450 nm, the expression pattern was mean ± SD. As for the differences with statistical significance between groups in the identical time point, (***) referred to ( p
    Figure Legend Snippet: Dynamic humoral immunoreaction of BALB/c mice under the induction of recombined TgTH protein. The BALB/c mice were classified into 4 groups at random and each of which contained 5 mice ( n = 5). In the three experimental groups, mice received the immunization of Freund adjuvant accompanied with the recombined protein TgTH (1:1), Freund adjuvant accompanied with pET-32a protein (1:1), and Freund adjuvant alone, and the fourth group was used as a blank control. The titers of IgG and the subclass IgG2a and IgG1 were detected on weeks 0, 2, 4, and 6. As for the absorption at 450 nm, the expression pattern was mean ± SD. As for the differences with statistical significance between groups in the identical time point, (***) referred to ( p

    Techniques Used: Mouse Assay, Positron Emission Tomography, Expressing

    30) Product Images from "The Molecular Characterization and Immunity Identification of Trichomonas vaginalis Adhesion Protein 33 (AP33)"

    Article Title: The Molecular Characterization and Immunity Identification of Trichomonas vaginalis Adhesion Protein 33 (AP33)

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2020.01433

    The dynamics of humoral response in BALB/c mice induced by recombinant TvAP33 protein. The BALB/c mice were randomly divided into four groups of five mice each ( n = 5). The BALB/c mice in group were immunized with recombinant protein TvAP33 mixed with Freund adjuvant (1:1), pET-32a protein mixed with Freund adjuvant (1:1), or Freund adjuvant alone, and the rest of the group as a blank control. The titers of IgG and the subclasses IgG1 and IgG2a were detected on days 0, 14, 28, and 42. The results were expressed as mean ± SD with respect to absorbance at 450 nm. Statistically significant differences ( P
    Figure Legend Snippet: The dynamics of humoral response in BALB/c mice induced by recombinant TvAP33 protein. The BALB/c mice were randomly divided into four groups of five mice each ( n = 5). The BALB/c mice in group were immunized with recombinant protein TvAP33 mixed with Freund adjuvant (1:1), pET-32a protein mixed with Freund adjuvant (1:1), or Freund adjuvant alone, and the rest of the group as a blank control. The titers of IgG and the subclasses IgG1 and IgG2a were detected on days 0, 14, 28, and 42. The results were expressed as mean ± SD with respect to absorbance at 450 nm. Statistically significant differences ( P

    Techniques Used: Mouse Assay, Recombinant, Positron Emission Tomography

    31) Product Images from "Kinetics of antibody response to influenza vaccination in renal transplant recipients"

    Article Title: Kinetics of antibody response to influenza vaccination in renal transplant recipients

    Journal: Transplant immunology

    doi: 10.1016/j.trim.2019.01.001

    Ig-ASC in control and transplant groups vaccinated with TIV 2007–08. ’ section. Figure 4A shows proportion of IgG-ASC at different time points post-vaccination. Figure 4B shows proportion of IgG-ASC in control group and transplant subjects at day 7 post-vaccination along with limit of detection of the ELISPOT assay. Hatched horizontal line indicates limit of detection of the ELISPOT assay. Data represent influenza-specific IgG-ASC per million PBMC.
    Figure Legend Snippet: Ig-ASC in control and transplant groups vaccinated with TIV 2007–08. ’ section. Figure 4A shows proportion of IgG-ASC at different time points post-vaccination. Figure 4B shows proportion of IgG-ASC in control group and transplant subjects at day 7 post-vaccination along with limit of detection of the ELISPOT assay. Hatched horizontal line indicates limit of detection of the ELISPOT assay. Data represent influenza-specific IgG-ASC per million PBMC.

    Techniques Used: Enzyme-linked Immunospot

    32) Product Images from "Tumor necrosis factor receptor superfamily members 1a and 1b contribute to exacerbation of atherosclerosis by Chlamydia pneumoniae in mice"

    Article Title: Tumor necrosis factor receptor superfamily members 1a and 1b contribute to exacerbation of atherosclerosis by Chlamydia pneumoniae in mice

    Journal: Microbes and infection

    doi: 10.1016/j.micinf.2018.09.003

    Cellular and humoral immune response to infection. Groups of 6–8-week-old male WT, TNF KO, TNFR1 KO, TNFR2 KO, or TNFR 1/2 DKO were infected on day 0, 15, and 30 with CPN. Separate groups of WT and TNF KO mice were infected with SPG buffer (mock). A B , Splenic Ag-specific IFN-γ ( A ) and TNF-α ( B ) response was measured on day 14 in four mice per group after primary inoculation. Mean ± SEM of cytokine concentrations are shown. * Significant (p≤0.05; ANOVA) between indicated group and mock-infected WT or TNF KO animals. ** Significant (p≤0.05; ANOVA) between indicated group and CPN-infected WT animals. *** Significant (p≤0.05; ANOVA) between indicated group and CPN-infected TNF KO animals. Results are representative of two independent experiments. C, Anti-CPN total Ab, IgG2c, and IgG1 responses analyzed in four to sixteen mice per group on day 90 after primary inoculation. Mean ± SEM of reciprocal 50% binding antibody titers are shown. * Significant (p≤0.05; ANOVA) between indicated group between indicated group and mock-infected WT or TNF KO animals. Results are representative of two independent experiments.
    Figure Legend Snippet: Cellular and humoral immune response to infection. Groups of 6–8-week-old male WT, TNF KO, TNFR1 KO, TNFR2 KO, or TNFR 1/2 DKO were infected on day 0, 15, and 30 with CPN. Separate groups of WT and TNF KO mice were infected with SPG buffer (mock). A B , Splenic Ag-specific IFN-γ ( A ) and TNF-α ( B ) response was measured on day 14 in four mice per group after primary inoculation. Mean ± SEM of cytokine concentrations are shown. * Significant (p≤0.05; ANOVA) between indicated group and mock-infected WT or TNF KO animals. ** Significant (p≤0.05; ANOVA) between indicated group and CPN-infected WT animals. *** Significant (p≤0.05; ANOVA) between indicated group and CPN-infected TNF KO animals. Results are representative of two independent experiments. C, Anti-CPN total Ab, IgG2c, and IgG1 responses analyzed in four to sixteen mice per group on day 90 after primary inoculation. Mean ± SEM of reciprocal 50% binding antibody titers are shown. * Significant (p≤0.05; ANOVA) between indicated group between indicated group and mock-infected WT or TNF KO animals. Results are representative of two independent experiments.

    Techniques Used: Infection, Mouse Assay, Binding Assay

    33) Product Images from "Uncoupling Splicing From Transcription Using Antisense Oligonucleotides Reveals a Dual Role for I Exon Donor Splice Sites in Antibody Class Switching"

    Article Title: Uncoupling Splicing From Transcription Using Antisense Oligonucleotides Reveals a Dual Role for I Exon Donor Splice Sites in Antibody Class Switching

