recombinant bovine il17a protein  (Kingfisher Biotech)


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    Name:
    Recombinant Bovine IL 17A
    Description:

    Catalog Number:
    RP0056B-100
    Price:
    750.0
    Source:
    Yeast
    Purity:
    98%
    Quantity:
    100 ug
    Molecular Weight:
    15.0 kDa
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    Structured Review

    Kingfisher Biotech recombinant bovine il17a protein
    Cellular immune responses following stimulation with LukM. IFNg ( a ) and <t>IL17a</t> ( c ) production following stimulation of whole blood with LukM for 48 h and 72 h, respectively. Proliferation measured as the percentage of CD4 ( b ) and CD8 ( d ) T-cells with diluted CFSE signal following 96 h stimulation with LukM. S/P = Sample to positive ratio. + = P

    https://www.bioz.com/result/recombinant bovine il17a protein/product/Kingfisher Biotech
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant bovine il17a protein - by Bioz Stars, 2021-09
    94/100 stars

    Images

    1) Product Images from "Immunization of young heifers with staphylococcal immune evasion proteins before natural exposure to Staphylococcus aureus induces a humoral immune response in serum and milk"

    Article Title: Immunization of young heifers with staphylococcal immune evasion proteins before natural exposure to Staphylococcus aureus induces a humoral immune response in serum and milk

    Journal: BMC Veterinary Research

    doi: 10.1186/s12917-018-1765-9

    Cellular immune responses following stimulation with LukM. IFNg ( a ) and IL17a ( c ) production following stimulation of whole blood with LukM for 48 h and 72 h, respectively. Proliferation measured as the percentage of CD4 ( b ) and CD8 ( d ) T-cells with diluted CFSE signal following 96 h stimulation with LukM. S/P = Sample to positive ratio. + = P
    Figure Legend Snippet: Cellular immune responses following stimulation with LukM. IFNg ( a ) and IL17a ( c ) production following stimulation of whole blood with LukM for 48 h and 72 h, respectively. Proliferation measured as the percentage of CD4 ( b ) and CD8 ( d ) T-cells with diluted CFSE signal following 96 h stimulation with LukM. S/P = Sample to positive ratio. + = P

    Techniques Used:

    Cellular immune responses following stimulation with EfB. IFNg ( a ) and IL17a ( c ) production following stimulation of whole blood with EfB for 48 h and 72 h, respectively. Proliferation measured as the percentage of CD4 ( b ) and CD8 ( d ) T-cells with diluted CFSE signal following 96 h stimulation with EfB. S/P = Sample to positive ratio. + = P
    Figure Legend Snippet: Cellular immune responses following stimulation with EfB. IFNg ( a ) and IL17a ( c ) production following stimulation of whole blood with EfB for 48 h and 72 h, respectively. Proliferation measured as the percentage of CD4 ( b ) and CD8 ( d ) T-cells with diluted CFSE signal following 96 h stimulation with EfB. S/P = Sample to positive ratio. + = P

    Techniques Used:

    2) Product Images from "Enhancing the toolbox to study IL-17A in cattle and sheep"

    Article Title: Enhancing the toolbox to study IL-17A in cattle and sheep

    Journal: Veterinary Research

    doi: 10.1186/s13567-017-0426-5

    Evaluation of commercial antibodies for the intracellular detection of recombinant bovine and ovine IL-17A. The eight commercial antibodies listed in Table 1 were tested against fixed, permeabilised untransfected (UTF) CHO cells and CHO cells transfected with cDNA encoding bovIL-17A or ovIL-17A for their capacity to detect intracellular recombinant IL-17A by flow cytometry. Results are shown for one polyclonal antibody (pab) produced against bovIL-17A ( A ) and seven monoclonal antibodies (mabs) produced against human or mouse IL-17A ( B – D ). Profiles of the relevant control antibodies listed in Table 2 are included in the overlapping histograms. Events were acquired on the MacsQuant according to the gating strategy described previously (in brief) and shown in Additional file 2 . Line colours representing different antibody treatments are given in parentheses: A Primary rabbit anti-bovine IL-17A pab PB0274B-100 at 1 μg/mL (A.1, red) or negative control primary anti-bovine CD34 pab (in-house) at an estimated 1 μg/mL equivalent (a, black) then detected with a secondary goat anti-rabbit alexafluor 488 at 1 μg/mL; B Directly conjugated mouse anti-human IL-17A eBio64DEC17-phycoerythrin (PE) mab (IgG1) at 2.5 μg/mL (B.1, red) and control IgG1 VPM21 mab (in-house) at an estimated 2.5 μg/mL equivalent (b, black) and detected with goat anti-mouse PE at 1 μg/mL; C Primary mouse anti-human IL-17A mabs MT44.6 (C.1, blue), MT241 (C.2, green), MT2770 (C.3, brown) and MT504 (C.4, red) [all IgG1] at 0.5 μg/mL and control IgG1 VPM21 mab (in-house) at an estimated 0.5 μg/mL equivalent (black), all detected with goat anti-mouse PE at 1 μg/mL; D Primary mouse anti-human IL-17A mabs #41809 (D.1, red) (IgG2b) and #41802 (D.2, blue) (IgG1) at 2.5 μg/mL and a mixture of control mabs VPM21 (IgG1) and VPM22 (IgG2b) at an estimated 2.5 μg/mL equivalent (d, black), all detected with goat anti-mouse PE at 1 μg/mL.
    Figure Legend Snippet: Evaluation of commercial antibodies for the intracellular detection of recombinant bovine and ovine IL-17A. The eight commercial antibodies listed in Table 1 were tested against fixed, permeabilised untransfected (UTF) CHO cells and CHO cells transfected with cDNA encoding bovIL-17A or ovIL-17A for their capacity to detect intracellular recombinant IL-17A by flow cytometry. Results are shown for one polyclonal antibody (pab) produced against bovIL-17A ( A ) and seven monoclonal antibodies (mabs) produced against human or mouse IL-17A ( B – D ). Profiles of the relevant control antibodies listed in Table 2 are included in the overlapping histograms. Events were acquired on the MacsQuant according to the gating strategy described previously (in brief) and shown in Additional file 2 . Line colours representing different antibody treatments are given in parentheses: A Primary rabbit anti-bovine IL-17A pab PB0274B-100 at 1 μg/mL (A.1, red) or negative control primary anti-bovine CD34 pab (in-house) at an estimated 1 μg/mL equivalent (a, black) then detected with a secondary goat anti-rabbit alexafluor 488 at 1 μg/mL; B Directly conjugated mouse anti-human IL-17A eBio64DEC17-phycoerythrin (PE) mab (IgG1) at 2.5 μg/mL (B.1, red) and control IgG1 VPM21 mab (in-house) at an estimated 2.5 μg/mL equivalent (b, black) and detected with goat anti-mouse PE at 1 μg/mL; C Primary mouse anti-human IL-17A mabs MT44.6 (C.1, blue), MT241 (C.2, green), MT2770 (C.3, brown) and MT504 (C.4, red) [all IgG1] at 0.5 μg/mL and control IgG1 VPM21 mab (in-house) at an estimated 0.5 μg/mL equivalent (black), all detected with goat anti-mouse PE at 1 μg/mL; D Primary mouse anti-human IL-17A mabs #41809 (D.1, red) (IgG2b) and #41802 (D.2, blue) (IgG1) at 2.5 μg/mL and a mixture of control mabs VPM21 (IgG1) and VPM22 (IgG2b) at an estimated 2.5 μg/mL equivalent (d, black), all detected with goat anti-mouse PE at 1 μg/mL.

