mouse iga  (Thermo Fisher)


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    Name:
    IgA Mouse ELISA Kit
    Description:
    The Mouse IgA ELISA research use only kit is an enzyme linked immunosorbent assay designed for the quantitation and detection of IgA in samples see sample types indicated using a microplate reader The assay will recognize both natural and recombinant forms of this target This complete ready to use kit has been developed and optimized to specifically detect IgA and includes antibody pre coated plate s and other components to perform the assay Using the supplied pre coated pre calibrated plate helps reduce variability issues associated with using other kits and assays that require you to coat plates with consistent levels of antibody Performance characteristics• Sensitivity 1 5 ng mL• Standard curve range 1 5 ng mL 500 ng mL• Sample type s cell culture supernatants plasma serum• Specificity This ELISA pair antibody detects mouse IgA Other species not determined • Sample volume 100 µL per sample after dilution• Total assay time 5 hoursRigorous validationTo help ensure sensitive accurate and consistent performance this ELISA kit is quality tested for criteria such as sensitivity specificity precision lot to lot consistency recovery linearity and parallelism See product inserts for further information on validation for this kit Learn more about our rigorous ELISA validation and quality testing process Principle of the methodThis IgA solid phase sandwich ELISA enzyme linked immunosorbent assay is designed to measure and detect the amount of the target bound between a matched antibody pair A target specific antibody has been pre coated in the wells of the supplied microplate Samples standards or controls are then added into these wells and bind to the immobilized capture antibody The sandwich is formed by the addition of the second detector antibody binding to the target on a different epitope from the capture antibody A conjugated enzyme has been incorporated into the assay After incubation periods and wash steps to remove unbound antibody from the plate a substrate solution is added that reacts with the enzyme antibody target complex to produce measurable signal The intensity of this signal is proportional to the concentration of target present in the original specimen Related linksLearn more about ELISA kits Learn more about other immunoassays
    Catalog Number:
    emiga
    Price:
    None
    Applications:
    Protein Assays and Analysis|Protein Biology
    Category:
    Kits and Assays
    Buy from Supplier


    Structured Review

    Thermo Fisher mouse iga
    Increased pneumococcal binding capacities by lung mucosal fluid in inflamed lungs. Pneumococci were co-incubated with bronchoalveolar lavage fluid (BALF) supernatants from SPC-HA and SPC-HAxTCR-HA mice. Bacteria were stained with anti-mouse <t>IgA</t> or <t>anti-IgM</t> antibodies and analyzed by flow cytometry (FACS). ( a ) Representative FACS plots of IgA+ pneumococci incubated with BALF from SPC-HA or SPC-HAxTCR-mice; control samples (CTRL) were stained with anti-IgA without prior incubation with BALF. ( b ) Percentages of IgA+ pneumococci and relative fluorescence intensities ( c ) of IgA+ pneumococci. ( d ) Representative FACS plots of IgM+ pneumococci incubated with BALF from SPC-HA or SPC-HAxTCR-mice; control samples (CTRL) were stained with anti-IgM without prior incubation with BALF. ( e ) Percentages of IgM+ pneumococci and relative fluorescence intensities ( f ) of IgM+ pneumococci. Relative fluorescence intensities are calculated by the ratio of the MFI of each individual sample over the mean MFI of the SPC-HA control group. Data are pooled from 2 independent experiments with similar results. *p
    The Mouse IgA ELISA research use only kit is an enzyme linked immunosorbent assay designed for the quantitation and detection of IgA in samples see sample types indicated using a microplate reader The assay will recognize both natural and recombinant forms of this target This complete ready to use kit has been developed and optimized to specifically detect IgA and includes antibody pre coated plate s and other components to perform the assay Using the supplied pre coated pre calibrated plate helps reduce variability issues associated with using other kits and assays that require you to coat plates with consistent levels of antibody Performance characteristics• Sensitivity 1 5 ng mL• Standard curve range 1 5 ng mL 500 ng mL• Sample type s cell culture supernatants plasma serum• Specificity This ELISA pair antibody detects mouse IgA Other species not determined • Sample volume 100 µL per sample after dilution• Total assay time 5 hoursRigorous validationTo help ensure sensitive accurate and consistent performance this ELISA kit is quality tested for criteria such as sensitivity specificity precision lot to lot consistency recovery linearity and parallelism See product inserts for further information on validation for this kit Learn more about our rigorous ELISA validation and quality testing process Principle of the methodThis IgA solid phase sandwich ELISA enzyme linked immunosorbent assay is designed to measure and detect the amount of the target bound between a matched antibody pair A target specific antibody has been pre coated in the wells of the supplied microplate Samples standards or controls are then added into these wells and bind to the immobilized capture antibody The sandwich is formed by the addition of the second detector antibody binding to the target on a different epitope from the capture antibody A conjugated enzyme has been incorporated into the assay After incubation periods and wash steps to remove unbound antibody from the plate a substrate solution is added that reacts with the enzyme antibody target complex to produce measurable signal The intensity of this signal is proportional to the concentration of target present in the original specimen Related linksLearn more about ELISA kits Learn more about other immunoassays
    https://www.bioz.com/result/mouse iga/product/Thermo Fisher
    Average 92 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    mouse iga - by Bioz Stars, 2020-07
    92/100 stars

    Images

    1) Product Images from "Chronic lung inflammation primes humoral immunity and augments antipneumococcal resistance"

    Article Title: Chronic lung inflammation primes humoral immunity and augments antipneumococcal resistance

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-05212-4

    Increased pneumococcal binding capacities by lung mucosal fluid in inflamed lungs. Pneumococci were co-incubated with bronchoalveolar lavage fluid (BALF) supernatants from SPC-HA and SPC-HAxTCR-HA mice. Bacteria were stained with anti-mouse IgA or anti-IgM antibodies and analyzed by flow cytometry (FACS). ( a ) Representative FACS plots of IgA+ pneumococci incubated with BALF from SPC-HA or SPC-HAxTCR-mice; control samples (CTRL) were stained with anti-IgA without prior incubation with BALF. ( b ) Percentages of IgA+ pneumococci and relative fluorescence intensities ( c ) of IgA+ pneumococci. ( d ) Representative FACS plots of IgM+ pneumococci incubated with BALF from SPC-HA or SPC-HAxTCR-mice; control samples (CTRL) were stained with anti-IgM without prior incubation with BALF. ( e ) Percentages of IgM+ pneumococci and relative fluorescence intensities ( f ) of IgM+ pneumococci. Relative fluorescence intensities are calculated by the ratio of the MFI of each individual sample over the mean MFI of the SPC-HA control group. Data are pooled from 2 independent experiments with similar results. *p
    Figure Legend Snippet: Increased pneumococcal binding capacities by lung mucosal fluid in inflamed lungs. Pneumococci were co-incubated with bronchoalveolar lavage fluid (BALF) supernatants from SPC-HA and SPC-HAxTCR-HA mice. Bacteria were stained with anti-mouse IgA or anti-IgM antibodies and analyzed by flow cytometry (FACS). ( a ) Representative FACS plots of IgA+ pneumococci incubated with BALF from SPC-HA or SPC-HAxTCR-mice; control samples (CTRL) were stained with anti-IgA without prior incubation with BALF. ( b ) Percentages of IgA+ pneumococci and relative fluorescence intensities ( c ) of IgA+ pneumococci. ( d ) Representative FACS plots of IgM+ pneumococci incubated with BALF from SPC-HA or SPC-HAxTCR-mice; control samples (CTRL) were stained with anti-IgM without prior incubation with BALF. ( e ) Percentages of IgM+ pneumococci and relative fluorescence intensities ( f ) of IgM+ pneumococci. Relative fluorescence intensities are calculated by the ratio of the MFI of each individual sample over the mean MFI of the SPC-HA control group. Data are pooled from 2 independent experiments with similar results. *p

    Techniques Used: Binding Assay, Incubation, Mouse Assay, Staining, Flow Cytometry, Cytometry, FACS, Fluorescence

    Increased mucosal transport of secretory antibodies in inflamed lungs. ( a ) anti-pIgR and anti-Gapdh immunoblot of 20 µg protein from whole lung homogenates of SPC-HA (n = 3) and SPC-HAxTCR-HA (n = 3) mice. Densitometric quantification of protein bands is stated in arbitrary units above each lane. Relative pIgR quantity was calculated normalizing densitometric pIgR value to the corresponding Gapdh value and subsequently comparing normalized pIgR values of the SPC-HAxTCR-HA group to the SPC-HA group. Data are representative for at least two individual experiments with similar results. ( b ) Lung tissue sections were stained with anti-pIgR (green), representative alveolar structures from n = 3/group are depicted. White circles illustrate representative densitometrically quantified tissue areas. Calculated total cell fluorescence (CTCF) was determined as: Integrated density of fluorescence-positive cell – (Area of fluorescence-positive cell × mean fluorescence intensity of background signal). Median CTCF of quantified areas in representative images are depicted as white numbers. IgA and IgM levels in ( c ) bronchoalveolar lavage fluid (BALF) and ( d ) serum of SPC-HA and SPC-HAxTCR-HA mice were determined by ELISA. ( e ) Relative secretory IgA concentrations in serial dilutions of BALF samples were determined by ELISA. Results are expressed as the mean optical density (OD) at 450 nm ± SEM, *p
    Figure Legend Snippet: Increased mucosal transport of secretory antibodies in inflamed lungs. ( a ) anti-pIgR and anti-Gapdh immunoblot of 20 µg protein from whole lung homogenates of SPC-HA (n = 3) and SPC-HAxTCR-HA (n = 3) mice. Densitometric quantification of protein bands is stated in arbitrary units above each lane. Relative pIgR quantity was calculated normalizing densitometric pIgR value to the corresponding Gapdh value and subsequently comparing normalized pIgR values of the SPC-HAxTCR-HA group to the SPC-HA group. Data are representative for at least two individual experiments with similar results. ( b ) Lung tissue sections were stained with anti-pIgR (green), representative alveolar structures from n = 3/group are depicted. White circles illustrate representative densitometrically quantified tissue areas. Calculated total cell fluorescence (CTCF) was determined as: Integrated density of fluorescence-positive cell – (Area of fluorescence-positive cell × mean fluorescence intensity of background signal). Median CTCF of quantified areas in representative images are depicted as white numbers. IgA and IgM levels in ( c ) bronchoalveolar lavage fluid (BALF) and ( d ) serum of SPC-HA and SPC-HAxTCR-HA mice were determined by ELISA. ( e ) Relative secretory IgA concentrations in serial dilutions of BALF samples were determined by ELISA. Results are expressed as the mean optical density (OD) at 450 nm ± SEM, *p

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

    2) Product Images from "Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity"