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.00780

    Treatment with Iγ1 exon dss ASO inhibited γ1 GLT constitutive splicing and IgG1 class switching in B cells. (A) Antisense oligonucleotide targeting the donor splice site of Iγ1 exon (Iγ1 dss ASO) was designed and synthetized as “ vivo -morpholino ASO” permitting passive administration of ASO in cells (Gene Tools, LLC). Targeted γ1 GLT (uppercase: exon sequence; lowercase: intron sequence) and Iγ1 dss ASO sequences are indicated. Iγ1, γ1 I exon; Sγ1, γ1 switch region; CH1γ1, γ1 constant exon 1; dss, donor splice site. (B–G) Splenic B cells were isolated from C57BL/6 mice, stimulated with LPS + IL4, and treated with 2 μM Iγ1 dss ASO (ASO) or an irrelevant ASO (control) for 2 days (B,C) or 3 days (D–G) . (B) Unspliced γ1 GLT expression relative to GAPDH mRNA expression was monitored by quantitative RT-PCR using Iγ1-for-Q and Sγ1U-rev-Q primers described in schema A . Expression of γ1 GLTs in control B cells was normalized to 1. ( C , top) RT-PCR was performed using Iγ1-for and Cγ1-rev primers (position described in schema C , middle) to identify constitutively and alternatively spliced transcripts. PCR products were analyzed on agarose gels. Expression of actin mRNA is also shown. Molecular markers in base pairs are indicated. Schematic representation of the different γ1 spliced transcripts is indicated on the right, and transcript sequences are given in Supplementary Figure 3 . One experiment out of three is shown. Relative quantification of amplification products was done using ImageJ software and expressed after normalization to actin band intensity. ( C , middle) Schematic representation of γ1 spliced transcripts detected in B cells from C57BL / 6 mice after treatment with an irrelevant ASO (control) or Iγ1 dss ASO (ASO). Gray hatched lines represent splicing events involving constitutive and alternative splice sites. Donor and acceptor splice sites are indicated in red and green, respectively. ( C , bottom) Consensus value (ranging from 0 to 100) of each predicted splice site determined using the HSF 3.1 tool. (D) Flow cytometry analysis of purified B-cell populations using the indicated cell surface markers. Plots are gated on live cells. The percentage of B220 + IgG1 + cells is indicated. One experiment out of five is shown. (E) Percentage of IgG1-positive cells determined by flow cytometry. (F) Quantification of IgG1 in culture supernatants by ELISA. (G) Post-switch Iμ-Cγ1 mRNA expression relative to GAPDH expression was monitored by quantitative RT-PCR. Expression of post-switch mRNAs in control B cells was normalized to 1. Sequences of primers used for RT-PCR and quantitative RT-PCR are indicated in Supplementary Table 1 . (B,C,E – G) Data are means ± SEM of two independent experiments, n = 3–8 for each group. Unpaired two-tailed Student's t test was used to determine significance. ** P
    Figure Legend Snippet: Treatment with Iγ1 exon dss ASO inhibited γ1 GLT constitutive splicing and IgG1 class switching in B cells. (A) Antisense oligonucleotide targeting the donor splice site of Iγ1 exon (Iγ1 dss ASO) was designed and synthetized as “ vivo -morpholino ASO” permitting passive administration of ASO in cells (Gene Tools, LLC). Targeted γ1 GLT (uppercase: exon sequence; lowercase: intron sequence) and Iγ1 dss ASO sequences are indicated. Iγ1, γ1 I exon; Sγ1, γ1 switch region; CH1γ1, γ1 constant exon 1; dss, donor splice site. (B–G) Splenic B cells were isolated from C57BL/6 mice, stimulated with LPS + IL4, and treated with 2 μM Iγ1 dss ASO (ASO) or an irrelevant ASO (control) for 2 days (B,C) or 3 days (D–G) . (B) Unspliced γ1 GLT expression relative to GAPDH mRNA expression was monitored by quantitative RT-PCR using Iγ1-for-Q and Sγ1U-rev-Q primers described in schema A . Expression of γ1 GLTs in control B cells was normalized to 1. ( C , top) RT-PCR was performed using Iγ1-for and Cγ1-rev primers (position described in schema C , middle) to identify constitutively and alternatively spliced transcripts. PCR products were analyzed on agarose gels. Expression of actin mRNA is also shown. Molecular markers in base pairs are indicated. Schematic representation of the different γ1 spliced transcripts is indicated on the right, and transcript sequences are given in Supplementary Figure 3 . One experiment out of three is shown. Relative quantification of amplification products was done using ImageJ software and expressed after normalization to actin band intensity. ( C , middle) Schematic representation of γ1 spliced transcripts detected in B cells from C57BL / 6 mice after treatment with an irrelevant ASO (control) or Iγ1 dss ASO (ASO). Gray hatched lines represent splicing events involving constitutive and alternative splice sites. Donor and acceptor splice sites are indicated in red and green, respectively. ( C , bottom) Consensus value (ranging from 0 to 100) of each predicted splice site determined using the HSF 3.1 tool. (D) Flow cytometry analysis of purified B-cell populations using the indicated cell surface markers. Plots are gated on live cells. The percentage of B220 + IgG1 + cells is indicated. One experiment out of five is shown. (E) Percentage of IgG1-positive cells determined by flow cytometry. (F) Quantification of IgG1 in culture supernatants by ELISA. (G) Post-switch Iμ-Cγ1 mRNA expression relative to GAPDH expression was monitored by quantitative RT-PCR. Expression of post-switch mRNAs in control B cells was normalized to 1. Sequences of primers used for RT-PCR and quantitative RT-PCR are indicated in Supplementary Table 1 . (B,C,E – G) Data are means ± SEM of two independent experiments, n = 3–8 for each group. Unpaired two-tailed Student's t test was used to determine significance. ** P

    Techniques Used: Allele-specific Oligonucleotide, Sequencing, Isolation, Mouse Assay, Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Software, Flow Cytometry, Purification, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    Specific IgG1 class switching inhibition in B cells treated by Iγ1 exon dss ASO. Splenic B cells were isolated from C57BL / 6 mice, stimulated with anti-CD40 + IL4 and treated with 2 μM Iγ1 dss ASO (ASO) or an irrelevant ASO (control) for 2 days (B,E) or 4 days (C,D,F,G) . (A) Schematic representation of unspliced ε GLT. Iε, ε I exon; Sε, ε switch region; CH1ε, ε constant exon 1; dss, donor splice site. (B,E) Unspliced γ1 (B) and ε (E) GLT expression monitored as described in Figure 2 . Iε-for-Q and SεU-rev-Q primers, described in schema A , were used for ε GLT expression determination. (C,F) Post-switch Iμ-Cγ1 and Iμ-Cε mRNA expressions monitored as described in Figure 2 . (D,G) Quantification of IgG1 and IgE in culture supernatants by ELISA. (B–G) Data are means ± SEM of two independent experiments, n = 3–4 for each group. Unpaired two-tailed Student's t test was used to determine significance. ns: non-significant, ** P
    Figure Legend Snippet: Specific IgG1 class switching inhibition in B cells treated by Iγ1 exon dss ASO. Splenic B cells were isolated from C57BL / 6 mice, stimulated with anti-CD40 + IL4 and treated with 2 μM Iγ1 dss ASO (ASO) or an irrelevant ASO (control) for 2 days (B,E) or 4 days (C,D,F,G) . (A) Schematic representation of unspliced ε GLT. Iε, ε I exon; Sε, ε switch region; CH1ε, ε constant exon 1; dss, donor splice site. (B,E) Unspliced γ1 (B) and ε (E) GLT expression monitored as described in Figure 2 . Iε-for-Q and SεU-rev-Q primers, described in schema A , were used for ε GLT expression determination. (C,F) Post-switch Iμ-Cγ1 and Iμ-Cε mRNA expressions monitored as described in Figure 2 . (D,G) Quantification of IgG1 and IgE in culture supernatants by ELISA. (B–G) Data are means ± SEM of two independent experiments, n = 3–4 for each group. Unpaired two-tailed Student's t test was used to determine significance. ns: non-significant, ** P

    Techniques Used: Inhibition, Allele-specific Oligonucleotide, Isolation, Mouse Assay, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    Impaired antibody class switching in B cells treated by Iμ exon dss ASO. (A) Antisense oligonucleotide targeting the donor splice site of Iμ exon (Iμ dss ASO) was designed and synthetized as “ vivo -morpholino ASO” permitting passive administration of ASO in the cells (Gene Tools, LLC). Targeted μ GLT (uppercase: exon sequence; lowercase: intron sequence) and Iμ dss ASO sequences are indicated. Iμ, μ I exon; Sμ, μ switch region; CH1μ, μ constant exon 1; dss, donor splice site. (B–K) Splenic B cells were isolated from C57BL / 6 mice, stimulated with LPS + IL4 (B–E) or LPS (F–K) and treated with 2 μM (B–E) , 3 μM (F,G,J) or 4 μM (H,I,K) Iμ dss ASO or an irrelevant control ASO for 2 days (B,E) or 3 days (C,D,F–K) . (B) Constitutively and alternatively spliced μ transcripts analyzed as described in Figure 2 using Iμ-for and Cμ-rev primers, described in schema A . One experiment out of three is shown. Relative quantification of amplification products was done using ImageJ software and expressed after normalization to actin band intensity. (C) Quantification of IgG1 in culture supernatants by ELISA. (D) Percentage of IgG1-positive cells determined by flow cytometry as described in Figure 2 . (E) Unspliced γ1 GLT expression monitored as described in Figure 2 . (F,I) Flow cytometry analysis of purified B-cell populations using the indicated cell surface markers. Plots are gated on live cells. The percentage of B220 + IgG3 + (F) or B220 + IgG2b + (I) cells is indicated. One experiment out of three is shown. (G,H,J,K) Percentage of IgG3-positive (G,H) or IgG2b-positive (J,K) cells determined by flow cytometry. (B–K) Data are means ± SEM, n = 3 for each group. Unpaired two-tailed Student's t test was used to determine significance. ns: non-significant, * P
    Figure Legend Snippet: Impaired antibody class switching in B cells treated by Iμ exon dss ASO. (A) Antisense oligonucleotide targeting the donor splice site of Iμ exon (Iμ dss ASO) was designed and synthetized as “ vivo -morpholino ASO” permitting passive administration of ASO in the cells (Gene Tools, LLC). Targeted μ GLT (uppercase: exon sequence; lowercase: intron sequence) and Iμ dss ASO sequences are indicated. Iμ, μ I exon; Sμ, μ switch region; CH1μ, μ constant exon 1; dss, donor splice site. (B–K) Splenic B cells were isolated from C57BL / 6 mice, stimulated with LPS + IL4 (B–E) or LPS (F–K) and treated with 2 μM (B–E) , 3 μM (F,G,J) or 4 μM (H,I,K) Iμ dss ASO or an irrelevant control ASO for 2 days (B,E) or 3 days (C,D,F–K) . (B) Constitutively and alternatively spliced μ transcripts analyzed as described in Figure 2 using Iμ-for and Cμ-rev primers, described in schema A . One experiment out of three is shown. Relative quantification of amplification products was done using ImageJ software and expressed after normalization to actin band intensity. (C) Quantification of IgG1 in culture supernatants by ELISA. (D) Percentage of IgG1-positive cells determined by flow cytometry as described in Figure 2 . (E) Unspliced γ1 GLT expression monitored as described in Figure 2 . (F,I) Flow cytometry analysis of purified B-cell populations using the indicated cell surface markers. Plots are gated on live cells. The percentage of B220 + IgG3 + (F) or B220 + IgG2b + (I) cells is indicated. One experiment out of three is shown. (G,H,J,K) Percentage of IgG3-positive (G,H) or IgG2b-positive (J,K) cells determined by flow cytometry. (B–K) Data are means ± SEM, n = 3 for each group. Unpaired two-tailed Student's t test was used to determine significance. ns: non-significant, * P