    Techniques Used: Recombinant, Transfection, Flow Cytometry, Cytometry, Produced, Negative Control

    Measurement and biological function of recombinant bovine and ovine IL-17A and detection of native ovine IL-17A by ELISA. A Detection of rbov and rovIL-17A by ELISA. The supernatants from transfected CHO cells expressing rbovIL-17A or rovIL-17A, or control parent untransfected line (UTF) were serially diluted (Log 3 dilutions) and evaluated using the commercial bovIL-17A ELISA. Data presented are optical density (OD) values from the Spectrophotometer at 450 nm. The X-axis displays Dilution 1/X and the Y-axis gives the OD value. Readings from UTF supernatant were below the limit of detection. B Functional activity of rbov and rovIL-17A on bovine embryonic lung cells. Bovine embryonic lung (EBL) cells were stimulated with 100 ng/mL CHO-expressed rbovIL-17A or rovIL-17A or UTF CHO negative control supernatant. Following 24 h incubation, culture supernatants were collected from triplicate cultures then tested for CXCL8 by ELISA. The X-axis displays the bioassay treatments and the Y-axis shows CXCL8 production in pg/mL. Data are the arithmetic mean of three technical replicates with error bars representing the standard error from one of three experiments. CXCL8 expression between treatments was statistically assessed using Kruskal–Wallis test. C Functional activity of rbov and rovIL-17A on ovine ST-6 cells. Ovine ST-6 cells were stimulated with 100 ng/mL CHO-expressed rbovIL-17A or rovIL-17A or UTF CHO supernatant. Following 24 h incubation and culture supernatants collected, tested and analysed as described in Figure 2B. CXCL8 expression between treatments was statistically assessed using Kruskal–Wallis test. D Detection of native ovIL-17A by ELISA. Ovine PBMC were cultured at 2 × 10 6 cells/mL with or without 5 μg/mL ConA. Culture supernatants were analysed for IL-17A using the bovIL-17A ELISA. Data represent the arithmetic mean of PBMC from six ewes and error bars represent standard error. Data were analysed statistically for significance using the two-tailed Mann–Whitney test.
    Figure Legend Snippet: Measurement and biological function of recombinant bovine and ovine IL-17A and detection of native ovine IL-17A by ELISA. A Detection of rbov and rovIL-17A by ELISA. The supernatants from transfected CHO cells expressing rbovIL-17A or rovIL-17A, or control parent untransfected line (UTF) were serially diluted (Log 3 dilutions) and evaluated using the commercial bovIL-17A ELISA. Data presented are optical density (OD) values from the Spectrophotometer at 450 nm. The X-axis displays Dilution 1/X and the Y-axis gives the OD value. Readings from UTF supernatant were below the limit of detection. B Functional activity of rbov and rovIL-17A on bovine embryonic lung cells. Bovine embryonic lung (EBL) cells were stimulated with 100 ng/mL CHO-expressed rbovIL-17A or rovIL-17A or UTF CHO negative control supernatant. Following 24 h incubation, culture supernatants were collected from triplicate cultures then tested for CXCL8 by ELISA. The X-axis displays the bioassay treatments and the Y-axis shows CXCL8 production in pg/mL. Data are the arithmetic mean of three technical replicates with error bars representing the standard error from one of three experiments. CXCL8 expression between treatments was statistically assessed using Kruskal–Wallis test. C Functional activity of rbov and rovIL-17A on ovine ST-6 cells. Ovine ST-6 cells were stimulated with 100 ng/mL CHO-expressed rbovIL-17A or rovIL-17A or UTF CHO supernatant. Following 24 h incubation and culture supernatants collected, tested and analysed as described in Figure 2B. CXCL8 expression between treatments was statistically assessed using Kruskal–Wallis test. D Detection of native ovIL-17A by ELISA. Ovine PBMC were cultured at 2 × 10 6 cells/mL with or without 5 μg/mL ConA. Culture supernatants were analysed for IL-17A using the bovIL-17A ELISA. Data represent the arithmetic mean of PBMC from six ewes and error bars represent standard error. Data were analysed statistically for significance using the two-tailed Mann–Whitney test.

    Techniques Used: Recombinant, Enzyme-linked Immunosorbent Assay, Transfection, Expressing, Spectrophotometry, Functional Assay, Activity Assay, Negative Control, Incubation, Cell Culture, Two Tailed Test, MANN-WHITNEY

    Relative intracellular expression of IL-17A and IFN-γ by activated bovine and ovine PBMC. The data sets described in “ Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section ” and presented in Figures 5 and 6 are summarised to compare overall intracellular expression of IL-17A ( A ) and IFN-γ ( B ) by PMA/ionomycin-stimulated bovine and ovine PBMC. Each bar represents the arithmetic mean of four cattle or four sheep and the error bars represent the standard error. The data for total percentage IFN-γ and IL-17A expression between species were assessed statistically using two-tailed Mann–Whitney tests allowing for ties.
    Figure Legend Snippet: Relative intracellular expression of IL-17A and IFN-γ by activated bovine and ovine PBMC. The data sets described in “ Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section ” and presented in Figures 5 and 6 are summarised to compare overall intracellular expression of IL-17A ( A ) and IFN-γ ( B ) by PMA/ionomycin-stimulated bovine and ovine PBMC. Each bar represents the arithmetic mean of four cattle or four sheep and the error bars represent the standard error. The data for total percentage IFN-γ and IL-17A expression between species were assessed statistically using two-tailed Mann–Whitney tests allowing for ties.