    Article Title: Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity

    Journal: Nature

    doi: 10.1038/nature24302

    NASH-induced accumulation of IgA + plasmocytes in human liver a , Serum IgA levels of patients with NAFLD/NASH (Newcastle cohort; n = 502) were analysed and compared with clinical data. b , Serum was collected from a second cohort of patients with NAFLD/NASH (San Diego cohort; n = 96), whose degree of liver fibrosis was measured by MRI and classified as grades 0 ( n = 51), 1–2 ( n = 33), and 3–4 ( n = 12). Serum concentrations of IgA, IgG, and IgM were determined by ELISA and plotted against the fibrosis grade. c , Single-cell suspensions were prepared from liver biopsies taken from patients with NASH ( n = 4) with fibrosis, stained with antibodies to IgA, CD19, and PD-L1, and analysed by flow cytometry. Blood collected from three healthy donors (used as a surrogate for liver biopsies that could not be obtained from such individuals; HD), stained with antibodies, as indicated. Shown are representative scatter plots from two patients and two healthy donors, and bar graphs compiling data from all samples and depicting PD-L1 expression by IgA + cells. d , Single-cell suspensions were prepared from liver biopsies as above ( n = 3 per group) and stained with antibodies to CD4, CD8, Tim-3, and PD-1. Shown are representative data from one patient and one healthy donor with bar graphs compiling results obtained from three patients and depicting frequencies of PD-1 + CD8 + T cells. χ 2 test ( a ) and two-sided t -test (means ± s.e.m.; a – d ) were used to determine significance. * P
    Figure Legend Snippet: NASH-induced accumulation of IgA + plasmocytes in human liver a , Serum IgA levels of patients with NAFLD/NASH (Newcastle cohort; n = 502) were analysed and compared with clinical data. b , Serum was collected from a second cohort of patients with NAFLD/NASH (San Diego cohort; n = 96), whose degree of liver fibrosis was measured by MRI and classified as grades 0 ( n = 51), 1–2 ( n = 33), and 3–4 ( n = 12). Serum concentrations of IgA, IgG, and IgM were determined by ELISA and plotted against the fibrosis grade. c , Single-cell suspensions were prepared from liver biopsies taken from patients with NASH ( n = 4) with fibrosis, stained with antibodies to IgA, CD19, and PD-L1, and analysed by flow cytometry. Blood collected from three healthy donors (used as a surrogate for liver biopsies that could not be obtained from such individuals; HD), stained with antibodies, as indicated. Shown are representative scatter plots from two patients and two healthy donors, and bar graphs compiling data from all samples and depicting PD-L1 expression by IgA + cells. d , Single-cell suspensions were prepared from liver biopsies as above ( n = 3 per group) and stained with antibodies to CD4, CD8, Tim-3, and PD-1. Shown are representative data from one patient and one healthy donor with bar graphs compiling results obtained from three patients and depicting frequencies of PD-1 + CD8 + T cells. χ 2 test ( a ) and two-sided t -test (means ± s.e.m.; a – d ) were used to determine significance. * P

    Techniques Used: Magnetic Resonance Imaging, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Cytometry, Expressing

    The response to PD-L1 blockade is dependent on CD8 + T cells and clonal expansion of HCC-directed CD8 + T cells MUP-uPA and MUP-uPA/Iga −/− mice were placed on HFD and treated with anti-PD-L1, as described in . a , At the end of the treatments, mouse weights were measured ( n = 3, 2, 4, 3, 3, 7, 3, 7). b , MUP-uPA mice treated with anti-PD-L1 were analysed for serum IgA by ELISA ( n = 10, 6). c , Liver/body weight ratio of indicated strains kept on HFD that received the indicated treatments and were of the indicated ages (6 months, 11 months) ( n = 4, 6, 9, 19, 6, 2, 4, 15, 11, 17, 3, 14, 7, 20). d , e , Paraffin-embedded and frozen liver sections from HCC-bearing MUP-uPA mice were stained with Oil Red O, haematoxylin and eosin, or Sirius Red and analysed ( n = 4 or 5). The experiments were repeated at least two times. Low and high magnifications are shown in e to demonstrate the absence of tumour-invading immune cells in a mouse that failed to respond to anti-PD-L1 and their presence within a tumour of a treatment responsive mouse. Non-responsiveness to anti-PD-L1 treatment correlates with a fibrotic tumour stroma. Scale bars: haematoxylin and eosin, 250 μm; Sirius Red, 250 μm; Oil Red O, 50 μm. f , Response to PD-L1 blockade is dependent on CD8 + T cells. HCC-bearing MUP-uPA/Cd8a −/− mice ( n = 3) were treated with anti-PD-L1 for 8 weeks and tumour multiplicity was determined. g , Two-dimensional plot showing the frequency of the top ten TCRα CDR3 sequences expressed by CD8 + T cells from spleens and livers of HCC-bearing MUP-uPA mice. h , CD8 + T cells were sorted from spleens and livers of normal-chow- and HFD-fed WT mice ( n = 5 mice), their RNA was extracted, and TCR α-chain (top) and β-chain (bottom) CDR3 sequences were amplified and analysed by deep sequencing (ten samples). The panels show the clonality, the frequency of the top 50 TCR α- and β-chain sequences, and the percentage of productive unique TCR α - and β-chain sequences. i – k , CD8 + T cells were sorted from spleens and livers of HCC-bearing mice of the indicated strains and treatments ( n = 13 mice, 26 samples as indicated) and their TCR α - and β-chain CDR3 sequence diversity was analysed. Shown are the clonality ( i ), the diversity (inverse Simpson’s index) ( j ), and percentage of productive unique TCR β-chain sequences in the indicated strains ( k ). Note that TCR sequencing data of WT, MUP-uPA , and MUP-uPA/Iga −/− and Extended Data Fig. 9h. l – n , Splenocytes from the indicated mice treated with or without anti- PD-L1 were stimulated overnight with alpha-fetoprotein, stained as indicated, and analysed by flow cytometry. Shown are the percentage of IFNγ + CD107a + cells ( n = 3, 3, 2, 5, 3, 2, 3, 4, 4) ( l ) and IFNγ + TNF + cells ( n = 4, 3, 5, 4) ( m ) gated on CD8 + T cells. n , The percentage of IFNγ + TNF + cells gated on CD4 + T cells ( n = 3, 3, 2, 4, 3, 2, 3, 6, 4). Two-sided t -test ( a – d , f , h – n ) and Mann–Whitney test ( l ) were used to determine significance. * P
    Figure Legend Snippet: The response to PD-L1 blockade is dependent on CD8 + T cells and clonal expansion of HCC-directed CD8 + T cells MUP-uPA and MUP-uPA/Iga −/− mice were placed on HFD and treated with anti-PD-L1, as described in . a , At the end of the treatments, mouse weights were measured ( n = 3, 2, 4, 3, 3, 7, 3, 7). b , MUP-uPA mice treated with anti-PD-L1 were analysed for serum IgA by ELISA ( n = 10, 6). c , Liver/body weight ratio of indicated strains kept on HFD that received the indicated treatments and were of the indicated ages (6 months, 11 months) ( n = 4, 6, 9, 19, 6, 2, 4, 15, 11, 17, 3, 14, 7, 20). d , e , Paraffin-embedded and frozen liver sections from HCC-bearing MUP-uPA mice were stained with Oil Red O, haematoxylin and eosin, or Sirius Red and analysed ( n = 4 or 5). The experiments were repeated at least two times. Low and high magnifications are shown in e to demonstrate the absence of tumour-invading immune cells in a mouse that failed to respond to anti-PD-L1 and their presence within a tumour of a treatment responsive mouse. Non-responsiveness to anti-PD-L1 treatment correlates with a fibrotic tumour stroma. Scale bars: haematoxylin and eosin, 250 μm; Sirius Red, 250 μm; Oil Red O, 50 μm. f , Response to PD-L1 blockade is dependent on CD8 + T cells. HCC-bearing MUP-uPA/Cd8a −/− mice ( n = 3) were treated with anti-PD-L1 for 8 weeks and tumour multiplicity was determined. g , Two-dimensional plot showing the frequency of the top ten TCRα CDR3 sequences expressed by CD8 + T cells from spleens and livers of HCC-bearing MUP-uPA mice. h , CD8 + T cells were sorted from spleens and livers of normal-chow- and HFD-fed WT mice ( n = 5 mice), their RNA was extracted, and TCR α-chain (top) and β-chain (bottom) CDR3 sequences were amplified and analysed by deep sequencing (ten samples). The panels show the clonality, the frequency of the top 50 TCR α- and β-chain sequences, and the percentage of productive unique TCR α - and β-chain sequences. i – k , CD8 + T cells were sorted from spleens and livers of HCC-bearing mice of the indicated strains and treatments ( n = 13 mice, 26 samples as indicated) and their TCR α - and β-chain CDR3 sequence diversity was analysed. Shown are the clonality ( i ), the diversity (inverse Simpson’s index) ( j ), and percentage of productive unique TCR β-chain sequences in the indicated strains ( k ). Note that TCR sequencing data of WT, MUP-uPA , and MUP-uPA/Iga −/− and Extended Data Fig. 9h. l – n , Splenocytes from the indicated mice treated with or without anti- PD-L1 were stimulated overnight with alpha-fetoprotein, stained as indicated, and analysed by flow cytometry. Shown are the percentage of IFNγ + CD107a + cells ( n = 3, 3, 2, 5, 3, 2, 3, 4, 4) ( l ) and IFNγ + TNF + cells ( n = 4, 3, 5, 4) ( m ) gated on CD8 + T cells. n , The percentage of IFNγ + TNF + cells gated on CD4 + T cells ( n = 3, 3, 2, 4, 3, 2, 3, 6, 4). Two-sided t -test ( a – d , f , h – n ) and Mann–Whitney test ( l ) were used to determine significance. * P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Staining, Amplification, Sequencing, Flow Cytometry, Cytometry, MANN-WHITNEY