    Techniques Used: Allele-specific Oligonucleotide, Sequencing, Isolation, Mouse Assay, Amplification, Software, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Purification, Two Tailed Test

    34) Product Images from "Murine lupus is neutrophil elastase-independent in the MRL.Faslpr model"

    Article Title: Murine lupus is neutrophil elastase-independent in the MRL.Faslpr model

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0226396

    Elane -genotype does not significantly alter the anti-self response or the AFC compartment. (A-D) Serum anti-RNA (A), anti-Sm (B), anti-nucleosome (C), and rheumatoid factor (D) antibody titers at 17 weeks of age. (E) Numbers of Ig κ + antibody forming cells (AFCs) per spleen as determined by ELISpot (left panel). Percentages of live cells that are TCRβ - CD44 + CD138 + intracellular κ + AFCs in spleens (right panel). (F-H) Numbers of IgG1 (F), IgG2a ( elane +/+ males n = 9, elane -/- females n = 17, elane +/+ females n = 11) (G), and IgM ( elane +/+ males n = 9, elane -/- females n = 14, elane +/+ females n = 10) (H) AFCs per spleen as determined by ELISpot ( elane -/- males n = 8; elane +/+ males n = 11; elane -/- females n = 19; elane +/+ females n = 13 mice per group unless otherwise indicated). Data representation and statistics are as in Fig 1 unless otherwise indicated. In panel E (right), bar graphs are represented as the mean with SEM and a two-tailed Welch’s T test was performed to determine statistical significance within each gender.
    Figure Legend Snippet: Elane -genotype does not significantly alter the anti-self response or the AFC compartment. (A-D) Serum anti-RNA (A), anti-Sm (B), anti-nucleosome (C), and rheumatoid factor (D) antibody titers at 17 weeks of age. (E) Numbers of Ig κ + antibody forming cells (AFCs) per spleen as determined by ELISpot (left panel). Percentages of live cells that are TCRβ - CD44 + CD138 + intracellular κ + AFCs in spleens (right panel). (F-H) Numbers of IgG1 (F), IgG2a ( elane +/+ males n = 9, elane -/- females n = 17, elane +/+ females n = 11) (G), and IgM ( elane +/+ males n = 9, elane -/- females n = 14, elane +/+ females n = 10) (H) AFCs per spleen as determined by ELISpot ( elane -/- males n = 8; elane +/+ males n = 11; elane -/- females n = 19; elane +/+ females n = 13 mice per group unless otherwise indicated). Data representation and statistics are as in Fig 1 unless otherwise indicated. In panel E (right), bar graphs are represented as the mean with SEM and a two-tailed Welch’s T test was performed to determine statistical significance within each gender.

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

    35) Product Images from "Autophagy in dendritic cells and B cells is critical for the inflammatory state of TLR7-mediated autoimmunity"

    Article Title: Autophagy in dendritic cells and B cells is critical for the inflammatory state of TLR7-mediated autoimmunity

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

    doi: 10.4049/jimmunol.1601307

    Auto-Abs against cardiolipin and high IgG1 are present in Tlr7.1 tg DBko mice ( A ) HEP2 slides used to detect IgG-ANA in 12–14 week old mice. Data are representative of 4 experiments. Controls (n=4), Tlr7.1 tg (n=5), Tlr7.1 tg Atg5 ko (n=5). Antibodies against cardiolipin ( B ) total serum IgM, IgG ( C ); IgG1 ( D ); IgG2a, IgG2b, IgG2c, and IgG3 ( E ); was determined by ELISA. Each point is representative of an individual animal. Mice were between 12–14 weeks of age. (*p
    Figure Legend Snippet: Auto-Abs against cardiolipin and high IgG1 are present in Tlr7.1 tg DBko mice ( A ) HEP2 slides used to detect IgG-ANA in 12–14 week old mice. Data are representative of 4 experiments. Controls (n=4), Tlr7.1 tg (n=5), Tlr7.1 tg Atg5 ko (n=5). Antibodies against cardiolipin ( B ) total serum IgM, IgG ( C ); IgG1 ( D ); IgG2a, IgG2b, IgG2c, and IgG3 ( E ); was determined by ELISA. Each point is representative of an individual animal. Mice were between 12–14 weeks of age. (*p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    36) Product Images from "Mannosylated Mucin-Type Immunoglobulin Fusion Proteins Enhance Antigen-Specific Antibody and T Lymphocyte Responses"

    Article Title: Mannosylated Mucin-Type Immunoglobulin Fusion Proteins Enhance Antigen-Specific Antibody and T Lymphocyte Responses

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0046959

    PSGL-1/mIgG 2b specific antibody titers. Serum IgG antibody titers against PPM, CP or mIgG Fc in groups of mice from study A and B. Titres were determined from pooled sera of mice in each group (8–10 mice/group) at week 8, and were defined as the reciprocal endpoint dilution giving an optical density at 450 nm of 3 times the background value.
    Figure Legend Snippet: PSGL-1/mIgG 2b specific antibody titers. Serum IgG antibody titers against PPM, CP or mIgG Fc in groups of mice from study A and B. Titres were determined from pooled sera of mice in each group (8–10 mice/group) at week 8, and were defined as the reciprocal endpoint dilution giving an optical density at 450 nm of 3 times the background value.

    Techniques Used: Mouse Assay

    OVA specific IgG antibody titers. Serum IgG antibody titers against OVA in study A ( A ) and B ( B ). Titres are given as mean +SD (n = 8–9 in study A and 8–10 in study B) and are defined as the reciprocal endpoint dilution giving an optical density at 450 nm of 3 times ( A ) or ≥0.2 above ( B ) the background value. In study A (A), the asterisk (**) indicates a significantly higher IgG titer compared to all other groups and (*) indicates a significantly higher IgG titer compared to all groups except OVA−PPM+AbISCO-100 at that time point. In study B, the asterisk (*) indicates a significantly higher IgG titer compared to all other groups. P
    Figure Legend Snippet: OVA specific IgG antibody titers. Serum IgG antibody titers against OVA in study A ( A ) and B ( B ). Titres are given as mean +SD (n = 8–9 in study A and 8–10 in study B) and are defined as the reciprocal endpoint dilution giving an optical density at 450 nm of 3 times ( A ) or ≥0.2 above ( B ) the background value. In study A (A), the asterisk (**) indicates a significantly higher IgG titer compared to all other groups and (*) indicates a significantly higher IgG titer compared to all groups except OVA−PPM+AbISCO-100 at that time point. In study B, the asterisk (*) indicates a significantly higher IgG titer compared to all other groups. P

    Techniques Used:

    37) Product Images from "Antigen activation and impaired Fas-induced death-inducing signaling complex formation in T-large-granular lymphocyte leukemia"

    Article Title: Antigen activation and impaired Fas-induced death-inducing signaling complex formation in T-large-granular lymphocyte leukemia