    Techniques Used: Expressing, Two Tailed Test, MANN-WHITNEY

    Detection of single-cell expression of ruminant IL-17A by ELISpot. Plates and PBMC were prepared and cultured as described in “ IL-17A ELISpot section ”. ELISpot images shown are representative of PBMC from one of three cattle ( A ) and one of three sheep ( B ) activated with ConA and PMA/ionomycin. The average number of spot-forming units (SFU) with standard errors are shown for 10 6 PBMC from all three cattle (grey bars) and sheep (black bars), stimulated under the different conditions ( C ). Data were modelled by fitting a Poisson generalised linear mixed model (GLMM) by maximum likelihood to the IL-17A SFU/10 6 values, using logarithmic link function and Laplace approximations to calculate log-likelihoods. The model included treatment (medium control, ConA and PMA/ionomycin), species (bovine, ovine) and their interaction as fixed effects and animal identification as a random effect in order to account for both within- and between-animal variability. An observation-level random effect term was specified to account for data over-dispersion. The statistical significance of the fixed effect terms was assessed using p values derived from type II Wald Chi square tests. Linear hypothesis tests were defined from the GLMM in order to conduct pair-wise comparisons of means between treatments and species. The associated p values were adjusted for false discovery rate (FDR) following Benjamini–Hochberg’s procedure.
    Figure Legend Snippet: Detection of single-cell expression of ruminant IL-17A by ELISpot. Plates and PBMC were prepared and cultured as described in “ IL-17A ELISpot section ”. ELISpot images shown are representative of PBMC from one of three cattle ( A ) and one of three sheep ( B ) activated with ConA and PMA/ionomycin. The average number of spot-forming units (SFU) with standard errors are shown for 10 6 PBMC from all three cattle (grey bars) and sheep (black bars), stimulated under the different conditions ( C ). Data were modelled by fitting a Poisson generalised linear mixed model (GLMM) by maximum likelihood to the IL-17A SFU/10 6 values, using logarithmic link function and Laplace approximations to calculate log-likelihoods. The model included treatment (medium control, ConA and PMA/ionomycin), species (bovine, ovine) and their interaction as fixed effects and animal identification as a random effect in order to account for both within- and between-animal variability. An observation-level random effect term was specified to account for data over-dispersion. The statistical significance of the fixed effect terms was assessed using p values derived from type II Wald Chi square tests. Linear hypothesis tests were defined from the GLMM in order to conduct pair-wise comparisons of means between treatments and species. The associated p values were adjusted for false discovery rate (FDR) following Benjamini–Hochberg’s procedure.

    Techniques Used: Expressing, Enzyme-linked Immunospot, Cell Culture, Derivative Assay

    Intracellular expression of IL-17A and IFN-γ by activated bovine T cell subsets. PBMC from four cattle were stimulated with phorbol 12-myristate 13 acetate, ionomycin and brefeldin A in RPMI culture medium for 4 h. Cells were harvested and stained for viability and with mabs specific for cell-surface phenotypic markers and intracellular cytokines as described in Table 3 and “ Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section ”. Cells were stained for CD4 with mab CC8-PE at 1:20 dilution ( A , D ), for CD8β with mab CC58-PE at 1:20 dilution ( B , D ) and for WC-1 (γδ T cells) with mab CC15-PE at 1:200 ( C , E ). Intracellular cytokine staining for IL-17A was conducted using mab eBioDEC17-APC at a 1:20 dilution ( A – C ) and for IFN-γ using mab CC302-Alexafluor 647 at a 1:200 dilution ( D – F ). Data are shown for PBMC from one representative animal of four.
    Figure Legend Snippet: Intracellular expression of IL-17A and IFN-γ by activated bovine T cell subsets. PBMC from four cattle were stimulated with phorbol 12-myristate 13 acetate, ionomycin and brefeldin A in RPMI culture medium for 4 h. Cells were harvested and stained for viability and with mabs specific for cell-surface phenotypic markers and intracellular cytokines as described in Table 3 and “ Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section ”. Cells were stained for CD4 with mab CC8-PE at 1:20 dilution ( A , D ), for CD8β with mab CC58-PE at 1:20 dilution ( B , D ) and for WC-1 (γδ T cells) with mab CC15-PE at 1:200 ( C , E ). Intracellular cytokine staining for IL-17A was conducted using mab eBioDEC17-APC at a 1:20 dilution ( A – C ) and for IFN-γ using mab CC302-Alexafluor 647 at a 1:200 dilution ( D – F ). Data are shown for PBMC from one representative animal of four.

    Techniques Used: Expressing, Staining

    Phylogenetic tree of mammalian IL-17A protein sequences. Evolutionary sequence comparisons were undertaken using 13 selected mammalian and other IL-17A sequences by initially conducting a multiple alignment using Clustal Omega (EMBL/EBI online, [ 21 ]). The evolutionary relationships between the sequences were inferred using Mr. Bayes launched from TOPALI v 2.5 using the Jones–Taylor–Thornton plus gamma (JTT + G) model with two runs each of 1 250 000 generations with a burn in period of 20% and sampling frequency of 1000. The horizontal lines are branches whose length represents the amount of genetic change over time. The scale bar shows the distance represented by 0.1 expected substitutions per site. The robustness of the clustering of sequences are shown by the Bayesian Posterior Probabilities at the nodes. Accession numbers of the sequences used for the comparison are: Human NP_002181.1; House mouse NP_034682.1; Cow NP_001008412.1; Sheep XP_004018936.1; Goat NP_001272654.1; Horse NP_001137264.1; Pig NP_001005729.1; Dog NP_001159350.1; Domestic guinea pig NP_001265697.1; Koala AHZ08738.1; Chicken NP_989791.1; EGW10039.1 Chinese hamster and European rabbit AMQ91106.1. The phylogenetic tree was annotated using Dendroscope.
    Figure Legend Snippet: Phylogenetic tree of mammalian IL-17A protein sequences. Evolutionary sequence comparisons were undertaken using 13 selected mammalian and other IL-17A sequences by initially conducting a multiple alignment using Clustal Omega (EMBL/EBI online, [ 21 ]). The evolutionary relationships between the sequences were inferred using Mr. Bayes launched from TOPALI v 2.5 using the Jones–Taylor–Thornton plus gamma (JTT + G) model with two runs each of 1 250 000 generations with a burn in period of 20% and sampling frequency of 1000. The horizontal lines are branches whose length represents the amount of genetic change over time. The scale bar shows the distance represented by 0.1 expected substitutions per site. The robustness of the clustering of sequences are shown by the Bayesian Posterior Probabilities at the nodes. Accession numbers of the sequences used for the comparison are: Human NP_002181.1; House mouse NP_034682.1; Cow NP_001008412.1; Sheep XP_004018936.1; Goat NP_001272654.1; Horse NP_001137264.1; Pig NP_001005729.1; Dog NP_001159350.1; Domestic guinea pig NP_001265697.1; Koala AHZ08738.1; Chicken NP_989791.1; EGW10039.1 Chinese hamster and European rabbit AMQ91106.1. The phylogenetic tree was annotated using Dendroscope.