    Gut microbes promote HCC development and microbial translocation does not account for the anti-tumorigenic effect of IgA ablation a – j , MUP-uPA and MUP-uPA/Iga −/− mice were placed on HFD and treated with broad spectrum antibiotics (Abx) as described in , from 3 to 6 months (all panels except right part of b , and d , h , i ) or 6 to 11 months (right part of b , and d , h , i ) of age. At the end of the treatments, the stool contents of the corresponding mice were subjected to eubacterial 16S rRNA encoding DNA sequencing. a , Principal coordinate analysis plot of microbiome data using unweighted UniFrac distances; antibiotic treatment was significant by PERMANOVA (pseudo- F statistic = 105.5, P = 0.001) (left: n = 181, 27 as indicated; right: n = 6, 3, 6, 3). Mouse weight (6 months: n = 5, 6, 13, 7; 11 months: n = 8, 6, 5, 7) ( b ) and circulating ALT ( n = 12, 5, 12, 6) ( e ) were measured. c , d , Paraffin-embedded and frozen liver sections from the above mice were stained with haematoxylin and eosin, Sirius Red, or Oil Red O, and were analysed for collagen deposition and lipid droplets as indicated. Scale bars, 50 μm for Oil Red O; 100 μm for haematoxylin and eosin, and Sirius Red (Sirius Red: n = 9, 6, 14, 7 for c and 4, 4, 5 for d ; Oil Red O: n = 5, 3, 6, 3 mice). The data were validated at least in two or three experiments. f – h , j , Liver cell suspensions were stained with antibodies as indicated, and analysed by flow cytometry. Each dot represents one mouse. Shown are percentages of CD8 + cells in total cells ( n = 11, 5, 9, 7), CD8 + CD44 + ( n = 10, 4, 11, 6) or CD8 + IFNγ + CD107a + TNF + ( n = 8, 6, 9, 7) cells in CD8 + T cells ( f ), CD19 + B220 + cells in CD45 + cells ( n = 9, 6, 11, 7) ( g ), IgA + cells in CD45 + cells ( n = 6, 6, 6, 3, 6, 6) ( h ), and CD4 + cells in CD45 + cells ( n = 9, 6, 11, 7) or IL-17 + cells in CD4 + T cells ( n = 9, 6, 10, 6) ( j ). i , MUP-uPA mice placed on HFD and treated with antibiotics were analysed for serum IgA by ELISA ( n = 4, 8, 5, 6, 5, 5). Note that flow cytometry data of MUP-uPA and MUP-uPA/Iga −/− . The data were validated at least in two or three experiments. k – r , Effects of HFD and immunological background on mouse intestinal microbiomes and metabolomes (total mouse number n = 288). Each dot represents one mouse. k , The most pronounced differences are engendered by HFD compared with normal chow. Left to right: principal coordinate analysis (PCoA) plot of microbiome data using unweighted UniFrac distances (PERMANOVA, pseudo- F statistic = 46, P = 0.001 comparing diet); barchart of relative abundances of bacterial phyla; principal component analysis plot of metabolome. k , l , Subsequent effect of immune status for the MUP-uPA HFD-fed mice: WT, Iga −/− , Cd8a −/− , μMT −/− , and Rag1 −/− groups. l , Left to right: PCoA plot of microbiome data using unweighted UniFrac distances (PERMANOVA, pseudo- F statistic = 4.37, P = 0.001 comparing immune status); principal component analysis plot of metabolome; partial least squares discriminant analysis (PLS-DA) plot (the tenfold cross validation Q 2 value was 0.817) of metabolome. Large differences between categories are evident. Subsequent juxtaposition of the ( m ) MUP and MUP;Iga −/− : PCoA of plot of microbiome using unweighted UniFrac distances (PERMANOVA, pseudo- F statistic = 7.31, P = 0.001 comparing immune status) (left); PLS-DA (the tenfold cross validation Q 2 value of 0.926) plot of metabolome (right) and ( n ) MUP and MUP;Cd8a −/− : PCoA plot of microbiome data using unweighted UniFrac distances (PERMANOVA, pseudo- F statistic = 4.61, P = 0.001 comparing immune status) (left); PLS-DA plot (the tenfold cross validation Q 2 value of 0.934) plot of metabolome (right) illustrates the discordance stemming from these specific immune status differences. o , Bacterial Faith’s phylogenetic diversity metric (alpha diversity, box plot with minimum to maximum) calculated with rarefaction at 4,500 sequences per sample using Faith’s phylogenetic diversity metric ( n = 14, 8, 14, 25, 16, 6, 44, 34). p , Heat map of abundant bacterial taxa by immune status, genetic background, and diet. Trends in significantly differing taxa (ANCOM) by immune status include increased Gammaproteobacteria in Iga −/− with HFD and increased Ruminococcaceae in WT versus Iga −/− with HFD. Mucispirillum schaedleri was elevated in Iga −/− for all groups except normal-chow-fed MUP-uPA . q , Discordance according to the immune status for the STAM model mice. Left to right: PCoA plot of microbiome data using unweighted UniFrac distances; principal component analysis plot of metabolome (the tenfold cross validation Q 2 value of the corresponding partial least squares discriminant analysis is 0.814). r , Box plot (minimum to maximum) of unweighted UniFrac distances comparing distances within Iga −/− , Pigr −/− , and IgHEL/MD4 strains with distances between these strains ( n = 44, 40, 56) and WT or Cd8a −/− STAM mice ( n = 16, 12, 12) for microbiome data. Two-sided t -test (means ± s.e.m.; a , b , i , r ) and Mann– Whitney test (median; c–h , j , o ) were used to determine significance. * P
    Figure Legend Snippet: Gut microbes promote HCC development and microbial translocation does not account for the anti-tumorigenic effect of IgA ablation a – j , MUP-uPA and MUP-uPA/Iga −/− mice were placed on HFD and treated with broad spectrum antibiotics (Abx) as described in , from 3 to 6 months (all panels except right part of b , and d , h , i ) or 6 to 11 months (right part of b , and d , h , i ) of age. At the end of the treatments, the stool contents of the corresponding mice were subjected to eubacterial 16S rRNA encoding DNA sequencing. a , Principal coordinate analysis plot of microbiome data using unweighted UniFrac distances; antibiotic treatment was significant by PERMANOVA (pseudo- F statistic = 105.5, P = 0.001) (left: n = 181, 27 as indicated; right: n = 6, 3, 6, 3). Mouse weight (6 months: n = 5, 6, 13, 7; 11 months: n = 8, 6, 5, 7) ( b ) and circulating ALT ( n = 12, 5, 12, 6) ( e ) were measured. c , d , Paraffin-embedded and frozen liver sections from the above mice were stained with haematoxylin and eosin, Sirius Red, or Oil Red O, and were analysed for collagen deposition and lipid droplets as indicated. Scale bars, 50 μm for Oil Red O; 100 μm for haematoxylin and eosin, and Sirius Red (Sirius Red: n = 9, 6, 14, 7 for c and 4, 4, 5 for d ; Oil Red O: n = 5, 3, 6, 3 mice). The data were validated at least in two or three experiments. f – h , j , Liver cell suspensions were stained with antibodies as indicated, and analysed by flow cytometry. Each dot represents one mouse. Shown are percentages of CD8 + cells in total cells ( n = 11, 5, 9, 7), CD8 + CD44 + ( n = 10, 4, 11, 6) or CD8 + IFNγ + CD107a + TNF + ( n = 8, 6, 9, 7) cells in CD8 + T cells ( f ), CD19 + B220 + cells in CD45 + cells ( n = 9, 6, 11, 7) ( g ), IgA + cells in CD45 + cells ( n = 6, 6, 6, 3, 6, 6) ( h ), and CD4 + cells in CD45 + cells ( n = 9, 6, 11, 7) or IL-17 + cells in CD4 + T cells ( n = 9, 6, 10, 6) ( j ). i , MUP-uPA mice placed on HFD and treated with antibiotics were analysed for serum IgA by ELISA ( n = 4, 8, 5, 6, 5, 5). Note that flow cytometry data of MUP-uPA and MUP-uPA/Iga −/− . The data were validated at least in two or three experiments. k – r , Effects of HFD and immunological background on mouse intestinal microbiomes and metabolomes (total mouse number n = 288). Each dot represents one mouse. k , The most pronounced differences are engendered by HFD compared with normal chow. Left to right: principal coordinate analysis (PCoA) plot of microbiome data using unweighted UniFrac distances (PERMANOVA, pseudo- F statistic = 46, P = 0.001 comparing diet); barchart of relative abundances of bacterial phyla; principal component analysis plot of metabolome. k , l , Subsequent effect of immune status for the MUP-uPA HFD-fed mice: WT, Iga −/− , Cd8a −/− , μMT −/− , and Rag1 −/− groups. l , Left to right: PCoA plot of microbiome data using unweighted UniFrac distances (PERMANOVA, pseudo- F statistic = 4.37, P = 0.001 comparing immune status); principal component analysis plot of metabolome; partial least squares discriminant analysis (PLS-DA) plot (the tenfold cross validation Q 2 value was 0.817) of metabolome. Large differences between categories are evident. Subsequent juxtaposition of the ( m ) MUP and MUP;Iga −/− : PCoA of plot of microbiome using unweighted UniFrac distances (PERMANOVA, pseudo- F statistic = 7.31, P = 0.001 comparing immune status) (left); PLS-DA (the tenfold cross validation Q 2 value of 0.926) plot of metabolome (right) and ( n ) MUP and MUP;Cd8a −/− : PCoA plot of microbiome data using unweighted UniFrac distances (PERMANOVA, pseudo- F statistic = 4.61, P = 0.001 comparing immune status) (left); PLS-DA plot (the tenfold cross validation Q 2 value of 0.934) plot of metabolome (right) illustrates the discordance stemming from these specific immune status differences. o , Bacterial Faith’s phylogenetic diversity metric (alpha diversity, box plot with minimum to maximum) calculated with rarefaction at 4,500 sequences per sample using Faith’s phylogenetic diversity metric ( n = 14, 8, 14, 25, 16, 6, 44, 34). p , Heat map of abundant bacterial taxa by immune status, genetic background, and diet. Trends in significantly differing taxa (ANCOM) by immune status include increased Gammaproteobacteria in Iga −/− with HFD and increased Ruminococcaceae in WT versus Iga −/− with HFD. Mucispirillum schaedleri was elevated in Iga −/− for all groups except normal-chow-fed MUP-uPA . q , Discordance according to the immune status for the STAM model mice. Left to right: PCoA plot of microbiome data using unweighted UniFrac distances; principal component analysis plot of metabolome (the tenfold cross validation Q 2 value of the corresponding partial least squares discriminant analysis is 0.814). r , Box plot (minimum to maximum) of unweighted UniFrac distances comparing distances within Iga −/− , Pigr −/− , and IgHEL/MD4 strains with distances between these strains ( n = 44, 40, 56) and WT or Cd8a −/− STAM mice ( n = 16, 12, 12) for microbiome data. Two-sided t -test (means ± s.e.m.; a , b , i , r ) and Mann– Whitney test (median; c–h , j , o ) were used to determine significance. * P