    Journal: Blood

    doi: 10.1182/blood-2007-06-093823

    DISC formation in PBMC from LGL leukemia patients and from healthy donors . (A) Fas-mediated DISC formation was determined after 45 minutes and after 6 hours of cross-linking with (500 ng/mL) anti-Fas (APO-1, IgG3) antibody (lanes 1, 3, 4, 6-9, 11-14) in samples from 5 patients with LGL leukemia. Cells were cultured for 24 hours with medium in the absence (−, lanes 1, 3, 6, 7, and 11) and presence of 500 IU/mL IL-2 (+, lanes 2, 4, 5, 8-10, 12, 13) for 24 hours, or PHA (1 μg/mL) plus IL-2 (500 IU/mL) for 5 to 7 days (indicated as PHA in lane 14). IgG3 isotype control antibody (500 ng/mL) was added to demonstrate specificity of protein interactions with the FasR (lanes 2, 5, and 10). After immunoprecipitation and gel electrophoresis, Western blot analysis was performed to detect caspase-8 (top panel) and FADD (bottom panel) in these FasR immunoprecipitates. Immunoprecipitation after anti-Fas antibody cross-linking of H9 cells (T-cell leukemia cell line) was used as a positive control. (B) Whole-cell lysates were prepared from a fraction of cells studied in these immunoprecipitation experiments after 1 and 6 hours of cross-linking with anti-Fas (Apo-1) antibody and isotype control (IgG3). Results shown represent the means (± SEM) of caspase-8, and -3/7 activities that were determined by a fluorometric enzyme activity assay. Protein bands for full-length caspase-8 (casp-8), FADD, and an activated cleaved product of caspase-8 (Ac casp-8) are indicated. NS = nonspecific band observed with the anti-caspase-8 antibody.
    Figure Legend Snippet: DISC formation in PBMC from LGL leukemia patients and from healthy donors . (A) Fas-mediated DISC formation was determined after 45 minutes and after 6 hours of cross-linking with (500 ng/mL) anti-Fas (APO-1, IgG3) antibody (lanes 1, 3, 4, 6-9, 11-14) in samples from 5 patients with LGL leukemia. Cells were cultured for 24 hours with medium in the absence (−, lanes 1, 3, 6, 7, and 11) and presence of 500 IU/mL IL-2 (+, lanes 2, 4, 5, 8-10, 12, 13) for 24 hours, or PHA (1 μg/mL) plus IL-2 (500 IU/mL) for 5 to 7 days (indicated as PHA in lane 14). IgG3 isotype control antibody (500 ng/mL) was added to demonstrate specificity of protein interactions with the FasR (lanes 2, 5, and 10). After immunoprecipitation and gel electrophoresis, Western blot analysis was performed to detect caspase-8 (top panel) and FADD (bottom panel) in these FasR immunoprecipitates. Immunoprecipitation after anti-Fas antibody cross-linking of H9 cells (T-cell leukemia cell line) was used as a positive control. (B) Whole-cell lysates were prepared from a fraction of cells studied in these immunoprecipitation experiments after 1 and 6 hours of cross-linking with anti-Fas (Apo-1) antibody and isotype control (IgG3). Results shown represent the means (± SEM) of caspase-8, and -3/7 activities that were determined by a fluorometric enzyme activity assay. Protein bands for full-length caspase-8 (casp-8), FADD, and an activated cleaved product of caspase-8 (Ac casp-8) are indicated. NS = nonspecific band observed with the anti-caspase-8 antibody.

    Techniques Used: Cell Culture, Immunoprecipitation, Nucleic Acid Electrophoresis, Western Blot, Positive Control, Enzyme Activity Assay

    38) Product Images from "An Immunogenic and Protective Alphavirus Replicon Particle-Based Dengue Vaccine Overcomes Maternal Antibody Interference in Weanling Mice ▿"

    Article Title: An Immunogenic and Protective Alphavirus Replicon Particle-Based Dengue Vaccine Overcomes Maternal Antibody Interference in Weanling Mice ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00512-07

    Kinetics of DENV2-specific serum antibodies in young mice after prime and boost immunizations. Groups of six weanling mice were immunized at 3 and 15 wks after birth with 10 6 IU of DENV2 VRP (squares) or 10 6 PFU of DENV2 NGC (triangles). The mice were bled at the indicated times for detection of serum-specific DENV2 antibodies. (A) Total serum IgG titers were determined by ELISA. The data represent IgG ELISA endpoint dilution GMTs (OD 450 > 0.2). The error bars represent standard deviations. (B) Neutralizing-antibody (Ab) titers were measured by flow cytometry-based neutralization tests on Vero cells (F-NEUT). The data represent the F-NEUT 50 GMTs. The error bars represent standard errors. *, significantly different at a P value of
    Figure Legend Snippet: Kinetics of DENV2-specific serum antibodies in young mice after prime and boost immunizations. Groups of six weanling mice were immunized at 3 and 15 wks after birth with 10 6 IU of DENV2 VRP (squares) or 10 6 PFU of DENV2 NGC (triangles). The mice were bled at the indicated times for detection of serum-specific DENV2 antibodies. (A) Total serum IgG titers were determined by ELISA. The data represent IgG ELISA endpoint dilution GMTs (OD 450 > 0.2). The error bars represent standard deviations. (B) Neutralizing-antibody (Ab) titers were measured by flow cytometry-based neutralization tests on Vero cells (F-NEUT). The data represent the F-NEUT 50 GMTs. The error bars represent standard errors. *, significantly different at a P value of

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

    Expression of DENV2 prM and E proteins in Vero cells infected with DENV2 VRP. (A) Immunofluorescence. Vero cells were mock infected or infected with DENV2 VRP (MOI = 10); 24 h p.i., the cells were rinsed with PBS, fixed in cold methanol for 10 min, and stained with MAb 3H5 (1:400) specific for DENV2 E protein. Texas Red-conjugated anti-mouse IgG was used as a secondary antibody. (B) Immunoprecipitation. Vero cells were mock infected, infected with DENV2 prM/E-VRP, or infected with DENV2 (MOI = 10). VRP-infected cells were starved in methionine/cysteine-free medium for 1 h, followed by metabolic radiolabeling with 35 S Pro-Mix (100 μCi/ml) for 4 h. DENV2-infected cells were starved at 49 h p.i. and labeled for 5 h. DENV-specific proteins were immunoprecipitated from the cell lysates and pelleted media using HMAF. Proteins were separated in a 10% SDS-PAGE gel.
    Figure Legend Snippet: Expression of DENV2 prM and E proteins in Vero cells infected with DENV2 VRP. (A) Immunofluorescence. Vero cells were mock infected or infected with DENV2 VRP (MOI = 10); 24 h p.i., the cells were rinsed with PBS, fixed in cold methanol for 10 min, and stained with MAb 3H5 (1:400) specific for DENV2 E protein. Texas Red-conjugated anti-mouse IgG was used as a secondary antibody. (B) Immunoprecipitation. Vero cells were mock infected, infected with DENV2 prM/E-VRP, or infected with DENV2 (MOI = 10). VRP-infected cells were starved in methionine/cysteine-free medium for 1 h, followed by metabolic radiolabeling with 35 S Pro-Mix (100 μCi/ml) for 4 h. DENV2-infected cells were starved at 49 h p.i. and labeled for 5 h. DENV-specific proteins were immunoprecipitated from the cell lysates and pelleted media using HMAF. Proteins were separated in a 10% SDS-PAGE gel.

    Techniques Used: Expressing, Infection, Immunofluorescence, Staining, Immunoprecipitation, Radioactivity, Labeling, SDS Page

    Kinetics of DENV-specific IgG subclass distribution as a marker for Th1/Th2 responses after DENV2 VRP immunization (A) and DENV2 immunization (B). Weanling mice were immunized at 3 weeks and 15 weeks after birth with 10 6 IU of DENV2 VRP or 10 6 PFU DENV2 NCG. The mice were bled at the times indicated on the x axis. The data represent mean DENV-specific IgG2a (solid bars) and IgG1 (empty bars) ELISA endpoint dilution titers. The error bars represent standard deviations. IgG2a/IgG1 ratios are shown above the histograms. *, below the level of detection.
    Figure Legend Snippet: Kinetics of DENV-specific IgG subclass distribution as a marker for Th1/Th2 responses after DENV2 VRP immunization (A) and DENV2 immunization (B). Weanling mice were immunized at 3 weeks and 15 weeks after birth with 10 6 IU of DENV2 VRP or 10 6 PFU DENV2 NCG. The mice were bled at the times indicated on the x axis. The data represent mean DENV-specific IgG2a (solid bars) and IgG1 (empty bars) ELISA endpoint dilution titers. The error bars represent standard deviations. IgG2a/IgG1 ratios are shown above the histograms. *, below the level of detection.