    Techniques Used: Sequencing, Sampling

    Intracellular expression of IL-17A and IFN-γ by activated ovine T cell subsets. PBMC from four sheep were stimulated with phorbol 12-myristate 13 acetate, ionomycin and brefeldin A in RPMI culture medium for 4 h. Cells were harvested and stained for viability and with mabs specific for cell-surface phenotypic markers and intracellular cytokines as described in Table 3 and “ Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section ”. Cells were then stained for CD4 with mab 44.38-PE at 1:20 dilution ( A , D ), CD8β with mab CC58-PE at 1:20 dilution ( B , D ) and WC-1 (γδ) with mab CC15-PE at 1:200 ( C , E ). Intracellular cytokine staining for IL-17A was conducted using mab eBio64DEC17-APC a 1:20 dilution ( A – C ) and for IFN-γ using mab CC302-alexafluor 647 at a 1:200 dilution ( D – F ). Data shown is for one representative animal out of four.
    Figure Legend Snippet: Intracellular expression of IL-17A and IFN-γ by activated ovine T cell subsets. PBMC from four sheep were stimulated with phorbol 12-myristate 13 acetate, ionomycin and brefeldin A in RPMI culture medium for 4 h. Cells were harvested and stained for viability and with mabs specific for cell-surface phenotypic markers and intracellular cytokines as described in Table 3 and “ Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section ”. Cells were then stained for CD4 with mab 44.38-PE at 1:20 dilution ( A , D ), CD8β with mab CC58-PE at 1:20 dilution ( B , D ) and WC-1 (γδ) with mab CC15-PE at 1:200 ( C , E ). Intracellular cytokine staining for IL-17A was conducted using mab eBio64DEC17-APC a 1:20 dilution ( A – C ) and for IFN-γ using mab CC302-alexafluor 647 at a 1:200 dilution ( D – F ). Data shown is for one representative animal out of four.

    Techniques Used: Expressing, Staining

    3) Product Images from "T Helper 17-Associated Cytokines Are Produced during Antigen-Specific Inflammation in the Mammary Gland"

    Article Title: T Helper 17-Associated Cytokines Are Produced during Antigen-Specific Inflammation in the Mammary Gland

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0063471

    Analysis by immunohistochemistry of representative sections of mammary tissue of ovalbumin-infused glands. A) Immunoreactivity of the epithelial lining the alveoli and of cells in the connective tissue (cow #4019) to antibody against N-terminal peptide of bovine IL-17A; B) Immunoreactivity of the mammary tissue of another cow (#1039) to the N-term antibody; C) Inhibition of labeling by Ab to N-terminal IL-17A peptide with the peptide antigen (cow #4019); D) Negative control with Ab to ovalbumin and second antibody conjugated to horseradish peroxidase; E, F) Immunoreactivity of the epithelial lining the alveoli and of cells in the connective tissue of cows #4019 and 1039 to the abcam antibody; G-H) Immunoreactivity of mammary tissue of cows #4019 and 1039 to the to the C-term and antibody. Scale bars indicate 25 µm.
    Figure Legend Snippet: Analysis by immunohistochemistry of representative sections of mammary tissue of ovalbumin-infused glands. A) Immunoreactivity of the epithelial lining the alveoli and of cells in the connective tissue (cow #4019) to antibody against N-terminal peptide of bovine IL-17A; B) Immunoreactivity of the mammary tissue of another cow (#1039) to the N-term antibody; C) Inhibition of labeling by Ab to N-terminal IL-17A peptide with the peptide antigen (cow #4019); D) Negative control with Ab to ovalbumin and second antibody conjugated to horseradish peroxidase; E, F) Immunoreactivity of the epithelial lining the alveoli and of cells in the connective tissue of cows #4019 and 1039 to the abcam antibody; G-H) Immunoreactivity of mammary tissue of cows #4019 and 1039 to the to the C-term and antibody. Scale bars indicate 25 µm.

    Techniques Used: Immunohistochemistry, Inhibition, Labeling, Negative Control

    Analysis by immunohistochemistry of representative tissue sections of uninfused, healthy mammary glands. A–B) Immunoreactivity of the apical side of the epithelial cells lining the alveoli to the N-term antibody to IL-17A of cows #1018 and 2014, respectively; C) Inhibition of labeling by Ab to N-terminal IL-17A peptide with the peptide antigen (cow #1018); D) Negative control with Ab to ovalbumin and second antibody conjugated to horseradish peroxidase (cow #1018); E–F) Immunoreactivity of the epithelial lining the alveoli to abcam antibody (cows #1018 and 2014, respectively); G–H) Immunoreactivity of mammary tissue of healthy uninfused quarters of cows 1018 and 2014 to the C-term antibody, respectively. Scale bars indicate 25 µm.
    Figure Legend Snippet: Analysis by immunohistochemistry of representative tissue sections of uninfused, healthy mammary glands. A–B) Immunoreactivity of the apical side of the epithelial cells lining the alveoli to the N-term antibody to IL-17A of cows #1018 and 2014, respectively; C) Inhibition of labeling by Ab to N-terminal IL-17A peptide with the peptide antigen (cow #1018); D) Negative control with Ab to ovalbumin and second antibody conjugated to horseradish peroxidase (cow #1018); E–F) Immunoreactivity of the epithelial lining the alveoli to abcam antibody (cows #1018 and 2014, respectively); G–H) Immunoreactivity of mammary tissue of healthy uninfused quarters of cows 1018 and 2014 to the C-term antibody, respectively. Scale bars indicate 25 µm.