    Techniques Used: Translocation Assay, Mouse Assay, DNA Sequencing, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    IgA + plasmocytes regulate tumour killing by CD8 + T cells a , Dih10 and dihXY HCC cells were transfected with an inducible ovalbumin (Ova) expression vector, and Ova expression and presentation were confirmed by flow cytometry, using an antibody that recognized the SIINFEKL peptide on the MHCI molecule H-2Kb. b – h , Ova-expressing dih cells or controls (dih–RFP) were starved overnight (5% cell death), after which their medium was changed and B cells from WT, Iga −/− , Pdl1/2 −/− , or SW-HEL mice were added in the presence of TGFβ (5 ng ml −1 ) and CTGF (3 ng ml −1 ), for an additional 24 h. Thereafter, the medium was replaced and CFSE-labelled OT-I T cells were added to the cultures that either contained or did not contain the B cells described above. After 4–6 days, the co-cultured cells were analysed by flow cytometry, while the secretory IgA was analysed by ELISA ( n = 2–4 wells per group per day). a – j , Experiments were repeated with two different Ova-expressing HCC and one prostate cancer cell lines. Shown are the representative flow cytometry histograms or plots depicting ( b ) OT-I CD8 + T-cell proliferation, ( c ) PD-L1 and SIINFEKL/H-2Kb expression on cancer cells, ( d ) PD-L1 expression on B cells, ( e ) cancer cell death. f , Relative dih-Ova–RFP killing by OT-I CD8 + T cells in the presence or absence of the indicated B cells. g , Total secretory IgA and anti-OVA-IgA antibody amounts in culture supernatants. h , Percentages of OT-I CD8 + cells in each culture, as indicated. i , j , TRC2-Ova–RFP cells or its control cell line (TRC2–RFP) were co-cultured with OT-I cells and splenic B cells (WT and Il10 −/− ), as described for a – h. i , Proliferation of OT-I cells was analysed using CFSE ( n = 3, 7, 5, 4). j , The amounts of secretory IgA were analysed using ELISA as indicated ( n = 3 per group). k – o , Liver cells from indicated 3-month-old mice were stained and analysed by flow cytometry. Experiments were repeated at least two or three times. Each dot represents one mouse. Shown are the percentage of CD8 + T cells among CD45 + cells ( n = 6, 5, 3, 7, 4) ( k ), absolute CD8 + T-cell number per gram of liver ( n = 3, 3, 7, 4) ( l ), the percentage of CD8 + CD44 + Ki-67 + T cells with representative scatter plots ( n = 3 or 4 per group) ( m , n ), and the representative scatter plots of perforin and GrzB among CD8 + CD44 + Ki-67 + T cells ( o ). p , q , Liver cell suspensions from the indicated mice were stained as shown and analysed by flow cytometry to determine the absolute CD8 + T-cell number in both STAM-BL6 and STAM-FVB mice ( n = 8, 4, 5, 3, 5, 5, 6, 7) ( p ), and the percentage of T EM cells using CD8, CD44, and CD62L ( n = 4, 4, 6, 9) ( q ). r , Liver cells from indicated 3-, 6-, and 11-month-old mice kept on HFD ( n = 4–10) were stained and analysed by flow cytometry. Shown are the percentage of CD8 + IFNγ + CD107a + T cells. Detailed n values are shown in . s – y , Liver cell suspensions from the indicated mice were stained as shown and analysed by flow cytometry to determine the percentage of Th17 cells using CD4 and IL-17a ( n = 4, 7, 9, 10, 4, 9, 11) ( s ), the percentage of regulatory T cells using CD4 and Foxp3 ( n = 4, 3, 8, 11, 7, 13) ( t ), the percentage of Tfh-like cells using CXCR5, PD1 and CD4 (3, 5, 6, 5) ( u ), the percentage of B220 + CD19 + B cells ( n = 4, 7, 7, 4, 5, 3, 9, 3, 11) ( v ), absolute B220 + CD19 + B-cell number per gram of liver ( n = 5, 7, 12, 27, 15, 7) ( w ), the percentage of IgG + cells ( n = 4, 3, 14, 11) ( x ), and CD138 + plasma cells ( n = 4, 7, 4, 7, 7, 8, 5, 8, 6, 6, 13, 27, 17) ( y ). Two-sided t -test (means ± s.e.m.; f – m ) and Mann–Whitney test (median; p – y ) were used to determine significance. * P
    Figure Legend Snippet: IgA + plasmocytes regulate tumour killing by CD8 + T cells a , Dih10 and dihXY HCC cells were transfected with an inducible ovalbumin (Ova) expression vector, and Ova expression and presentation were confirmed by flow cytometry, using an antibody that recognized the SIINFEKL peptide on the MHCI molecule H-2Kb. b – h , Ova-expressing dih cells or controls (dih–RFP) were starved overnight (5% cell death), after which their medium was changed and B cells from WT, Iga −/− , Pdl1/2 −/− , or SW-HEL mice were added in the presence of TGFβ (5 ng ml −1 ) and CTGF (3 ng ml −1 ), for an additional 24 h. Thereafter, the medium was replaced and CFSE-labelled OT-I T cells were added to the cultures that either contained or did not contain the B cells described above. After 4–6 days, the co-cultured cells were analysed by flow cytometry, while the secretory IgA was analysed by ELISA ( n = 2–4 wells per group per day). a – j , Experiments were repeated with two different Ova-expressing HCC and one prostate cancer cell lines. Shown are the representative flow cytometry histograms or plots depicting ( b ) OT-I CD8 + T-cell proliferation, ( c ) PD-L1 and SIINFEKL/H-2Kb expression on cancer cells, ( d ) PD-L1 expression on B cells, ( e ) cancer cell death. f , Relative dih-Ova–RFP killing by OT-I CD8 + T cells in the presence or absence of the indicated B cells. g , Total secretory IgA and anti-OVA-IgA antibody amounts in culture supernatants. h , Percentages of OT-I CD8 + cells in each culture, as indicated. i , j , TRC2-Ova–RFP cells or its control cell line (TRC2–RFP) were co-cultured with OT-I cells and splenic B cells (WT and Il10 −/− ), as described for a – h. i , Proliferation of OT-I cells was analysed using CFSE ( n = 3, 7, 5, 4). j , The amounts of secretory IgA were analysed using ELISA as indicated ( n = 3 per group). k – o , Liver cells from indicated 3-month-old mice were stained and analysed by flow cytometry. Experiments were repeated at least two or three times. Each dot represents one mouse. Shown are the percentage of CD8 + T cells among CD45 + cells ( n = 6, 5, 3, 7, 4) ( k ), absolute CD8 + T-cell number per gram of liver ( n = 3, 3, 7, 4) ( l ), the percentage of CD8 + CD44 + Ki-67 + T cells with representative scatter plots ( n = 3 or 4 per group) ( m , n ), and the representative scatter plots of perforin and GrzB among CD8 + CD44 + Ki-67 + T cells ( o ). p , q , Liver cell suspensions from the indicated mice were stained as shown and analysed by flow cytometry to determine the absolute CD8 + T-cell number in both STAM-BL6 and STAM-FVB mice ( n = 8, 4, 5, 3, 5, 5, 6, 7) ( p ), and the percentage of T EM cells using CD8, CD44, and CD62L ( n = 4, 4, 6, 9) ( q ). r , Liver cells from indicated 3-, 6-, and 11-month-old mice kept on HFD ( n = 4–10) were stained and analysed by flow cytometry. Shown are the percentage of CD8 + IFNγ + CD107a + T cells. Detailed n values are shown in . s – y , Liver cell suspensions from the indicated mice were stained as shown and analysed by flow cytometry to determine the percentage of Th17 cells using CD4 and IL-17a ( n = 4, 7, 9, 10, 4, 9, 11) ( s ), the percentage of regulatory T cells using CD4 and Foxp3 ( n = 4, 3, 8, 11, 7, 13) ( t ), the percentage of Tfh-like cells using CXCR5, PD1 and CD4 (3, 5, 6, 5) ( u ), the percentage of B220 + CD19 + B cells ( n = 4, 7, 7, 4, 5, 3, 9, 3, 11) ( v ), absolute B220 + CD19 + B-cell number per gram of liver ( n = 5, 7, 12, 27, 15, 7) ( w ), the percentage of IgG + cells ( n = 4, 3, 14, 11) ( x ), and CD138 + plasma cells ( n = 4, 7, 4, 7, 7, 8, 5, 8, 6, 6, 13, 27, 17) ( y ). Two-sided t -test (means ± s.e.m.; f – m ) and Mann–Whitney test (median; p – y ) were used to determine significance. * P

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Flow Cytometry, Cytometry, Mouse Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, MANN-WHITNEY