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

    39) Product Images from "Macrophage Migration Inhibitory Factor (MIF) Is Essential for Type 2 Effector Cell Immunity to an Intestinal Helminth Parasite"

    Article Title: Macrophage Migration Inhibitory Factor (MIF) Is Essential for Type 2 Effector Cell Immunity to an Intestinal Helminth Parasite

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.02375

    Intact adaptive and regulatory responses in MIF-deficient mice. (A) Comparable anti-helminth humoral immunity in BALB/c and MIF −/− mice. Titers of HES-specific IgG1 serum antibodies from naïve and day 28-infected BALB/c and MIF −/− mice, assessed by ELISA. Data are representative of two independent experiments. (B) Comparable anti-helminth humoral immunity in HES-vaccinated BALB/c and MIF −/− mice. Parasite-specific antibody responses in vaccinated BALB/c and MIF −/− mice. HES-specific serum IgG1 levels were measured by ELISA on the day of challenge infection. Data are representative of two independent experiments. (C,D) Comparable adaptive type 2 immune responses in BALB/c and MIF −/− mice. Th2 cytokines from culture medium of MLNC from naïve and day 14 H. polygyrus -infected BALB/c and MIF −/− mice, restimulated with 1 μg/ml HES or media for 72 hours. Levels of IL-4 (C) and IL-13 (D) were measured by ELISA. Data are representative of two independent experiments. (E) Regulatory cell induction by helminth infection is comparable between BALB/c and MIF −/− mice. Numbers of CD4 + Foxp3 + Treg cells within MLNs from BALB/c and MIF −/− mice at days 14 and 28 post-infection with H. polygyrus . Data are representative of two independent experiments. n.s., not significant, ** p
    Figure Legend Snippet: Intact adaptive and regulatory responses in MIF-deficient mice. (A) Comparable anti-helminth humoral immunity in BALB/c and MIF −/− mice. Titers of HES-specific IgG1 serum antibodies from naïve and day 28-infected BALB/c and MIF −/− mice, assessed by ELISA. Data are representative of two independent experiments. (B) Comparable anti-helminth humoral immunity in HES-vaccinated BALB/c and MIF −/− mice. Parasite-specific antibody responses in vaccinated BALB/c and MIF −/− mice. HES-specific serum IgG1 levels were measured by ELISA on the day of challenge infection. Data are representative of two independent experiments. (C,D) Comparable adaptive type 2 immune responses in BALB/c and MIF −/− mice. Th2 cytokines from culture medium of MLNC from naïve and day 14 H. polygyrus -infected BALB/c and MIF −/− mice, restimulated with 1 μg/ml HES or media for 72 hours. Levels of IL-4 (C) and IL-13 (D) were measured by ELISA. Data are representative of two independent experiments. (E) Regulatory cell induction by helminth infection is comparable between BALB/c and MIF −/− mice. Numbers of CD4 + Foxp3 + Treg cells within MLNs from BALB/c and MIF −/− mice at days 14 and 28 post-infection with H. polygyrus . Data are representative of two independent experiments. n.s., not significant, ** p

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

    Myeloid cell infiltration around tissue larvae of H. polygyrus with expression of Arginase-1 and MIF. (A) M2 macrophages infiltrate the infection site of H. polygyrus larvae in the small intestine. Arginase, CD68 and EpCam staining of granulomas around larval parasites in the small intestinal submucosa at days 4 and 6 of H. polygyrus infection in BALB/c and MIF −/− mice. Images are representative of two experiments with similar results. (B,C) Analysis of intensity of Arginase staining in granulomas at days 4 and 6 after H. polygyrus infection. For each group at each time point, granulomas were analyzed from 4 individual animals, and intensity data pooled. Each data point represents an individual granuloma. (D) MIF is expressed in the infection site of H. polygyrus larvae in the small intestine. Intense staining of polyclonal rabbit anti-MIF antibody is observed in the granulomas around larval parasites in the small intestinal submucosa at day 6 of H. polygyrus infection in BALB/c mice, but not in MIF −/− animals or in sections stained with isotype control IgG. In addition, widespread specific antibody staining is seen throughout the submucosal tissue. Images were collected on a Leica compound microscope. Scale bars represent 500 and 250 μm.
    Figure Legend Snippet: Myeloid cell infiltration around tissue larvae of H. polygyrus with expression of Arginase-1 and MIF. (A) M2 macrophages infiltrate the infection site of H. polygyrus larvae in the small intestine. Arginase, CD68 and EpCam staining of granulomas around larval parasites in the small intestinal submucosa at days 4 and 6 of H. polygyrus infection in BALB/c and MIF −/− mice. Images are representative of two experiments with similar results. (B,C) Analysis of intensity of Arginase staining in granulomas at days 4 and 6 after H. polygyrus infection. For each group at each time point, granulomas were analyzed from 4 individual animals, and intensity data pooled. Each data point represents an individual granuloma. (D) MIF is expressed in the infection site of H. polygyrus larvae in the small intestine. Intense staining of polyclonal rabbit anti-MIF antibody is observed in the granulomas around larval parasites in the small intestinal submucosa at day 6 of H. polygyrus infection in BALB/c mice, but not in MIF −/− animals or in sections stained with isotype control IgG. In addition, widespread specific antibody staining is seen throughout the submucosal tissue. Images were collected on a Leica compound microscope. Scale bars represent 500 and 250 μm.

    Techniques Used: Expressing, Infection, Staining, Mouse Assay, Microscopy

    40) Product Images from "Robust Immunity and Heterologous Protection against Influenza in Mice Elicited by a Novel Recombinant NP-M2e Fusion Protein Expressed in E. coli"

    Article Title: Robust Immunity and Heterologous Protection against Influenza in Mice Elicited by a Novel Recombinant NP-M2e Fusion Protein Expressed in E. coli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0052488

    Antibody response trend and long-term humoral immune response induced by NM2e protein in mice. (A and B) Mice were immunized intramuscularly with 10 µg of NM2e protein three times at 2-week intervals. Al(OH) 3 and/or CpG 1826 were used as adjuvants. Mice immunized with normal saline (NS) or adjuvant alone was used as negative controls. Serum was obtained from each mouse on days 14, 28, and 38, respectively, and analyzed for the presence of IgG antibodies specific for NP (left) or M2e (right), in an ELISA, as described in the Materials and Methods. Antibody response trends after three immunizations are presented in A, and the comparison of results on day 38 are presented in B. Columns show geometric mean antibody titers, and bars indicate the 95% confidence interval in each group. Plots in B show the NP- and M2e-specific IgG titers of all of the mice in each treatment group on day 38, and bars indicate the geometric mean antibody titers of each treatment group ( n = 6 mice per experimental group, except n = 5 mice in the NS group). Lines above two or more groups indicate that they have the same comparative results. *, p ≤0.05; **, p ≤0.01; ***, p ≤0.001 by one-way ANOVA. (C) Mice were immunized intramuscularly with 10 µg of NM2e protein formulated with Al(OH) 3 three times at 2-week intervals or immunized with a single dose of 10 µg of NM2e formulated with Al(OH) 3 . Serum was prepared from each mouse at the indicated times, and NP- and M2e-specific IgG antibodies were analyzed by ELISA, as described in the Materials and Methods.
    Figure Legend Snippet: Antibody response trend and long-term humoral immune response induced by NM2e protein in mice. (A and B) Mice were immunized intramuscularly with 10 µg of NM2e protein three times at 2-week intervals. Al(OH) 3 and/or CpG 1826 were used as adjuvants. Mice immunized with normal saline (NS) or adjuvant alone was used as negative controls. Serum was obtained from each mouse on days 14, 28, and 38, respectively, and analyzed for the presence of IgG antibodies specific for NP (left) or M2e (right), in an ELISA, as described in the Materials and Methods. Antibody response trends after three immunizations are presented in A, and the comparison of results on day 38 are presented in B. Columns show geometric mean antibody titers, and bars indicate the 95% confidence interval in each group. Plots in B show the NP- and M2e-specific IgG titers of all of the mice in each treatment group on day 38, and bars indicate the geometric mean antibody titers of each treatment group ( n = 6 mice per experimental group, except n = 5 mice in the NS group). Lines above two or more groups indicate that they have the same comparative results. *, p ≤0.05; **, p ≤0.01; ***, p ≤0.001 by one-way ANOVA. (C) Mice were immunized intramuscularly with 10 µg of NM2e protein formulated with Al(OH) 3 three times at 2-week intervals or immunized with a single dose of 10 µg of NM2e formulated with Al(OH) 3 . Serum was prepared from each mouse at the indicated times, and NP- and M2e-specific IgG antibodies were analyzed by ELISA, as described in the Materials and Methods.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Correlations between survival percentage and immune responses in mice. A, Correlation analysis was conducted to determine the relationships of the survival percentage data from Fig. 6 with the NP-, M2e-specific IgG (left) ELISA data from Fig. 3, IgG1 (middle) and IgG2a (right) ELISA data in Fig. 4. Log conversion was performed for the murine serum antibody titers. B, Correlation analysis was conducted to determine the relationships of the survival percentage data in Fig. 6 with the IFN-γ- (left) IL-4- (middle), and IL-10-secreting (right) SMNCs stimulated with NP147-155, NP55-69, or M2e peptide pool based on the ELISPOT data in Fig. 5.
    Figure Legend Snippet: Correlations between survival percentage and immune responses in mice. A, Correlation analysis was conducted to determine the relationships of the survival percentage data from Fig. 6 with the NP-, M2e-specific IgG (left) ELISA data from Fig. 3, IgG1 (middle) and IgG2a (right) ELISA data in Fig. 4. Log conversion was performed for the murine serum antibody titers. B, Correlation analysis was conducted to determine the relationships of the survival percentage data in Fig. 6 with the IFN-γ- (left) IL-4- (middle), and IL-10-secreting (right) SMNCs stimulated with NP147-155, NP55-69, or M2e peptide pool based on the ELISPOT data in Fig. 5.