    Techniques Used: Immunohistochemistry, Inhibition, Labeling, Negative Control

    Concentrations of chemoattractants and cytokines in milk samples of the 9 responsive cows. Concentrations were measured by ELISA in the milk samples of the 9 responder cows. Quarters were infused with 25 µg ovalbumin at time 0 and milk samples taken at indicated times. Median values (Q1, Q3) are shown. Concentrations varied significantly (Friedman test) as a function of hpi for C5a, CXCL8, IL-1β, IL-6, IFN-γ, IL-17A (p
    Figure Legend Snippet: Concentrations of chemoattractants and cytokines in milk samples of the 9 responsive cows. Concentrations were measured by ELISA in the milk samples of the 9 responder cows. Quarters were infused with 25 µg ovalbumin at time 0 and milk samples taken at indicated times. Median values (Q1, Q3) are shown. Concentrations varied significantly (Friedman test) as a function of hpi for C5a, CXCL8, IL-1β, IL-6, IFN-γ, IL-17A (p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    4) Product Images from "Comparison of innate immune agonists for induction of tracheal antimicrobial peptide gene expression in tracheal epithelial cells of cattle"

    Article Title: Comparison of innate immune agonists for induction of tracheal antimicrobial peptide gene expression in tracheal epithelial cells of cattle

    Journal: Veterinary Research

    doi: 10.1186/s13567-014-0105-8

    Effects of CpG oligodinucleotide, interleukin-17A and interferon-α on tracheal antimicrobial peptide gene expression. Dose- and time-dependent effects were measured for CpG oligodinucleotide (A,B) , a TLR9 agonist; interleukin-17A (C,D) ; and interferon-α (E,F) . For the dose–response studies (A,C,E) , confluent bTEC were stimulated in triplicate with various concentrations of agonist for 16 h. For the time-course studies (B,D,F) , confluent bTEC were stimulated in triplicate for 4, 8, 16 or 24 h with the doses of agonist shown. Gene expression of TAP relative to that of GAPDH was measured using real-time RT-qPCR. Lipopolysaccharide (LPS, 0.1 μg/mL) was used in all assays as a positive control and standard. *, significantly different from unstimulated cells ( P
    Figure Legend Snippet: Effects of CpG oligodinucleotide, interleukin-17A and interferon-α on tracheal antimicrobial peptide gene expression. Dose- and time-dependent effects were measured for CpG oligodinucleotide (A,B) , a TLR9 agonist; interleukin-17A (C,D) ; and interferon-α (E,F) . For the dose–response studies (A,C,E) , confluent bTEC were stimulated in triplicate with various concentrations of agonist for 16 h. For the time-course studies (B,D,F) , confluent bTEC were stimulated in triplicate for 4, 8, 16 or 24 h with the doses of agonist shown. Gene expression of TAP relative to that of GAPDH was measured using real-time RT-qPCR. Lipopolysaccharide (LPS, 0.1 μg/mL) was used in all assays as a positive control and standard. *, significantly different from unstimulated cells ( P

    Techniques Used: Expressing, Quantitative RT-PCR, Positive Control

    Comparison of the effects of IL-17A, Pam3CSK4 and LPS on tracheal antimicrobial peptide gene expression. Confluent cultures of tracheal epithelial cells from 4 different calves were non-stimulated (NS) or stimulated with 1 μg/mL Pam3CSK4, 316 ng/mL IL-17A, or 0.1 μg/mL LPS for 8 h (A) and 16 h (B) in triplicate. Gene expression was assessed using real-time RT-qPCR. Pam3CSK4 induced significantly higher tracheal antimicrobial peptide gene expression than IL-17A and LPS at both 8 and 16 h ( P
    Figure Legend Snippet: Comparison of the effects of IL-17A, Pam3CSK4 and LPS on tracheal antimicrobial peptide gene expression. Confluent cultures of tracheal epithelial cells from 4 different calves were non-stimulated (NS) or stimulated with 1 μg/mL Pam3CSK4, 316 ng/mL IL-17A, or 0.1 μg/mL LPS for 8 h (A) and 16 h (B) in triplicate. Gene expression was assessed using real-time RT-qPCR. Pam3CSK4 induced significantly higher tracheal antimicrobial peptide gene expression than IL-17A and LPS at both 8 and 16 h ( P

    Techniques Used: Expressing, Quantitative RT-PCR

    Effect of stimulation with single agonists compared to combined agonists. Cultured bovine tracheal epithelial cells were stimulated for 16 h in triplicate with various combinations of 1 μg/mL Pam3CSK4, 316 ng/mL IL-17A, or 0.1 μg/mL LPS. Tracheal antimicrobial peptide gene expression was measured as above. The effects of combined agonists were greater than that of interleukin-17A (IL-17A) alone, but minimally or not different than that of lipopolysaccharide (LPS) or Pam3CSK4 alone. The data shown (panels A, B and C) represent 3 studies conducted on different days using cells from different calves.
    Figure Legend Snippet: Effect of stimulation with single agonists compared to combined agonists. Cultured bovine tracheal epithelial cells were stimulated for 16 h in triplicate with various combinations of 1 μg/mL Pam3CSK4, 316 ng/mL IL-17A, or 0.1 μg/mL LPS. Tracheal antimicrobial peptide gene expression was measured as above. The effects of combined agonists were greater than that of interleukin-17A (IL-17A) alone, but minimally or not different than that of lipopolysaccharide (LPS) or Pam3CSK4 alone. The data shown (panels A, B and C) represent 3 studies conducted on different days using cells from different calves.

    Techniques Used: Cell Culture, Expressing

    5) Product Images from "Enhancing the toolbox to study IL-17A in cattle and sheep"