    IgA ablation inhibits, while CD8 deficiency accelerates HCC development a , b , Total DNA was extracted from HCC nodules of 11-month-old mice ( n = 16) of the indicated genotypes with or without anti-PD-L1 treatment, and subjected to exome sequencing. Shown are the number of point mutations identified per sample ( a ) and mutational signatures ( b ). Horizontal axis shows the 96-substitution patterns with substitution subtypes on top, and vertical axis indicates the probability of each pattern in b. c , Top 20 hallmark gene sets sorted by normalized enrichment score (NES) are shown to depict HCC progression in MUP-uPA and MUP-uPA/Iga −/− for human HCC are marked with a red asterisk. d , Total RNA was extracted from livers of 6-month-old mice and from HCC nodules of 11-month-old mice of the indicated genotypes and subjected to RNA-seq analysis. Hierarchical clustering of gene expression profiles comparing HCC and non-HCC mouse samples according to the RNA-seq. The top three enriched pathways/functional categories from Metascape are reported for major clusters of genes. e , Representative images of liver histology at different time points and indicated strains are shown with detailed n values. Scale bars, 100 μm (3 months), 250 μm (6, 11 months). f , Total tumour numbers in 3-month-old MUP-uPA mice ( n = 11, 7, 8). g , Heat map depicting differential expression of 17 liver-specific genes and 33 HCC-related genes in the indicated strains, illustrating the upregulation of some HCC-related genes in MUP-uPA/Cd8a −/− livers at 6 months of age (total mice number 29; n = 3 or 4 per group). h – j , BL6 mice of the indicated phenotype were subjected to the STAM protocol and their tumour volumes ( n = 14, 6, 6, 4, 3, 9, 3) ( h ) and histopathology ( i , j ) were evaluated at 25 weeks of age. The data were validated at least in two or three experiments. Paraffin-embedded and frozen liver sections from these mice were stained with haematoxylin and eosin, Sirius Red, or Oil Red O, as indicated. Shown are typical images of tumour-containing and tumour-free areas, the borders between which are marked by the black lines. Scale bars: haematoxylin and eosin, 100 μm; Sirius Red, 100, 250, or 500 μm; Oil Red O, 50 μm. Oil Red O-positive areas were quantitated and are shown on the right. The Sirius Red-stained areas for each mouse were calculated by image analysis of the whole-tissue scan and normalized to the haematoxylin and eosin stain ( n = 3 or 4 per group). k , Tumour volumes are shown for STAM-WT ( n = 10), STAM- Iga −/− ( n = 13), and STAM- Iga −/− after CD8 depletion ( n = 3). l , MUP-uPA/Iga −/− -HFD mice were injected weekly with anti-CD8 for 6 weeks and tumour multiplicity was determined ( n = 3). k , l , CD8 depletion experiments were repeated using two different HCC models (MUP and STAM). m , Heat map depicting the differential expression of 59 genes involved in allograft rejection, IFNγ response, and inflammation (total mouse number 41; 6 months: n = 3 and 11 months: 3 or 4 per group). n , o , Paraffin-embedded and frozen liver sections from 11-month-old mice ( n ) and adoptively transferred mice ( o ) were stained with haematoxylin and eosin, Sirius Red, or Oil Red O as indicated and analysed (Sirius Red: n = 9, 16, 14, 6; Oil Red O: n = 8, 5, 5, 8 for n ). Shown are typical images of tumourcontaining and tumour-free areas, the borders between which are marked by the black lines. p , Liver cells from MUP-uPA/Rag1 −/− mice 1 week after being adoptively transferred with CFSE-labelled T cells with or without B cells as indicated ( n = 3 in each group) were stained and analysed by flow cytometry. Shown are the percentage of CD8 + T cells among CD45 + cells (left), and histogram of proliferating CFSE-labelled T cells with the corresponding mean fluorescence intensity (right). q , Liver sections for MUP-uPA/Rag1 −/− and the corresponding adoptive lymphocyte transfer mice (4 weeks after adoptive transfer (AT)) were stained with alpha-SMA, IgA, CD3, and CD8 antibodies, counterstained with DAPI and examined by fluorescent microscopy (scale bars, 50 μm). For CD3/CD8 staining, images with higher magnification are shown (scale bars, 20 μm). Single-cell suspensions were prepared from the corresponding liver, stained with antibodies to CD45, IgA, CD19, B220, CD8, and CD4, and analysed by flow cytometry. Shown are representative scatter plots. MUP;Rag1 −/− mouse livers have been used for validation of CD4, CD8, IgA, and CD19, both for flow cytometry and for immunofluorescence analyses. The data were validated in at least two experiments. r , STAM-BL6 mice of the indicated phenotypes were analysed for IgA serum amounts by ELISA ( n = 11, 4, 4, 4, 4). s , Absolute IgA + cell number in livers of indicated STAM-BL6 mice ( n = 13, 4, 4, 9, 8). Each dot represents a mouse. Two-sided t -test (means ± s.e.m.; f , j , l , n , r ) and Mann–Whitney test (median; h , k , n , s ) were used to determine significance. * P
    Figure Legend Snippet: IgA ablation inhibits, while CD8 deficiency accelerates HCC development a , b , Total DNA was extracted from HCC nodules of 11-month-old mice ( n = 16) of the indicated genotypes with or without anti-PD-L1 treatment, and subjected to exome sequencing. Shown are the number of point mutations identified per sample ( a ) and mutational signatures ( b ). Horizontal axis shows the 96-substitution patterns with substitution subtypes on top, and vertical axis indicates the probability of each pattern in b. c , Top 20 hallmark gene sets sorted by normalized enrichment score (NES) are shown to depict HCC progression in MUP-uPA and MUP-uPA/Iga −/− for human HCC are marked with a red asterisk. d , Total RNA was extracted from livers of 6-month-old mice and from HCC nodules of 11-month-old mice of the indicated genotypes and subjected to RNA-seq analysis. Hierarchical clustering of gene expression profiles comparing HCC and non-HCC mouse samples according to the RNA-seq. The top three enriched pathways/functional categories from Metascape are reported for major clusters of genes. e , Representative images of liver histology at different time points and indicated strains are shown with detailed n values. Scale bars, 100 μm (3 months), 250 μm (6, 11 months). f , Total tumour numbers in 3-month-old MUP-uPA mice ( n = 11, 7, 8). g , Heat map depicting differential expression of 17 liver-specific genes and 33 HCC-related genes in the indicated strains, illustrating the upregulation of some HCC-related genes in MUP-uPA/Cd8a −/− livers at 6 months of age (total mice number 29; n = 3 or 4 per group). h – j , BL6 mice of the indicated phenotype were subjected to the STAM protocol and their tumour volumes ( n = 14, 6, 6, 4, 3, 9, 3) ( h ) and histopathology ( i , j ) were evaluated at 25 weeks of age. The data were validated at least in two or three experiments. Paraffin-embedded and frozen liver sections from these mice were stained with haematoxylin and eosin, Sirius Red, or Oil Red O, as indicated. Shown are typical images of tumour-containing and tumour-free areas, the borders between which are marked by the black lines. Scale bars: haematoxylin and eosin, 100 μm; Sirius Red, 100, 250, or 500 μm; Oil Red O, 50 μm. Oil Red O-positive areas were quantitated and are shown on the right. The Sirius Red-stained areas for each mouse were calculated by image analysis of the whole-tissue scan and normalized to the haematoxylin and eosin stain ( n = 3 or 4 per group). k , Tumour volumes are shown for STAM-WT ( n = 10), STAM- Iga −/− ( n = 13), and STAM- Iga −/− after CD8 depletion ( n = 3). l , MUP-uPA/Iga −/− -HFD mice were injected weekly with anti-CD8 for 6 weeks and tumour multiplicity was determined ( n = 3). k , l , CD8 depletion experiments were repeated using two different HCC models (MUP and STAM). m , Heat map depicting the differential expression of 59 genes involved in allograft rejection, IFNγ response, and inflammation (total mouse number 41; 6 months: n = 3 and 11 months: 3 or 4 per group). n , o , Paraffin-embedded and frozen liver sections from 11-month-old mice ( n ) and adoptively transferred mice ( o ) were stained with haematoxylin and eosin, Sirius Red, or Oil Red O as indicated and analysed (Sirius Red: n = 9, 16, 14, 6; Oil Red O: n = 8, 5, 5, 8 for n ). Shown are typical images of tumourcontaining and tumour-free areas, the borders between which are marked by the black lines. p , Liver cells from MUP-uPA/Rag1 −/− mice 1 week after being adoptively transferred with CFSE-labelled T cells with or without B cells as indicated ( n = 3 in each group) were stained and analysed by flow cytometry. Shown are the percentage of CD8 + T cells among CD45 + cells (left), and histogram of proliferating CFSE-labelled T cells with the corresponding mean fluorescence intensity (right). q , Liver sections for MUP-uPA/Rag1 −/− and the corresponding adoptive lymphocyte transfer mice (4 weeks after adoptive transfer (AT)) were stained with alpha-SMA, IgA, CD3, and CD8 antibodies, counterstained with DAPI and examined by fluorescent microscopy (scale bars, 50 μm). For CD3/CD8 staining, images with higher magnification are shown (scale bars, 20 μm). Single-cell suspensions were prepared from the corresponding liver, stained with antibodies to CD45, IgA, CD19, B220, CD8, and CD4, and analysed by flow cytometry. Shown are representative scatter plots. MUP;Rag1 −/− mouse livers have been used for validation of CD4, CD8, IgA, and CD19, both for flow cytometry and for immunofluorescence analyses. The data were validated in at least two experiments. r , STAM-BL6 mice of the indicated phenotypes were analysed for IgA serum amounts by ELISA ( n = 11, 4, 4, 4, 4). s , Absolute IgA + cell number in livers of indicated STAM-BL6 mice ( n = 13, 4, 4, 9, 8). Each dot represents a mouse. Two-sided t -test (means ± s.e.m.; f , j , l , n , r ) and Mann–Whitney test (median; h , k , n , s ) were used to determine significance. * P

    Techniques Used: Mouse Assay, Sequencing, RNA Sequencing Assay, Expressing, Functional Assay, Histopathology, Staining, H&E Stain, Injection, Flow Cytometry, Cytometry, Fluorescence, Adoptive Transfer Assay, Microscopy, Immunofluorescence, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    3) Product Images from "Intranasal Vaccination with the Recombinant Listeria monocytogenes ?actA prfA* Mutant Elicits Robust Systemic and Pulmonary Cellular Responses and Secretory Mucosal IgA ▿"

    Article Title: Intranasal Vaccination with the Recombinant Listeria monocytogenes ?actA prfA* Mutant Elicits Robust Systemic and Pulmonary Cellular Responses and Secretory Mucosal IgA ▿

    Journal: Clinical and Vaccine Immunology : CVI

    doi: 10.1128/CVI.00254-10

    Intranasal vaccination with r- Listeria Δ actA prfA */PA appears to elicit higher secretory mucosal IgA Ab titers than s.c. or i.v. immunizations, but the i.v. route induced much greater systemic IgG responses. (A) Mean anti-PA IgG Ab titers in sera
    Figure Legend Snippet: Intranasal vaccination with r- Listeria Δ actA prfA */PA appears to elicit higher secretory mucosal IgA Ab titers than s.c. or i.v. immunizations, but the i.v. route induced much greater systemic IgG responses. (A) Mean anti-PA IgG Ab titers in sera

    Techniques Used:

    4) Product Images from "Cooperation of ETV6/RUNX1 and BCL2 enhances immunoglobulin production and accelerates glomerulonephritis in transgenic mice"

    Article Title: Cooperation of ETV6/RUNX1 and BCL2 enhances immunoglobulin production and accelerates glomerulonephritis in transgenic mice

    Journal: Oncotarget

    doi: 10.18632/oncotarget.7687

    E/R tg ;BCL2 tg mice have higher B cell numbers and immunoglobulin titers than BCL2 tg mice A Fluorescence immunohistochemistry showing representative IgG deposits (green) in glomeruli, nuclei counterstained with DAPI (blue). Glomeruli are indicated by arrows, scale bars, 50 μm. B Quantification of mean IgG intensity in glomeruli ( n = 19 - 29 glomeruli per group) of at least three mice per group, including early and late disease mice in each group. Data are shown as means ± SEM. C ELISA of Ig Lambda (left panel) and Ig Kappa (right panel), normalized to a wt control. Relative fold change to the control mouse is indicated for Ig Lambda and Kappa, n = 4-7 mice per group, including both, early and late disease mice. D Quantification of serum IgA ( n = 4-6 mice per group) and IgG ( n = 8-10) levels. Data are presented as scatter dot plots with means indicated by a line. ( E - F ) Flow cytometric analysis of B cells (E), plasma cells and plasmablasts (F) is shown. (E) Percentage of CD19 + B of living cells in bone marrow are shown, n = 4-7 mice per group. (F) CD138 + cell, plasmablast- and plasma cell levels in spleen are indicated, n = 4-8 mice per group. (C, E, F) Data are visualized as box plots, whiskers indicating min and max. For all: Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison post-test, p values are considered as follows: * p
    Figure Legend Snippet: E/R tg ;BCL2 tg mice have higher B cell numbers and immunoglobulin titers than BCL2 tg mice A Fluorescence immunohistochemistry showing representative IgG deposits (green) in glomeruli, nuclei counterstained with DAPI (blue). Glomeruli are indicated by arrows, scale bars, 50 μm. B Quantification of mean IgG intensity in glomeruli ( n = 19 - 29 glomeruli per group) of at least three mice per group, including early and late disease mice in each group. Data are shown as means ± SEM. C ELISA of Ig Lambda (left panel) and Ig Kappa (right panel), normalized to a wt control. Relative fold change to the control mouse is indicated for Ig Lambda and Kappa, n = 4-7 mice per group, including both, early and late disease mice. D Quantification of serum IgA ( n = 4-6 mice per group) and IgG ( n = 8-10) levels. Data are presented as scatter dot plots with means indicated by a line. ( E - F ) Flow cytometric analysis of B cells (E), plasma cells and plasmablasts (F) is shown. (E) Percentage of CD19 + B of living cells in bone marrow are shown, n = 4-7 mice per group. (F) CD138 + cell, plasmablast- and plasma cell levels in spleen are indicated, n = 4-8 mice per group. (C, E, F) Data are visualized as box plots, whiskers indicating min and max. For all: Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison post-test, p values are considered as follows: * p

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

    Related Articles

    Enzyme-linked Immunosorbent Assay:

    Article Title: Myeloid ATG16L1 facilitates host-bacteria interactions in maintaining intestinal homeostasis
    Article Snippet: .. Total murine IgA was measured using the Mouse IgA ELISA kit per manufacturer’s protocol (eBioscience). .. For immunofluorescent staining, 50,000 cells were plated on 12mm diameter cover slips in 24-well plates overnight.

    Article Title: The comparative immunology of wild and laboratory mice, Mus musculus domesticus
    Article Snippet: .. The IgA concentration of these supernatants was measured using a commercially available mouse IgA ELISA kit following the manufacturer's instructions (Mouse IgA ELISA Ready-SET-Go, 88-50450, eBioscience). ..

    Article Title: The protection role of Atg16l1 in CD11c+ dendritic cells in murine colitis
    Article Snippet: .. Total murine IgA was measured using the Mouse IgA ELISA kit per manufacturer’s protocol (eBioscience). .. Isolation and culture of lamina propria mononuclear cells (LPMC) and mesenteric lymph node cells (MLN) were carried out as previously reported ( ) MLN and LPMC were plated at 2.5 × 105 cells/well in 96 well plates in RPMI 1640 medium (cellgro) with 10% fetal calf serum, 100 U/ml penicillin, 100 mg/ml gentamycin, 2 mM L-glutamine and stimulated with 2 µL/ml LK Activation cocktail for 4 h. The expression of TNF-α, IL-6 and IL-1β in cell supernatants were assayed by ELISA according to manufacturer’s instructions (Mouse TNF-α ELISA Ready-Set-Go, eBioscience, 88–7324-77; Mouse IL-1β ELISA Ready-SET-Go, eBioscience, 88–7013-77; Mouse IL-6 ELISA Ready-SET-Go, eBioscience, 88–7064-77).