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

    IgG1 and IgG2a isotypes in serum from NM2e-immunized mice. Mice were treated as described in Fig. 3. NP- and M2e-specific IgG isotypes in mouse serum were analyzed by ELISA. The plots show the (A) NP- (left) and M2e-specific (right) IgG1 isotypes and (B) the respective IgG2a isotypes. The scatter dot plots show the results for every mouse in each group, and the bars show the geometric mean of each group. The plots in (C) present the NP-(left) and M2e-specific (right) IgG1/IgG2a ratios, and the bars show the means with SD. Lines above two or more groups indicate that they have the same comparative results. *, p ≤0.05; **, p ≤0.01; ***, p ≤0.001 by one-way ANOVA.
    Figure Legend Snippet: IgG1 and IgG2a isotypes in serum from NM2e-immunized mice. Mice were treated as described in Fig. 3. NP- and M2e-specific IgG isotypes in mouse serum were analyzed by ELISA. The plots show the (A) NP- (left) and M2e-specific (right) IgG1 isotypes and (B) the respective IgG2a isotypes. The scatter dot plots show the results for every mouse in each group, and the bars show the geometric mean of each group. The plots in (C) present the NP-(left) and M2e-specific (right) IgG1/IgG2a ratios, and the bars show the means with SD. Lines above two or more groups indicate that they have the same comparative results. *, p ≤0.05; **, p ≤0.01; ***, p ≤0.001 by one-way ANOVA.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

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    SouthernBiotech igg1
    Production of Sm29-specific antibodies in immunized mice. Sera from mice were obtained 15 days after each immunization dose and were assessed to determine the levels of IgG (A) , <t>IgG1</t> (B) , IgG2a (C) , and IgE (D) antibodies against rSm29 in the animals inoculated with alum (open bars), MPLA (bright-gray bars), Sm29Alum (black bars) or Sm29/MPLA (dark-gray bar). Significant differences between adjuvant control and experimental groups are indicated by an asterisk ( p
    Igg1, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 94/100, based on 899 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SouthernBiotech anti falvac 1a immunoglobulin g igg
    Antibody responses to <t>FALVAC-1A</t> vaccine formulations in congenic mice. Mice were immunized with 10 μg FALVAC-1A/dose formulated in copolymer CRL-1005 (Copol/FAL), Montanide ISA-720 (ISA-720/FAL), QS-21 (QS-21/FAL), aluminum (AlPO4/FAL), or PBS (FALVAC-1A) on days 0, 14, and 28. Total <t>IgG</t> antibody titers (geometric mean of 10 mice) were determined as the geometric mean of the reciprocal dilution where the OD 450 of the titration curve equaled 0.1 (the maximum value of preimmune sera at 1:100).
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    SouthernBiotech anti mouse igg2a
    Contribution of type I IFN signaling. Ifnar1 −/− (gray bars) and 129sv/ew (black bars) mice were immunized with β-Gal alone or with indicated adjuvant. 0.2 µg of soluble flagellin and 10 6 IU of VREP constructs were used. Control mice were given VREP-OVA. Each immunized group consisted of five mice, and two control mice were used. Serum was assayed for anti-β-Gal IgG, IgG1 and <t>IgG2a</t> by ELISA. A one-way ANOVA with Bonferroni post-hoc test was used to compare the response between WT and Ifnar1 −/− mice given the same vaccination. * P
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    SouthernBiotech hel binding igg
    Islet-reactive T cells cause germinal center formation, autoantibodies, and diabetes in NOD k mice. (a) Pancreatic islets stained with H E from TCR/InsHEL mice of the indicated genotypes. Right panel is a frozen section stained with peanut agglutinin (blue) and anti-IgD (brown). GC, germinal center. (b) Onset of diabetes in TCR/insHEL female mice of the indicated genotypes. Data represent > 20 mice per group. (c) <t>HEL-binding</t> <t>IgG</t> titre in serum from diabetic double transgenic mice, collected within 2 wk of diabetes onset. Titres are expressed in arbitrary units relative to a reference immune sera from HEL-immunized nontransgenic mice set at 100 units.
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    Production of Sm29-specific antibodies in immunized mice. Sera from mice were obtained 15 days after each immunization dose and were assessed to determine the levels of IgG (A) , IgG1 (B) , IgG2a (C) , and IgE (D) antibodies against rSm29 in the animals inoculated with alum (open bars), MPLA (bright-gray bars), Sm29Alum (black bars) or Sm29/MPLA (dark-gray bar). Significant differences between adjuvant control and experimental groups are indicated by an asterisk ( p

    Journal: Frontiers in Immunology

    Article Title: A Strong Humoral Immune Response Induced by a Vaccine Formulation Containing rSm29 Adsorbed to Alum Is Associated With Protection Against Schistosoma mansoni Reinfection in Mice

    doi: 10.3389/fimmu.2018.02488

    Figure Lengend Snippet: Production of Sm29-specific antibodies in immunized mice. Sera from mice were obtained 15 days after each immunization dose and were assessed to determine the levels of IgG (A) , IgG1 (B) , IgG2a (C) , and IgE (D) antibodies against rSm29 in the animals inoculated with alum (open bars), MPLA (bright-gray bars), Sm29Alum (black bars) or Sm29/MPLA (dark-gray bar). Significant differences between adjuvant control and experimental groups are indicated by an asterisk ( p

    Article Snippet: One hundred microliters of each serum sample was diluted 1:1,000 (for IgG, IgG1, and IgG2a) or 1:40 (for IgE) and added to the plates for 1 h. Finally, the plates were incubated with peroxidase-conjugated anti-mouse IgG (1:10,000), IgG1, (1:10,000), or IgG2a (1:8,000) (Southern Biotech, Birmingham, AL, United States), for 1 h at 25°C.

    Techniques: Mouse Assay

    Antibody responses to FALVAC-1A vaccine formulations in congenic mice. Mice were immunized with 10 μg FALVAC-1A/dose formulated in copolymer CRL-1005 (Copol/FAL), Montanide ISA-720 (ISA-720/FAL), QS-21 (QS-21/FAL), aluminum (AlPO4/FAL), or PBS (FALVAC-1A) on days 0, 14, and 28. Total IgG antibody titers (geometric mean of 10 mice) were determined as the geometric mean of the reciprocal dilution where the OD 450 of the titration curve equaled 0.1 (the maximum value of preimmune sera at 1:100).

    Journal: Clinical and Vaccine Immunology : CVI

    Article Title: Immune Responses of Mice with Different Genetic Backgrounds to Improved Multiepitope, Multitarget Malaria Vaccine Candidate Antigen FALVAC-1A ▿

    doi: 10.1128/CVI.00164-08

    Figure Lengend Snippet: Antibody responses to FALVAC-1A vaccine formulations in congenic mice. Mice were immunized with 10 μg FALVAC-1A/dose formulated in copolymer CRL-1005 (Copol/FAL), Montanide ISA-720 (ISA-720/FAL), QS-21 (QS-21/FAL), aluminum (AlPO4/FAL), or PBS (FALVAC-1A) on days 0, 14, and 28. Total IgG antibody titers (geometric mean of 10 mice) were determined as the geometric mean of the reciprocal dilution where the OD 450 of the titration curve equaled 0.1 (the maximum value of preimmune sera at 1:100).