    Article Title: Enhancing the toolbox to study IL-17A in cattle and sheep

    Journal: Veterinary Research

    doi: 10.1186/s13567-017-0426-5

    Evaluation of commercial antibodies for the intracellular detection of recombinant bovine and ovine IL-17A. The eight commercial antibodies listed in Table 1 were tested against fixed, permeabilised untransfected (UTF) CHO cells and CHO cells transfected with cDNA encoding bovIL-17A or ovIL-17A for their capacity to detect intracellular recombinant IL-17A by flow cytometry. Results are shown for one polyclonal antibody (pab) produced against bovIL-17A ( A ) and seven monoclonal antibodies (mabs) produced against human or mouse IL-17A ( B – D ). Profiles of the relevant control antibodies listed in Table 2 are included in the overlapping histograms. Events were acquired on the MacsQuant according to the gating strategy described previously (in brief) and shown in Additional file 2 . Line colours representing different antibody treatments are given in parentheses: A Primary rabbit anti-bovine IL-17A pab PB0274B-100 at 1 μg/mL (A.1, red) or negative control primary anti-bovine CD34 pab (in-house) at an estimated 1 μg/mL equivalent (a, black) then detected with a secondary goat anti-rabbit alexafluor 488 at 1 μg/mL; B Directly conjugated mouse anti-human IL-17A eBio64DEC17-phycoerythrin (PE) mab (IgG1) at 2.5 μg/mL (B.1, red) and control IgG1 VPM21 mab (in-house) at an estimated 2.5 μg/mL equivalent (b, black) and detected with goat anti-mouse PE at 1 μg/mL; C Primary mouse anti-human IL-17A mabs MT44.6 (C.1, blue), MT241 (C.2, green), MT2770 (C.3, brown) and MT504 (C.4, red) [all IgG1] at 0.5 μg/mL and control IgG1 VPM21 mab (in-house) at an estimated 0.5 μg/mL equivalent (black), all detected with goat anti-mouse PE at 1 μg/mL; D Primary mouse anti-human IL-17A mabs #41809 (D.1, red) (IgG2b) and #41802 (D.2, blue) (IgG1) at 2.5 μg/mL and a mixture of control mabs VPM21 (IgG1) and VPM22 (IgG2b) at an estimated 2.5 μg/mL equivalent (d, black), all detected with goat anti-mouse PE at 1 μg/mL.
    Figure Legend Snippet: Evaluation of commercial antibodies for the intracellular detection of recombinant bovine and ovine IL-17A. The eight commercial antibodies listed in Table 1 were tested against fixed, permeabilised untransfected (UTF) CHO cells and CHO cells transfected with cDNA encoding bovIL-17A or ovIL-17A for their capacity to detect intracellular recombinant IL-17A by flow cytometry. Results are shown for one polyclonal antibody (pab) produced against bovIL-17A ( A ) and seven monoclonal antibodies (mabs) produced against human or mouse IL-17A ( B – D ). Profiles of the relevant control antibodies listed in Table 2 are included in the overlapping histograms. Events were acquired on the MacsQuant according to the gating strategy described previously (in brief) and shown in Additional file 2 . Line colours representing different antibody treatments are given in parentheses: A Primary rabbit anti-bovine IL-17A pab PB0274B-100 at 1 μg/mL (A.1, red) or negative control primary anti-bovine CD34 pab (in-house) at an estimated 1 μg/mL equivalent (a, black) then detected with a secondary goat anti-rabbit alexafluor 488 at 1 μg/mL; B Directly conjugated mouse anti-human IL-17A eBio64DEC17-phycoerythrin (PE) mab (IgG1) at 2.5 μg/mL (B.1, red) and control IgG1 VPM21 mab (in-house) at an estimated 2.5 μg/mL equivalent (b, black) and detected with goat anti-mouse PE at 1 μg/mL; C Primary mouse anti-human IL-17A mabs MT44.6 (C.1, blue), MT241 (C.2, green), MT2770 (C.3, brown) and MT504 (C.4, red) [all IgG1] at 0.5 μg/mL and control IgG1 VPM21 mab (in-house) at an estimated 0.5 μg/mL equivalent (black), all detected with goat anti-mouse PE at 1 μg/mL; D Primary mouse anti-human IL-17A mabs #41809 (D.1, red) (IgG2b) and #41802 (D.2, blue) (IgG1) at 2.5 μg/mL and a mixture of control mabs VPM21 (IgG1) and VPM22 (IgG2b) at an estimated 2.5 μg/mL equivalent (d, black), all detected with goat anti-mouse PE at 1 μg/mL.

    Techniques Used: Recombinant, Transfection, Flow Cytometry, Cytometry, Produced, Negative Control

    Measurement and biological function of recombinant bovine and ovine IL-17A and detection of native ovine IL-17A by ELISA. A Detection of rbov and rovIL-17A by ELISA. The supernatants from transfected CHO cells expressing rbovIL-17A or rovIL-17A, or control parent untransfected line (UTF) were serially diluted (Log 3 dilutions) and evaluated using the commercial bovIL-17A ELISA. Data presented are optical density (OD) values from the Spectrophotometer at 450 nm. The X-axis displays Dilution 1/X and the Y-axis gives the OD value. Readings from UTF supernatant were below the limit of detection. B Functional activity of rbov and rovIL-17A on bovine embryonic lung cells. Bovine embryonic lung (EBL) cells were stimulated with 100 ng/mL CHO-expressed rbovIL-17A or rovIL-17A or UTF CHO negative control supernatant. Following 24 h incubation, culture supernatants were collected from triplicate cultures then tested for CXCL8 by ELISA. The X-axis displays the bioassay treatments and the Y-axis shows CXCL8 production in pg/mL. Data are the arithmetic mean of three technical replicates with error bars representing the standard error from one of three experiments. CXCL8 expression between treatments was statistically assessed using Kruskal–Wallis test. C Functional activity of rbov and rovIL-17A on ovine ST-6 cells. Ovine ST-6 cells were stimulated with 100 ng/mL CHO-expressed rbovIL-17A or rovIL-17A or UTF CHO supernatant. Following 24 h incubation and culture supernatants collected, tested and analysed as described in Figure 2B. CXCL8 expression between treatments was statistically assessed using Kruskal–Wallis test. D Detection of native ovIL-17A by ELISA. Ovine PBMC were cultured at 2 × 10 6 cells/mL with or without 5 μg/mL ConA. Culture supernatants were analysed for IL-17A using the bovIL-17A ELISA. Data represent the arithmetic mean of PBMC from six ewes and error bars represent standard error. Data were analysed statistically for significance using the two-tailed Mann–Whitney test.
    Figure Legend Snippet: Measurement and biological function of recombinant bovine and ovine IL-17A and detection of native ovine IL-17A by ELISA. A Detection of rbov and rovIL-17A by ELISA. The supernatants from transfected CHO cells expressing rbovIL-17A or rovIL-17A, or control parent untransfected line (UTF) were serially diluted (Log 3 dilutions) and evaluated using the commercial bovIL-17A ELISA. Data presented are optical density (OD) values from the Spectrophotometer at 450 nm. The X-axis displays Dilution 1/X and the Y-axis gives the OD value. Readings from UTF supernatant were below the limit of detection. B Functional activity of rbov and rovIL-17A on bovine embryonic lung cells. Bovine embryonic lung (EBL) cells were stimulated with 100 ng/mL CHO-expressed rbovIL-17A or rovIL-17A or UTF CHO negative control supernatant. Following 24 h incubation, culture supernatants were collected from triplicate cultures then tested for CXCL8 by ELISA. The X-axis displays the bioassay treatments and the Y-axis shows CXCL8 production in pg/mL. Data are the arithmetic mean of three technical replicates with error bars representing the standard error from one of three experiments. CXCL8 expression between treatments was statistically assessed using Kruskal–Wallis test. C Functional activity of rbov and rovIL-17A on ovine ST-6 cells. Ovine ST-6 cells were stimulated with 100 ng/mL CHO-expressed rbovIL-17A or rovIL-17A or UTF CHO supernatant. Following 24 h incubation and culture supernatants collected, tested and analysed as described in Figure 2B. CXCL8 expression between treatments was statistically assessed using Kruskal–Wallis test. D Detection of native ovIL-17A by ELISA. Ovine PBMC were cultured at 2 × 10 6 cells/mL with or without 5 μg/mL ConA. Culture supernatants were analysed for IL-17A using the bovIL-17A ELISA. Data represent the arithmetic mean of PBMC from six ewes and error bars represent standard error. Data were analysed statistically for significance using the two-tailed Mann–Whitney test.