    Article Title: Chronic lung inflammation primes humoral immunity and augments antipneumococcal resistance
    Article Snippet: .. ELISA ELISA kits for mouse IgA and IgM (eBioscience, Frankfurt am Main, Germany), mouse IL-6 and TNF-α (BioLegend, London, United Kingdom), mouse C5a (R & D Systems, Minneapolis, MN) and mouse albumin (Bethyl Laboratories Inc., Montogomery, TX) were used according to manufacturer’s instructions. .. For SIgA-detection Nunc MaxiSorp plates were coated overnight with monoclonal rat anti-mouse IgA (SouthernBiotech, Birmingham, 2 µg/mL).

    Article Title: Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity
    Article Snippet: .. Mouse IgA and IgG ELISA kits from eBioscience were used according to the manufacturer’s protocols. ..

    Article Title: MHC variation sculpts individualized microbial communities that control susceptibility to enteric infection
    Article Snippet: .. Faecal Ig ELISA The concentration of IgA in faecal pellets were quantified using an IgA-specific ELISA kit (eBioscience: Mouse IgA Ready-SET-go kit (cat#88-90450-88)) following kit protocols. .. All concentration estimates are standardized by faecal weight and depicted as concentration per gram of faeces.

    Article Title: Endothelial Cell-Specific Overexpression of Endothelial Nitric Oxide Synthase in Ins2Akita Mice Exacerbates Diabetic Nephropathy
    Article Snippet: .. IgA levels were determined using a mouse IgA ELISA kit purchased from Thermoscientific (Frederick, MD). ..

    Concentration Assay:

    Article Title: The comparative immunology of wild and laboratory mice, Mus musculus domesticus
    Article Snippet: .. The IgA concentration of these supernatants was measured using a commercially available mouse IgA ELISA kit following the manufacturer's instructions (Mouse IgA ELISA Ready-SET-Go, 88-50450, eBioscience). ..

    Article Title: MHC variation sculpts individualized microbial communities that control susceptibility to enteric infection
    Article Snippet: .. Faecal Ig ELISA The concentration of IgA in faecal pellets were quantified using an IgA-specific ELISA kit (eBioscience: Mouse IgA Ready-SET-go kit (cat#88-90450-88)) following kit protocols. .. All concentration estimates are standardized by faecal weight and depicted as concentration per gram of faeces.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: MHC variation sculpts individualized microbial communities that control susceptibility to enteric infection
    Article Snippet: .. Faecal Ig ELISA The concentration of IgA in faecal pellets were quantified using an IgA-specific ELISA kit (eBioscience: Mouse IgA Ready-SET-go kit (cat88-90450-88)) following kit protocols. .. All concentration estimates are standardized by faecal weight and depicted as concentration per gram of faeces.

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    Thermo Fisher ready set go mouse iga kit
    <t>IgA</t> + and CD 11b + IgA + plasma cells and the IgA response to E. faecium in the colon. (a) In immuno fluorescence experiments, IgA + plasma cells were stained in all four mouse groups on days −2, 0, and 1. Green arrows indicate IgA + plasma cells. White arrows indicate agglutinated IgA. In green: goat anti‐mouse IgA + donkey anti‐goat Alexa Fluor 488. (b) CD 11b + IgA + plasma cells were stained, similar as in (A). Green arrows indicate CD 11b + IgA + plasma cells. For both (a) and (b), in red: TO ‐ PRO ‐3 nucleic acid stain. The images are representative of two animals per group. (C) Quantification of IgA + plasma cells (white bars) and CD 11b + IgA + plasma cells (blue bars) per crypt from colon sections from all mice (n = 24). (d) Quantification of IgA by a specific <t>ELISA</t> from fecal extracts of mice from the groups of mice in this experiment, similar as in (a). IgA concentration was corrected for the amount of feces obtained per mouse to yield ng IgA per gram mouse feces. The ELISA was repeated three times, data averaged, and plotted. IgA concentrations for mice from groups 1 to 4 from day ‐2 was averaged and termed “untreated”, since mouse feces was obtained on each sampling day at 10 am (See Figure 1 b). For (C) and (D): ns , not significant; P
    Ready Set Go Mouse Iga Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ready set go mouse iga kit - by Bioz Stars, 2020-07
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    94
    Thermo Fisher 594 labeled donkey anti mouse immunoglobulin
    Transfection of I/1Ki and Vero E6 cells with pCAGGS-SDeV-FWD and pCAGGS-SDeV-REV constructs results in SDeV replication. (A) I/1Ki (top) and Vero E6 (bottom) cells transfected with Δ-fwd (pCAGGS-SDeV-FWD) and Δ-rev (pCAGGS-SDeV-REV) were stained for SDAg (anti-SDAg antiserum [1:7,500] and Alexa Fluor <t>594-labeled</t> donkey anti-rabbit immunoglobulin [1:1,000]) at 1, 2, 3, and 4 days posttransfection (from left to right). The images were taken at ×400 magnification using a Zeiss Axioplan 2 microscope. (B) Western blot of I/1Ki (left panel) and Vero E6 (right panel) cell pellets at 1, 2, 3, and 4 days posttransfection with Δ-fwd and Δ-rev constructs. Precision Plus Protein Dual Color Standards (Bio-Rad) served as the marker, and the results were recorded using the Odyssey infrared imaging system (Li-Cor).
    594 Labeled Donkey Anti Mouse Immunoglobulin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti mouse iga
    HD induces the expression of all isotypes of anti-HA antibodies in the serum. (A) HD was conducted in BALB/c mice and after 1 day, serum was obtained. The expression of each antibody isotype was determined by ELISA using <t>HRP-conjugated</t> got anti-mouse <t>IgA,</t> IgE, or APC-conjugated rat anti-mouse IgD followed by HRP-conjugated anti-rat IgG. (B) The expression level of each isotype was determined by competitive ELISA. A 96-well plate coated with A/PR8-HA, was incubated with the serum samples, followed by biotin-conjugated anti-HA IgG. Data were analyzed using a non-parametric Kruskal–Wallis test ( p = 0.0457). Horizontal bar represents the median. The indicated data are representative of two independent experiments.
    Anti Mouse Iga, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    IgA + and CD 11b + IgA + plasma cells and the IgA response to E. faecium in the colon. (a) In immuno fluorescence experiments, IgA + plasma cells were stained in all four mouse groups on days −2, 0, and 1. Green arrows indicate IgA + plasma cells. White arrows indicate agglutinated IgA. In green: goat anti‐mouse IgA + donkey anti‐goat Alexa Fluor 488. (b) CD 11b + IgA + plasma cells were stained, similar as in (A). Green arrows indicate CD 11b + IgA + plasma cells. For both (a) and (b), in red: TO ‐ PRO ‐3 nucleic acid stain. The images are representative of two animals per group. (C) Quantification of IgA + plasma cells (white bars) and CD 11b + IgA + plasma cells (blue bars) per crypt from colon sections from all mice (n = 24). (d) Quantification of IgA by a specific ELISA from fecal extracts of mice from the groups of mice in this experiment, similar as in (a). IgA concentration was corrected for the amount of feces obtained per mouse to yield ng IgA per gram mouse feces. The ELISA was repeated three times, data averaged, and plotted. IgA concentrations for mice from groups 1 to 4 from day ‐2 was averaged and termed “untreated”, since mouse feces was obtained on each sampling day at 10 am (See Figure 1 b). For (C) and (D): ns , not significant; P

    Journal: MicrobiologyOpen

    Article Title: Primary murine mucosal response during cephalosporin‐induced intestinal colonization by Enterococcus faecium. Primary murine mucosal response during cephalosporin‐induced intestinal colonization by Enterococcus faecium

    doi: 10.1002/mbo3.602

    Figure Lengend Snippet: IgA + and CD 11b + IgA + plasma cells and the IgA response to E. faecium in the colon. (a) In immuno fluorescence experiments, IgA + plasma cells were stained in all four mouse groups on days −2, 0, and 1. Green arrows indicate IgA + plasma cells. White arrows indicate agglutinated IgA. In green: goat anti‐mouse IgA + donkey anti‐goat Alexa Fluor 488. (b) CD 11b + IgA + plasma cells were stained, similar as in (A). Green arrows indicate CD 11b + IgA + plasma cells. For both (a) and (b), in red: TO ‐ PRO ‐3 nucleic acid stain. The images are representative of two animals per group. (C) Quantification of IgA + plasma cells (white bars) and CD 11b + IgA + plasma cells (blue bars) per crypt from colon sections from all mice (n = 24). (d) Quantification of IgA by a specific ELISA from fecal extracts of mice from the groups of mice in this experiment, similar as in (a). IgA concentration was corrected for the amount of feces obtained per mouse to yield ng IgA per gram mouse feces. The ELISA was repeated three times, data averaged, and plotted. IgA concentrations for mice from groups 1 to 4 from day ‐2 was averaged and termed “untreated”, since mouse feces was obtained on each sampling day at 10 am (See Figure 1 b). For (C) and (D): ns , not significant; P

    Article Snippet: 2.10 Immunoglobulin A ELISA An Enzyme‐linked immunoabsorbent assay (ELISA) was performed using a Ready‐Set‐Go mouse IgA kit (Thermofischer, The Netherlands).