    Article Snippet: To determine the titers of total anti-FALVAC-1A immunoglobulin G (IgG), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Southern Biotech, Birmingham, AL) was used at a dilution of 1:5,000.

    Techniques: Mouse Assay, Titration

    Reactivity of mice anti-FALVAC-1A sera with P. falciparum sporozoites and blood-stage parasites. Day 42 sera were tested by IFA. Each bar represents the geometric mean titer of the total IgG antibody titers for 10 mice induced by the given adjuvant formulation.

    Journal: Clinical and Vaccine Immunology : CVI

    Article Title: Immune Responses of Mice with Different Genetic Backgrounds to Improved Multiepitope, Multitarget Malaria Vaccine Candidate Antigen FALVAC-1A ▿

    doi: 10.1128/CVI.00164-08

    Figure Lengend Snippet: Reactivity of mice anti-FALVAC-1A sera with P. falciparum sporozoites and blood-stage parasites. Day 42 sera were tested by IFA. Each bar represents the geometric mean titer of the total IgG antibody titers for 10 mice induced by the given adjuvant formulation.

    Article Snippet: To determine the titers of total anti-FALVAC-1A immunoglobulin G (IgG), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Southern Biotech, Birmingham, AL) was used at a dilution of 1:5,000.

    Techniques: Mouse Assay, Immunofluorescence

    Contribution of type I IFN signaling. Ifnar1 −/− (gray bars) and 129sv/ew (black bars) mice were immunized with β-Gal alone or with indicated adjuvant. 0.2 µg of soluble flagellin and 10 6 IU of VREP constructs were used. Control mice were given VREP-OVA. Each immunized group consisted of five mice, and two control mice were used. Serum was assayed for anti-β-Gal IgG, IgG1 and IgG2a by ELISA. A one-way ANOVA with Bonferroni post-hoc test was used to compare the response between WT and Ifnar1 −/− mice given the same vaccination. * P

    Journal: PLoS ONE

    Article Title: The Adjuvant Activity of Alphavirus Replicons Is Enhanced by Incorporating the Microbial Molecule Flagellin into the Replicon

    doi: 10.1371/journal.pone.0065964

    Figure Lengend Snippet: Contribution of type I IFN signaling. Ifnar1 −/− (gray bars) and 129sv/ew (black bars) mice were immunized with β-Gal alone or with indicated adjuvant. 0.2 µg of soluble flagellin and 10 6 IU of VREP constructs were used. Control mice were given VREP-OVA. Each immunized group consisted of five mice, and two control mice were used. Serum was assayed for anti-β-Gal IgG, IgG1 and IgG2a by ELISA. A one-way ANOVA with Bonferroni post-hoc test was used to compare the response between WT and Ifnar1 −/− mice given the same vaccination. * P

    Article Snippet: After 2 h incubation at room temperature, plates were washed five times with PBS plus 0.05% Tween, and horseradish peroxidase-conjugated anti-mouse-IgG, anti-mouse-IgG1, anti-mouse-IgG2a or anti-mouse-IgG2c (all Southern Biotech, Birmingham, AL) was added and incubated for 1.5 h. Plates were subsequently washed five times with PBS plus 0.05% Tween and the o-phenylenediamine dihydrochloride substrate (Sigma) was added for detection of antibodies.

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

    Antibody responses after multiple immunizations. C57/BL6 mice were immunized two times with OVA protein alone or mixed with 10 6 IU VREP-FliC-D3. Control mice were given PBS. Each immunized group consisted of five mice, and two control mice were used. Serum was assayed for anti-OVA IgG by ELISA after one (black bars) or two (gray bars) immunizations. A Student’s t -test was used to compare the response after one and two administrations of the same vaccine. * P

    Journal: PLoS ONE

    Article Title: The Adjuvant Activity of Alphavirus Replicons Is Enhanced by Incorporating the Microbial Molecule Flagellin into the Replicon

    doi: 10.1371/journal.pone.0065964

    Figure Lengend Snippet: Antibody responses after multiple immunizations. C57/BL6 mice were immunized two times with OVA protein alone or mixed with 10 6 IU VREP-FliC-D3. Control mice were given PBS. Each immunized group consisted of five mice, and two control mice were used. Serum was assayed for anti-OVA IgG by ELISA after one (black bars) or two (gray bars) immunizations. A Student’s t -test was used to compare the response after one and two administrations of the same vaccine. * P

    Article Snippet: After 2 h incubation at room temperature, plates were washed five times with PBS plus 0.05% Tween, and horseradish peroxidase-conjugated anti-mouse-IgG, anti-mouse-IgG1, anti-mouse-IgG2a or anti-mouse-IgG2c (all Southern Biotech, Birmingham, AL) was added and incubated for 1.5 h. Plates were subsequently washed five times with PBS plus 0.05% Tween and the o-phenylenediamine dihydrochloride substrate (Sigma) was added for detection of antibodies.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Antibody responses induced by VREP and sFliC-D3. 129sv/ew mice were immunized with β-Gal alone or with indicated adjuvant. VREP-OVA is indicated as ‘VREP’, and the dose used was 10 6 IU. Doses of sFliC-D3 are indicated in the figure. Control mice were given PBS. Each immunized group consisted of five mice, and two control mice were used. Serum was assayed for anti-β-Gal IgG, IgG1 and IgG2a by ELISA. A one-way ANOVA with Bonferroni post-hoc test was used to compare the response between mice given the same dose of sFliC-D3, between mice given VREP+sFliC-D3 and mice given VREP, as well as all adjuvanted groups and mice given β-Gal alone. ** P

    Journal: PLoS ONE

    Article Title: The Adjuvant Activity of Alphavirus Replicons Is Enhanced by Incorporating the Microbial Molecule Flagellin into the Replicon

    doi: 10.1371/journal.pone.0065964

    Figure Lengend Snippet: Antibody responses induced by VREP and sFliC-D3. 129sv/ew mice were immunized with β-Gal alone or with indicated adjuvant. VREP-OVA is indicated as ‘VREP’, and the dose used was 10 6 IU. Doses of sFliC-D3 are indicated in the figure. Control mice were given PBS. Each immunized group consisted of five mice, and two control mice were used. Serum was assayed for anti-β-Gal IgG, IgG1 and IgG2a by ELISA. A one-way ANOVA with Bonferroni post-hoc test was used to compare the response between mice given the same dose of sFliC-D3, between mice given VREP+sFliC-D3 and mice given VREP, as well as all adjuvanted groups and mice given β-Gal alone. ** P

    Article Snippet: After 2 h incubation at room temperature, plates were washed five times with PBS plus 0.05% Tween, and horseradish peroxidase-conjugated anti-mouse-IgG, anti-mouse-IgG1, anti-mouse-IgG2a or anti-mouse-IgG2c (all Southern Biotech, Birmingham, AL) was added and incubated for 1.5 h. Plates were subsequently washed five times with PBS plus 0.05% Tween and the o-phenylenediamine dihydrochloride substrate (Sigma) was added for detection of antibodies.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Contribution of TLR5 signaling. Tlr5 −/− (gray bars) and C57BL/6N (black bars) mice were immunized with β-Gal alone or with indicated adjuvant. 0.2 µg of soluble flagellin and 10 6 IU of VREP constructs were used. Control mice were given VREP-OVA. Each immunized group consisted of five (WT) or six ( Tlr5 −/− ) mice, and two-three control mice were used. Serum was assayed for anti-β-Gal IgG, IgG1 and IgG2c by ELISA. A one-way ANOVA with Bonferroni post-hoc test was used to compare the response between WT and Tlr5 −/− mice given the same vaccination. * P

    Journal: PLoS ONE

    Article Title: The Adjuvant Activity of Alphavirus Replicons Is Enhanced by Incorporating the Microbial Molecule Flagellin into the Replicon

    doi: 10.1371/journal.pone.0065964

    Figure Lengend Snippet: Contribution of TLR5 signaling. Tlr5 −/− (gray bars) and C57BL/6N (black bars) mice were immunized with β-Gal alone or with indicated adjuvant. 0.2 µg of soluble flagellin and 10 6 IU of VREP constructs were used. Control mice were given VREP-OVA. Each immunized group consisted of five (WT) or six ( Tlr5 −/− ) mice, and two-three control mice were used. Serum was assayed for anti-β-Gal IgG, IgG1 and IgG2c by ELISA. A one-way ANOVA with Bonferroni post-hoc test was used to compare the response between WT and Tlr5 −/− mice given the same vaccination. * P

    Article Snippet: After 2 h incubation at room temperature, plates were washed five times with PBS plus 0.05% Tween, and horseradish peroxidase-conjugated anti-mouse-IgG, anti-mouse-IgG1, anti-mouse-IgG2a or anti-mouse-IgG2c (all Southern Biotech, Birmingham, AL) was added and incubated for 1.5 h. Plates were subsequently washed five times with PBS plus 0.05% Tween and the o-phenylenediamine dihydrochloride substrate (Sigma) was added for detection of antibodies.