    Techniques Used: Recombinant, Enzyme-linked Immunosorbent Assay, Transfection, Expressing, Spectrophotometry, Functional Assay, Activity Assay, Negative Control, Incubation, Cell Culture, Two Tailed Test, MANN-WHITNEY

    Relative intracellular expression of IL-17A and IFN-γ by activated bovine and ovine PBMC. The data sets described in “ Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section ” and presented in Figures 5 and 6 are summarised to compare overall intracellular expression of IL-17A ( A ) and IFN-γ ( B ) by PMA/ionomycin-stimulated bovine and ovine PBMC. Each bar represents the arithmetic mean of four cattle or four sheep and the error bars represent the standard error. The data for total percentage IFN-γ and IL-17A expression between species were assessed statistically using two-tailed Mann–Whitney tests allowing for ties.
    Figure Legend Snippet: Relative intracellular expression of IL-17A and IFN-γ by activated bovine and ovine PBMC. The data sets described in “ Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section ” and presented in Figures 5 and 6 are summarised to compare overall intracellular expression of IL-17A ( A ) and IFN-γ ( B ) by PMA/ionomycin-stimulated bovine and ovine PBMC. Each bar represents the arithmetic mean of four cattle or four sheep and the error bars represent the standard error. The data for total percentage IFN-γ and IL-17A expression between species were assessed statistically using two-tailed Mann–Whitney tests allowing for ties.

    Techniques Used: Expressing, Two Tailed Test, MANN-WHITNEY

    Detection of single-cell expression of ruminant IL-17A by ELISpot. Plates and PBMC were prepared and cultured as described in “ IL-17A ELISpot section ”. ELISpot images shown are representative of PBMC from one of three cattle ( A ) and one of three sheep ( B ) activated with ConA and PMA/ionomycin. The average number of spot-forming units (SFU) with standard errors are shown for 10 6 PBMC from all three cattle (grey bars) and sheep (black bars), stimulated under the different conditions ( C ). Data were modelled by fitting a Poisson generalised linear mixed model (GLMM) by maximum likelihood to the IL-17A SFU/10 6 values, using logarithmic link function and Laplace approximations to calculate log-likelihoods. The model included treatment (medium control, ConA and PMA/ionomycin), species (bovine, ovine) and their interaction as fixed effects and animal identification as a random effect in order to account for both within- and between-animal variability. An observation-level random effect term was specified to account for data over-dispersion. The statistical significance of the fixed effect terms was assessed using p values derived from type II Wald Chi square tests. Linear hypothesis tests were defined from the GLMM in order to conduct pair-wise comparisons of means between treatments and species. The associated p values were adjusted for false discovery rate (FDR) following Benjamini–Hochberg’s procedure.
    Figure Legend Snippet: Detection of single-cell expression of ruminant IL-17A by ELISpot. Plates and PBMC were prepared and cultured as described in “ IL-17A ELISpot section ”. ELISpot images shown are representative of PBMC from one of three cattle ( A ) and one of three sheep ( B ) activated with ConA and PMA/ionomycin. The average number of spot-forming units (SFU) with standard errors are shown for 10 6 PBMC from all three cattle (grey bars) and sheep (black bars), stimulated under the different conditions ( C ). Data were modelled by fitting a Poisson generalised linear mixed model (GLMM) by maximum likelihood to the IL-17A SFU/10 6 values, using logarithmic link function and Laplace approximations to calculate log-likelihoods. The model included treatment (medium control, ConA and PMA/ionomycin), species (bovine, ovine) and their interaction as fixed effects and animal identification as a random effect in order to account for both within- and between-animal variability. An observation-level random effect term was specified to account for data over-dispersion. The statistical significance of the fixed effect terms was assessed using p values derived from type II Wald Chi square tests. Linear hypothesis tests were defined from the GLMM in order to conduct pair-wise comparisons of means between treatments and species. The associated p values were adjusted for false discovery rate (FDR) following Benjamini–Hochberg’s procedure.

    Techniques Used: Expressing, Enzyme-linked Immunospot, Cell Culture, Derivative Assay

    Intracellular expression of IL-17A and IFN-γ by activated bovine T cell subsets. PBMC from four cattle were stimulated with phorbol 12-myristate 13 acetate, ionomycin and brefeldin A in RPMI culture medium for 4 h. Cells were harvested and stained for viability and with mabs specific for cell-surface phenotypic markers and intracellular cytokines as described in Table 3 and “ Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section ”. Cells were stained for CD4 with mab CC8-PE at 1:20 dilution ( A , D ), for CD8β with mab CC58-PE at 1:20 dilution ( B , D ) and for WC-1 (γδ T cells) with mab CC15-PE at 1:200 ( C , E ). Intracellular cytokine staining for IL-17A was conducted using mab eBioDEC17-APC at a 1:20 dilution ( A – C ) and for IFN-γ using mab CC302-Alexafluor 647 at a 1:200 dilution ( D – F ). Data are shown for PBMC from one representative animal of four.
    Figure Legend Snippet: Intracellular expression of IL-17A and IFN-γ by activated bovine T cell subsets. PBMC from four cattle were stimulated with phorbol 12-myristate 13 acetate, ionomycin and brefeldin A in RPMI culture medium for 4 h. Cells were harvested and stained for viability and with mabs specific for cell-surface phenotypic markers and intracellular cytokines as described in Table 3 and “ Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section ”. Cells were stained for CD4 with mab CC8-PE at 1:20 dilution ( A , D ), for CD8β with mab CC58-PE at 1:20 dilution ( B , D ) and for WC-1 (γδ T cells) with mab CC15-PE at 1:200 ( C , E ). Intracellular cytokine staining for IL-17A was conducted using mab eBioDEC17-APC at a 1:20 dilution ( A – C ) and for IFN-γ using mab CC302-Alexafluor 647 at a 1:200 dilution ( D – F ). Data are shown for PBMC from one representative animal of four.