    Techniques: Fluorescence, Staining, Mouse Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Sampling

    The reduction of IgD + and IgM + B cells of colon in IMQ mice was not caused by migration to skin or skin draining lymph nodes, or by systemic B-cell depletion. ( A ) Representative plots of flow cytometry analysis for lymphocytes in CD45 + colonic lamina propria. ( B and C ) Percentage ( left ) and absolute number ( right ) of ( B ) T cells and ( C ) B cells in CD45 + colonic lamina propria as shown in panel A . ( D ) Representative plots of flow cytometry analysis for IFN-γ and IL17A in CD3 + CD4 + cells from colonic lamina propria, skin dLN. and mesenteric lymph node (MLN), stimulated with PMA, ionomycin, and IL23 for 4 hours. ( E ) Percentage of IFN-γ + ( top ) and IL17A + ( bottom ) cells in CD3 + CD4 + lymphocytes from each organ. ( F and G ) Intracellular staining and the ratio of Foxp3 in CD3 + CD4 + lymphocytes from each organ. ( H ) Absolute number of IgA + B cells ( left ), IgD + B cells ( middle ), and IgM + B cells ( right ). ( I ) Percentage in CD45 + cells of IgA + B220 - plasma cells. ( J ) Concentration of IgA in feces measured by enzyme-linked immunosorbent assay. ( K ) Percentage of IgA-coated bacteria in feces. ( L ) Representative plots of flow cytometry analysis for B cells in epidermis, dermis, skin dLNs, spleen, and MLN after topical IMQ treatment. The numbers within plots indicate the percentages of gated cells. The plots are gated on CD45 + cells. ( M ) Percentages of B cells in CD45 + cells in epidermis, dermis, skin dLN, spleen, and MLN as shown in panel L . ( N ) Absolute number of B cells ( top ), IgD + B cells ( middle ), and IgM + B cells ( bottom ) in skin dLN and MLN. Each symbol represents an individual mouse (n = 4–8). Statistical analyses were performed with the Student t test. * P

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Toll-Like Receptor 7 Agonist–Induced Dermatitis Causes Severe Dextran Sulfate Sodium Colitis by Altering the Gut Microbiome and Immune Cells

    doi: 10.1016/j.jcmgh.2018.09.010

    Figure Lengend Snippet: The reduction of IgD + and IgM + B cells of colon in IMQ mice was not caused by migration to skin or skin draining lymph nodes, or by systemic B-cell depletion. ( A ) Representative plots of flow cytometry analysis for lymphocytes in CD45 + colonic lamina propria. ( B and C ) Percentage ( left ) and absolute number ( right ) of ( B ) T cells and ( C ) B cells in CD45 + colonic lamina propria as shown in panel A . ( D ) Representative plots of flow cytometry analysis for IFN-γ and IL17A in CD3 + CD4 + cells from colonic lamina propria, skin dLN. and mesenteric lymph node (MLN), stimulated with PMA, ionomycin, and IL23 for 4 hours. ( E ) Percentage of IFN-γ + ( top ) and IL17A + ( bottom ) cells in CD3 + CD4 + lymphocytes from each organ. ( F and G ) Intracellular staining and the ratio of Foxp3 in CD3 + CD4 + lymphocytes from each organ. ( H ) Absolute number of IgA + B cells ( left ), IgD + B cells ( middle ), and IgM + B cells ( right ). ( I ) Percentage in CD45 + cells of IgA + B220 - plasma cells. ( J ) Concentration of IgA in feces measured by enzyme-linked immunosorbent assay. ( K ) Percentage of IgA-coated bacteria in feces. ( L ) Representative plots of flow cytometry analysis for B cells in epidermis, dermis, skin dLNs, spleen, and MLN after topical IMQ treatment. The numbers within plots indicate the percentages of gated cells. The plots are gated on CD45 + cells. ( M ) Percentages of B cells in CD45 + cells in epidermis, dermis, skin dLN, spleen, and MLN as shown in panel L . ( N ) Absolute number of B cells ( top ), IgD + B cells ( middle ), and IgM + B cells ( bottom ) in skin dLN and MLN. Each symbol represents an individual mouse (n = 4–8). Statistical analyses were performed with the Student t test. * P

    Article Snippet: The supernatant was collected and the IgA concentration was measured by IgA Mouse Enzyme-Linked Immunosorbent Assay kit (88-50450-88, lot 146856016; Thermo Fisher Scientific) according to the manufacturer’s instructions.

    Techniques: Mouse Assay, Migration, Flow Cytometry, Cytometry, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Transfection of I/1Ki and Vero E6 cells with pCAGGS-SDeV-FWD and pCAGGS-SDeV-REV constructs results in SDeV replication. (A) I/1Ki (top) and Vero E6 (bottom) cells transfected with Δ-fwd (pCAGGS-SDeV-FWD) and Δ-rev (pCAGGS-SDeV-REV) were stained for SDAg (anti-SDAg antiserum [1:7,500] and Alexa Fluor 594-labeled donkey anti-rabbit immunoglobulin [1:1,000]) at 1, 2, 3, and 4 days posttransfection (from left to right). The images were taken at ×400 magnification using a Zeiss Axioplan 2 microscope. (B) Western blot of I/1Ki (left panel) and Vero E6 (right panel) cell pellets at 1, 2, 3, and 4 days posttransfection with Δ-fwd and Δ-rev constructs. Precision Plus Protein Dual Color Standards (Bio-Rad) served as the marker, and the results were recorded using the Odyssey infrared imaging system (Li-Cor).

    Journal: mBio

    Article Title: Snake Deltavirus Utilizes Envelope Proteins of Different Viruses To Generate Infectious Particles

    doi: 10.1128/mBio.03250-19

    Figure Lengend Snippet: Transfection of I/1Ki and Vero E6 cells with pCAGGS-SDeV-FWD and pCAGGS-SDeV-REV constructs results in SDeV replication. (A) I/1Ki (top) and Vero E6 (bottom) cells transfected with Δ-fwd (pCAGGS-SDeV-FWD) and Δ-rev (pCAGGS-SDeV-REV) were stained for SDAg (anti-SDAg antiserum [1:7,500] and Alexa Fluor 594-labeled donkey anti-rabbit immunoglobulin [1:1,000]) at 1, 2, 3, and 4 days posttransfection (from left to right). The images were taken at ×400 magnification using a Zeiss Axioplan 2 microscope. (B) Western blot of I/1Ki (left panel) and Vero E6 (right panel) cell pellets at 1, 2, 3, and 4 days posttransfection with Δ-fwd and Δ-rev constructs. Precision Plus Protein Dual Color Standards (Bio-Rad) served as the marker, and the results were recorded using the Odyssey infrared imaging system (Li-Cor).

    Article Snippet: For secondary antibodies, we used the following dilutions: 1:1,000 for Alexa Fluor 488- or 594-labeled donkey anti-rabbit immunoglobulin (ThermoFisher Scientific) and 1:1,000 for Alexa Fluor 488- or 594-labeled donkey anti-mouse immunoglobulin (ThermoFisher Scientific).

    Techniques: Transfection, Construct, Staining, Labeling, Microscopy, Western Blot, Marker, Imaging

    SDeV-infected cells can be superinfected with reptarenaviruses (UHV-2 and UGV-1) and hartmanivirus (HISV-1). (A) Mock-infected I/1Ki cells (boa kidney) and mock-, UGV-1-, and UHV-2-infected I/1Ki-Δ cells were stained for SDAg (anti-SDAg-AF488 [α-SDAg-AF488], left panels, green) and reptarenavirus NP (α-NP-AF594, second column, red). Hoechst 33342 was used to visualize the nuclei. The panels on the right show an overlay of the three images. (B) Mock-, UGV-1-, and HISV-1-infected V/1Ki-Δ cells (boa kidney) were stained for SDAg (α-SDAg-AF488, left panels, green), reptarenavirus NP (α-NP-AF594, second column, except bottom, red), or hartmanivirus NP (anti-HISV NP [1:3,000] and Alexa Fluor 594-labeled donkey anti-rabbit immunoglobulin [1:1,000], second column bottom panel, red). Hoechst 33342 was used to visualize the nuclei. The panels on the right show an overlay of the three images. (C) Mock-, UGV-1-, and HISV-1-infected V/1Liv-Δ cells (boa liver) were stained for SDAg (α-SDAg-AF488, left panels, green), reptarenavirus NP (α-NP-AF594, second column, except bottom, red), or hartmanivirus NP (anti-HISV NP [1:3,000] and Alexa Fluor 594-labeled donkey anti-rabbit immunoglobulin [1:1,000], second column bottom panel, red). Hoechst 33342 was used to visualize the nuclei. The panels on the right show an overlay of the three images. (D) Mock-, UGV-1-, and HISV-1-infected V/2Hz-Δ cells (boa heart) were stained for SDAg (α-SDAg-AF488, left panels, green), reptarenavirus NP (α-NP-AF594, second column, except bottom, red), or hartmanivirus NP (anti-HISV NP [1:3,000] and Alexa Fluor 594-labeled donkey anti-rabbit immunoglobulin [1:1,000], second column bottom panel, red). Hoechst 33342 was used to visualize the nuclei. The panels on the right show an overlay of the three images. All images were taken at ×400 magnification using a Zeiss Axioplan 2 microscope, and a 30-μm bar is shown in the bottom right corner of each panel.

    Journal: mBio

    Article Title: Snake Deltavirus Utilizes Envelope Proteins of Different Viruses To Generate Infectious Particles

    doi: 10.1128/mBio.03250-19

    Figure Lengend Snippet: SDeV-infected cells can be superinfected with reptarenaviruses (UHV-2 and UGV-1) and hartmanivirus (HISV-1). (A) Mock-infected I/1Ki cells (boa kidney) and mock-, UGV-1-, and UHV-2-infected I/1Ki-Δ cells were stained for SDAg (anti-SDAg-AF488 [α-SDAg-AF488], left panels, green) and reptarenavirus NP (α-NP-AF594, second column, red). Hoechst 33342 was used to visualize the nuclei. The panels on the right show an overlay of the three images. (B) Mock-, UGV-1-, and HISV-1-infected V/1Ki-Δ cells (boa kidney) were stained for SDAg (α-SDAg-AF488, left panels, green), reptarenavirus NP (α-NP-AF594, second column, except bottom, red), or hartmanivirus NP (anti-HISV NP [1:3,000] and Alexa Fluor 594-labeled donkey anti-rabbit immunoglobulin [1:1,000], second column bottom panel, red). Hoechst 33342 was used to visualize the nuclei. The panels on the right show an overlay of the three images. (C) Mock-, UGV-1-, and HISV-1-infected V/1Liv-Δ cells (boa liver) were stained for SDAg (α-SDAg-AF488, left panels, green), reptarenavirus NP (α-NP-AF594, second column, except bottom, red), or hartmanivirus NP (anti-HISV NP [1:3,000] and Alexa Fluor 594-labeled donkey anti-rabbit immunoglobulin [1:1,000], second column bottom panel, red). Hoechst 33342 was used to visualize the nuclei. The panels on the right show an overlay of the three images. (D) Mock-, UGV-1-, and HISV-1-infected V/2Hz-Δ cells (boa heart) were stained for SDAg (α-SDAg-AF488, left panels, green), reptarenavirus NP (α-NP-AF594, second column, except bottom, red), or hartmanivirus NP (anti-HISV NP [1:3,000] and Alexa Fluor 594-labeled donkey anti-rabbit immunoglobulin [1:1,000], second column bottom panel, red). Hoechst 33342 was used to visualize the nuclei. The panels on the right show an overlay of the three images. All images were taken at ×400 magnification using a Zeiss Axioplan 2 microscope, and a 30-μm bar is shown in the bottom right corner of each panel.

    Article Snippet: For secondary antibodies, we used the following dilutions: 1:1,000 for Alexa Fluor 488- or 594-labeled donkey anti-rabbit immunoglobulin (ThermoFisher Scientific) and 1:1,000 for Alexa Fluor 488- or 594-labeled donkey anti-mouse immunoglobulin (ThermoFisher Scientific).