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

    Antibody responses induced by different doses of VREP-FliC-D3. 129sv/ew mice were immunized with β-Gal alone or with adjuvant, as indicated. Three different doses of VREP-FliC-D3 and VREP-OVA were used: 10 6 IU, 10 5 IU and 10 4 IU. Control mice were given PBS. Each immunized group consisted of five mice, and four control mice were used. Serum was assayed for anti-β-Gal IgG, IgG1 and IgG2a by ELISA. A one-way ANOVA with Bonferroni post-hoc test was used to compare the response between mice given the same dose of adjuvant as well as between groups given adjuvant and the control group given β-Gal without adjuvant. * P

    Journal: PLoS ONE

    Article Title: The Adjuvant Activity of Alphavirus Replicons Is Enhanced by Incorporating the Microbial Molecule Flagellin into the Replicon

    doi: 10.1371/journal.pone.0065964

    Figure Lengend Snippet: Antibody responses induced by different doses of VREP-FliC-D3. 129sv/ew mice were immunized with β-Gal alone or with adjuvant, as indicated. Three different doses of VREP-FliC-D3 and VREP-OVA were used: 10 6 IU, 10 5 IU and 10 4 IU. Control mice were given PBS. Each immunized group consisted of five mice, and four control mice were used. Serum was assayed for anti-β-Gal IgG, IgG1 and IgG2a by ELISA. A one-way ANOVA with Bonferroni post-hoc test was used to compare the response between mice given the same dose of adjuvant as well as between groups given adjuvant and the control group given β-Gal without adjuvant. * P

    Article Snippet: After 2 h incubation at room temperature, plates were washed five times with PBS plus 0.05% Tween, and horseradish peroxidase-conjugated anti-mouse-IgG, anti-mouse-IgG1, anti-mouse-IgG2a or anti-mouse-IgG2c (all Southern Biotech, Birmingham, AL) was added and incubated for 1.5 h. Plates were subsequently washed five times with PBS plus 0.05% Tween and the o-phenylenediamine dihydrochloride substrate (Sigma) was added for detection of antibodies.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Adjuvant effect of VREP encoding FliC-WT or FliC-D3. 129sv/ew mice were immunized with β-Gal alone or with indicated adjuvant. 0.2 µg of soluble flagellin and 10 6 IU of VREP constructs were used. Control mice were given PBS. Each immunized group consisted of five to six mice, and two control mice were used. Serum was assayed for anti-β-Gal IgG, IgG1 and IgG2a by ELISA. A one-way ANOVA with Bonferroni post-hoc test was used to compare the response between mice given sFliC-WT and VREP-FliC-WT, between mice given sFliC-D3 and VREP-FliC-D3, between mice given VREP-FliC-WT or VREP-FliC-D3 and VREP, as well as all adjuvanted groups and mice given β-Gal alone. * P

    Journal: PLoS ONE

    Article Title: The Adjuvant Activity of Alphavirus Replicons Is Enhanced by Incorporating the Microbial Molecule Flagellin into the Replicon

    doi: 10.1371/journal.pone.0065964

    Figure Lengend Snippet: Adjuvant effect of VREP encoding FliC-WT or FliC-D3. 129sv/ew mice were immunized with β-Gal alone or with indicated adjuvant. 0.2 µg of soluble flagellin and 10 6 IU of VREP constructs were used. Control mice were given PBS. Each immunized group consisted of five to six mice, and two control mice were used. Serum was assayed for anti-β-Gal IgG, IgG1 and IgG2a by ELISA. A one-way ANOVA with Bonferroni post-hoc test was used to compare the response between mice given sFliC-WT and VREP-FliC-WT, between mice given sFliC-D3 and VREP-FliC-D3, between mice given VREP-FliC-WT or VREP-FliC-D3 and VREP, as well as all adjuvanted groups and mice given β-Gal alone. * P

    Article Snippet: After 2 h incubation at room temperature, plates were washed five times with PBS plus 0.05% Tween, and horseradish peroxidase-conjugated anti-mouse-IgG, anti-mouse-IgG1, anti-mouse-IgG2a or anti-mouse-IgG2c (all Southern Biotech, Birmingham, AL) was added and incubated for 1.5 h. Plates were subsequently washed five times with PBS plus 0.05% Tween and the o-phenylenediamine dihydrochloride substrate (Sigma) was added for detection of antibodies.

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

    Antibody responses against β-Gal expressed from VREP. 129sv/ew (A) or BALB/c (B) mice were immunized with the indicated regimen. Doses used were: 10 6 infectious units (IU) of VREP particles, 0.2 µg sFliC-D3 and 1 µg sFliC-WT. When two different VREP particles were given to the same mouse, 5×10 5 IU of each VREP was given. Each immunized group consisted of five mice, and one to two control mice was used. Serum was assayed for anti-β-Gal IgG by ELISA. A one-way ANOVA with Bonferroni post-hoc test of the response between vaccinated groups revealed no significant differences.

    Journal: PLoS ONE

    Article Title: The Adjuvant Activity of Alphavirus Replicons Is Enhanced by Incorporating the Microbial Molecule Flagellin into the Replicon

    doi: 10.1371/journal.pone.0065964

    Figure Lengend Snippet: Antibody responses against β-Gal expressed from VREP. 129sv/ew (A) or BALB/c (B) mice were immunized with the indicated regimen. Doses used were: 10 6 infectious units (IU) of VREP particles, 0.2 µg sFliC-D3 and 1 µg sFliC-WT. When two different VREP particles were given to the same mouse, 5×10 5 IU of each VREP was given. Each immunized group consisted of five mice, and one to two control mice was used. Serum was assayed for anti-β-Gal IgG by ELISA. A one-way ANOVA with Bonferroni post-hoc test of the response between vaccinated groups revealed no significant differences.

    Article Snippet: After 2 h incubation at room temperature, plates were washed five times with PBS plus 0.05% Tween, and horseradish peroxidase-conjugated anti-mouse-IgG, anti-mouse-IgG1, anti-mouse-IgG2a or anti-mouse-IgG2c (all Southern Biotech, Birmingham, AL) was added and incubated for 1.5 h. Plates were subsequently washed five times with PBS plus 0.05% Tween and the o-phenylenediamine dihydrochloride substrate (Sigma) was added for detection of antibodies.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Islet-reactive T cells cause germinal center formation, autoantibodies, and diabetes in NOD k mice. (a) Pancreatic islets stained with H E from TCR/InsHEL mice of the indicated genotypes. Right panel is a frozen section stained with peanut agglutinin (blue) and anti-IgD (brown). GC, germinal center. (b) Onset of diabetes in TCR/insHEL female mice of the indicated genotypes. Data represent > 20 mice per group. (c) HEL-binding IgG titre in serum from diabetic double transgenic mice, collected within 2 wk of diabetes onset. Titres are expressed in arbitrary units relative to a reference immune sera from HEL-immunized nontransgenic mice set at 100 units.

    Journal: The Journal of Experimental Medicine

    Article Title: Failure to Censor Forbidden Clones of CD4 T Cells in Autoimmune Diabetes

    doi: 10.1084/jem.20020735

    Figure Lengend Snippet: Islet-reactive T cells cause germinal center formation, autoantibodies, and diabetes in NOD k mice. (a) Pancreatic islets stained with H E from TCR/InsHEL mice of the indicated genotypes. Right panel is a frozen section stained with peanut agglutinin (blue) and anti-IgD (brown). GC, germinal center. (b) Onset of diabetes in TCR/insHEL female mice of the indicated genotypes. Data represent > 20 mice per group. (c) HEL-binding IgG titre in serum from diabetic double transgenic mice, collected within 2 wk of diabetes onset. Titres are expressed in arbitrary units relative to a reference immune sera from HEL-immunized nontransgenic mice set at 100 units.

    Article Snippet: HEL-binding IgG was measured in serum by ELISA on Nunc maxisorp plates coated with 100 μg/ml HEL, developed with sheep anti–mouse IgG-alkaline phosphatase (Southern Biotech Associates, Inc.) and Sigma Substrate 104.

    Techniques: Mouse Assay, Staining, Binding Assay, Transgenic Assay