    Techniques: Infection, Staining, Labeling, Microscopy

    Infectious SDeV particles are formed when I/1Ki-Δ cells are transfected with viral glycoproteins. (A) I/1Ki-Δ cells transfected with HISV-1 GPC (top row), Puumala virus glycoproteins (PUUV Gn Gc, second row), UGV-1 GPC (third row), UGV-1 ZP and GPC (fourth row), HBV S-Ag (fifth row), and empty pCAGGS-MCS plasmid (bottom row) were stained for HA tag (anti-HA [1:4,000] and Alexa Fluor 594-labeled donkey anti-mouse immunoglobulin [1:1,000], left panels, red) and SDAg (α-SDAg-AF488, middle panels, green). Hoechst 33342 was used to visualize the nuclei. The panels on the right show an overlay of the two images. A 30-μm bar is shown in the bottom right corner. All images were taken at ×400 magnification using a Zeiss Axioplan 2 microscope. (B) Supernatants collected from I/1Ki-Δ cells transfected with empty pCAGGS-MCS plasmid, UGV-1 ZP, UGV-1 GPC and ZP, UGV-1 GPC, HISV-1 GPC, LCMV GPC, JUNV GPC, PUUV glycoproteins, and HBV S-Ag were pelleted by ultracentrifugation and analyzed by Western blotting. The left panel shows anti-SDAg staining, and the right panel shows anti-SDAg and anti-HA staining. (C) Supernatants collected from I/1Ki-Δ cells transfected with empty pCAGGS-MCS plasmid, UGV-1 ZP, UGV-1 GPC and ZP, UGV-1 GPC, HISV-1 GPC, LCMV GPC, JUNV GPC, PUUV glycoproteins, and HBV S-Ag were titrated on clean I/1Ki cells. The plasmid used for transfection is shown in the left column, and the corresponding SDeV titer (in fluorescent focus-forming units [fffus] per milliliter) is shown in the right column.

    Journal: mBio

    Article Title: Snake Deltavirus Utilizes Envelope Proteins of Different Viruses To Generate Infectious Particles

    doi: 10.1128/mBio.03250-19

    Figure Lengend Snippet: Infectious SDeV particles are formed when I/1Ki-Δ cells are transfected with viral glycoproteins. (A) I/1Ki-Δ cells transfected with HISV-1 GPC (top row), Puumala virus glycoproteins (PUUV Gn Gc, second row), UGV-1 GPC (third row), UGV-1 ZP and GPC (fourth row), HBV S-Ag (fifth row), and empty pCAGGS-MCS plasmid (bottom row) were stained for HA tag (anti-HA [1:4,000] and Alexa Fluor 594-labeled donkey anti-mouse immunoglobulin [1:1,000], left panels, red) and SDAg (α-SDAg-AF488, middle panels, green). Hoechst 33342 was used to visualize the nuclei. The panels on the right show an overlay of the two images. A 30-μm bar is shown in the bottom right corner. All images were taken at ×400 magnification using a Zeiss Axioplan 2 microscope. (B) Supernatants collected from I/1Ki-Δ cells transfected with empty pCAGGS-MCS plasmid, UGV-1 ZP, UGV-1 GPC and ZP, UGV-1 GPC, HISV-1 GPC, LCMV GPC, JUNV GPC, PUUV glycoproteins, and HBV S-Ag were pelleted by ultracentrifugation and analyzed by Western blotting. The left panel shows anti-SDAg staining, and the right panel shows anti-SDAg and anti-HA staining. (C) Supernatants collected from I/1Ki-Δ cells transfected with empty pCAGGS-MCS plasmid, UGV-1 ZP, UGV-1 GPC and ZP, UGV-1 GPC, HISV-1 GPC, LCMV GPC, JUNV GPC, PUUV glycoproteins, and HBV S-Ag were titrated on clean I/1Ki cells. The plasmid used for transfection is shown in the left column, and the corresponding SDeV titer (in fluorescent focus-forming units [fffus] per milliliter) is shown in the right column.

    Article Snippet: For secondary antibodies, we used the following dilutions: 1:1,000 for Alexa Fluor 488- or 594-labeled donkey anti-rabbit immunoglobulin (ThermoFisher Scientific) and 1:1,000 for Alexa Fluor 488- or 594-labeled donkey anti-mouse immunoglobulin (ThermoFisher Scientific).

    Techniques: Transfection, Gel Permeation Chromatography, Plasmid Preparation, Staining, Labeling, Microscopy, Western Blot

    HD induces the expression of all isotypes of anti-HA antibodies in the serum. (A) HD was conducted in BALB/c mice and after 1 day, serum was obtained. The expression of each antibody isotype was determined by ELISA using HRP-conjugated got anti-mouse IgA, IgE, or APC-conjugated rat anti-mouse IgD followed by HRP-conjugated anti-rat IgG. (B) The expression level of each isotype was determined by competitive ELISA. A 96-well plate coated with A/PR8-HA, was incubated with the serum samples, followed by biotin-conjugated anti-HA IgG. Data were analyzed using a non-parametric Kruskal–Wallis test ( p = 0.0457). Horizontal bar represents the median. The indicated data are representative of two independent experiments.

    Journal: Frontiers in Immunology

    Article Title: Neutralizing Antibodies Induced by Gene-Based Hydrodynamic Injection Have a Therapeutic Effect in Lethal Influenza Infection

    doi: 10.3389/fimmu.2018.00047

    Figure Lengend Snippet: HD induces the expression of all isotypes of anti-HA antibodies in the serum. (A) HD was conducted in BALB/c mice and after 1 day, serum was obtained. The expression of each antibody isotype was determined by ELISA using HRP-conjugated got anti-mouse IgA, IgE, or APC-conjugated rat anti-mouse IgD followed by HRP-conjugated anti-rat IgG. (B) The expression level of each isotype was determined by competitive ELISA. A 96-well plate coated with A/PR8-HA, was incubated with the serum samples, followed by biotin-conjugated anti-HA IgG. Data were analyzed using a non-parametric Kruskal–Wallis test ( p = 0.0457). Horizontal bar represents the median. The indicated data are representative of two independent experiments.

    Article Snippet: HRP-conjugated goat anti-mouse IgG (Southern Biotech), anti-mouse IgA (Thermo Fisher Scientific), anti-mouse IgM (Southern Biotech), and anti-mouse IgE (Southern Biotech) were purchased to measure anti-HA IgG, anti-HA IgA, anti-HA IgM, and anti-HA IgE in serum and nasal wash, respectively.

    Techniques: Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay, Competitive ELISA, Incubation

    Anti-HA IgG, IgA, and IgM neutralize influenza virus in vitro . (A–F) HEK293T cells were co-transfected with pCADEST1-empty (vector) or pCADEST1-anti-HA kappa and anti-HA IgG, anti-HA IgA and Joining chain, anti-HA IgM and Joining chain, anti-HA IgD, or anti-HA IgE. Supernatants were collected after 1 week. (B,D,F) The supernatants were treated with receptor destroying enzyme (RDE) for overnight at 37°C. (A,B) The supernatants were processed by western blotting under non-reducing conditions followed by probing with HRP-conjugated goat anti-mouse IgG, IgA, IgM, and IgE. For detecting IgD, the blotted supernatants were probed with APC-conjugated rat anti-mouse IgD followed by HRP-conjugated anti-rat IgG. The indicated data are representative of two independent experiments. (C,D) The concentration of anti-HA antibodies in the supernatants was measured by competitive ELISA. A 96-well plate coated with HA was incubated with the supernatants, followed by addition of biotin-conjugated anti-HA IgG monoclonal antibody (mAb). (E,F) The neutralizing antibody titer was measured by a micro-neutralization assay. ND, not detected.

    Journal: Frontiers in Immunology

    Article Title: Neutralizing Antibodies Induced by Gene-Based Hydrodynamic Injection Have a Therapeutic Effect in Lethal Influenza Infection

    doi: 10.3389/fimmu.2018.00047

    Figure Lengend Snippet: Anti-HA IgG, IgA, and IgM neutralize influenza virus in vitro . (A–F) HEK293T cells were co-transfected with pCADEST1-empty (vector) or pCADEST1-anti-HA kappa and anti-HA IgG, anti-HA IgA and Joining chain, anti-HA IgM and Joining chain, anti-HA IgD, or anti-HA IgE. Supernatants were collected after 1 week. (B,D,F) The supernatants were treated with receptor destroying enzyme (RDE) for overnight at 37°C. (A,B) The supernatants were processed by western blotting under non-reducing conditions followed by probing with HRP-conjugated goat anti-mouse IgG, IgA, IgM, and IgE. For detecting IgD, the blotted supernatants were probed with APC-conjugated rat anti-mouse IgD followed by HRP-conjugated anti-rat IgG. The indicated data are representative of two independent experiments. (C,D) The concentration of anti-HA antibodies in the supernatants was measured by competitive ELISA. A 96-well plate coated with HA was incubated with the supernatants, followed by addition of biotin-conjugated anti-HA IgG monoclonal antibody (mAb). (E,F) The neutralizing antibody titer was measured by a micro-neutralization assay. ND, not detected.

    Article Snippet: HRP-conjugated goat anti-mouse IgG (Southern Biotech), anti-mouse IgA (Thermo Fisher Scientific), anti-mouse IgM (Southern Biotech), and anti-mouse IgE (Southern Biotech) were purchased to measure anti-HA IgG, anti-HA IgA, anti-HA IgM, and anti-HA IgE in serum and nasal wash, respectively.

    Techniques: In Vitro, Transfection, Plasmid Preparation, Western Blot, Concentration Assay, Competitive ELISA, Incubation, Neutralization

    HD protects mice from influenza virus infection in the upper respiratory tract. (A) HD was conducted in BALB/c mice 8 h after intranasal infection (1,000 PFU/2 μl, each nostril). At 3 days post-infection, the virus titer in nasal wash specimens was determined by an MDCK plaque assay. Data were analyzed using a non-parametric Kruskal–Wallis test ( p = 0.0091). (B) HD for anti-HA IgG was conducted in BALB/c mice and after 1 day, nasal wash specimens were obtained under serum-excluding conditions and analyzed for the concentration of anti-HA IgG by ELISA. (C) HD for anti-HA IgA was conducted in BALB/c mice after 8 h of virus infection. At 3 days post-infection, the nasal wash was obtained and the secreted HA-specific IgA (anti-HA sIgA) level was measured by ELISA using rabbit anti-mouse polymeric Ig receptor (pIgR). Horizontal bar represents the median. The indicated data are representative of two independent experiments.

    Journal: Frontiers in Immunology

    Article Title: Neutralizing Antibodies Induced by Gene-Based Hydrodynamic Injection Have a Therapeutic Effect in Lethal Influenza Infection

    doi: 10.3389/fimmu.2018.00047

    Figure Lengend Snippet: HD protects mice from influenza virus infection in the upper respiratory tract. (A) HD was conducted in BALB/c mice 8 h after intranasal infection (1,000 PFU/2 μl, each nostril). At 3 days post-infection, the virus titer in nasal wash specimens was determined by an MDCK plaque assay. Data were analyzed using a non-parametric Kruskal–Wallis test ( p = 0.0091). (B) HD for anti-HA IgG was conducted in BALB/c mice and after 1 day, nasal wash specimens were obtained under serum-excluding conditions and analyzed for the concentration of anti-HA IgG by ELISA. (C) HD for anti-HA IgA was conducted in BALB/c mice after 8 h of virus infection. At 3 days post-infection, the nasal wash was obtained and the secreted HA-specific IgA (anti-HA sIgA) level was measured by ELISA using rabbit anti-mouse polymeric Ig receptor (pIgR). Horizontal bar represents the median. The indicated data are representative of two independent experiments.

    Article Snippet: HRP-conjugated goat anti-mouse IgG (Southern Biotech), anti-mouse IgA (Thermo Fisher Scientific), anti-mouse IgM (Southern Biotech), and anti-mouse IgE (Southern Biotech) were purchased to measure anti-HA IgG, anti-HA IgA, anti-HA IgM, and anti-HA IgE in serum and nasal wash, respectively.

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