    Techniques Used: Expressing, Staining

    Phylogenetic tree of mammalian IL-17A protein sequences. Evolutionary sequence comparisons were undertaken using 13 selected mammalian and other IL-17A sequences by initially conducting a multiple alignment using Clustal Omega (EMBL/EBI online, [ 21 ]). The evolutionary relationships between the sequences were inferred using Mr. Bayes launched from TOPALI v 2.5 using the Jones–Taylor–Thornton plus gamma (JTT + G) model with two runs each of 1 250 000 generations with a burn in period of 20% and sampling frequency of 1000. The horizontal lines are branches whose length represents the amount of genetic change over time. The scale bar shows the distance represented by 0.1 expected substitutions per site. The robustness of the clustering of sequences are shown by the Bayesian Posterior Probabilities at the nodes. Accession numbers of the sequences used for the comparison are: Human NP_002181.1; House mouse NP_034682.1; Cow NP_001008412.1; Sheep XP_004018936.1; Goat NP_001272654.1; Horse NP_001137264.1; Pig NP_001005729.1; Dog NP_001159350.1; Domestic guinea pig NP_001265697.1; Koala AHZ08738.1; Chicken NP_989791.1; EGW10039.1 Chinese hamster and European rabbit AMQ91106.1. The phylogenetic tree was annotated using Dendroscope.
    Figure Legend Snippet: Phylogenetic tree of mammalian IL-17A protein sequences. Evolutionary sequence comparisons were undertaken using 13 selected mammalian and other IL-17A sequences by initially conducting a multiple alignment using Clustal Omega (EMBL/EBI online, [ 21 ]). The evolutionary relationships between the sequences were inferred using Mr. Bayes launched from TOPALI v 2.5 using the Jones–Taylor–Thornton plus gamma (JTT + G) model with two runs each of 1 250 000 generations with a burn in period of 20% and sampling frequency of 1000. The horizontal lines are branches whose length represents the amount of genetic change over time. The scale bar shows the distance represented by 0.1 expected substitutions per site. The robustness of the clustering of sequences are shown by the Bayesian Posterior Probabilities at the nodes. Accession numbers of the sequences used for the comparison are: Human NP_002181.1; House mouse NP_034682.1; Cow NP_001008412.1; Sheep XP_004018936.1; Goat NP_001272654.1; Horse NP_001137264.1; Pig NP_001005729.1; Dog NP_001159350.1; Domestic guinea pig NP_001265697.1; Koala AHZ08738.1; Chicken NP_989791.1; EGW10039.1 Chinese hamster and European rabbit AMQ91106.1. The phylogenetic tree was annotated using Dendroscope.

    Techniques Used: Sequencing, Sampling

    Intracellular expression of IL-17A and IFN-γ by activated ovine T cell subsets. PBMC from four sheep were stimulated with phorbol 12-myristate 13 acetate, ionomycin and brefeldin A in RPMI culture medium for 4 h. Cells were harvested and stained for viability and with mabs specific for cell-surface phenotypic markers and intracellular cytokines as described in Table 3 and “ Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section ”. Cells were then stained for CD4 with mab 44.38-PE at 1:20 dilution ( A , D ), CD8β with mab CC58-PE at 1:20 dilution ( B , D ) and WC-1 (γδ) with mab CC15-PE at 1:200 ( C , E ). Intracellular cytokine staining for IL-17A was conducted using mab eBio64DEC17-APC a 1:20 dilution ( A – C ) and for IFN-γ using mab CC302-alexafluor 647 at a 1:200 dilution ( D – F ). Data shown is for one representative animal out of four.
    Figure Legend Snippet: Intracellular expression of IL-17A and IFN-γ by activated ovine T cell subsets. PBMC from four sheep were stimulated with phorbol 12-myristate 13 acetate, ionomycin and brefeldin A in RPMI culture medium for 4 h. Cells were harvested and stained for viability and with mabs specific for cell-surface phenotypic markers and intracellular cytokines as described in Table 3 and “ Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section ”. Cells were then stained for CD4 with mab 44.38-PE at 1:20 dilution ( A , D ), CD8β with mab CC58-PE at 1:20 dilution ( B , D ) and WC-1 (γδ) with mab CC15-PE at 1:200 ( C , E ). Intracellular cytokine staining for IL-17A was conducted using mab eBio64DEC17-APC a 1:20 dilution ( A – C ) and for IFN-γ using mab CC302-alexafluor 647 at a 1:200 dilution ( D – F ). Data shown is for one representative animal out of four.

    Techniques Used: Expressing, Staining

    6) Product Images from "Enhancing the toolbox to study IL-17A in cattle and sheep"

    Article Title: Enhancing the toolbox to study IL-17A in cattle and sheep

    Journal: Veterinary Research

    doi: 10.1186/s13567-017-0426-5

    Evaluation of commercial antibodies for the intracellular detection of recombinant bovine and ovine IL-17A. The eight commercial antibodies listed in Table 1 were tested against fixed, permeabilised untransfected (UTF) CHO cells and CHO cells transfected with cDNA encoding bovIL-17A or ovIL-17A for their capacity to detect intracellular recombinant IL-17A by flow cytometry. Results are shown for one polyclonal antibody (pab) produced against bovIL-17A ( A ) and seven monoclonal antibodies (mabs) produced against human or mouse IL-17A ( B – D ). Profiles of the relevant control antibodies listed in Table 2 are included in the overlapping histograms. Events were acquired on the MacsQuant according to the gating strategy described previously (in brief) and shown in Additional file 2 . Line colours representing different antibody treatments are given in parentheses: A Primary rabbit anti-bovine IL-17A pab PB0274B-100 at 1 μg/mL (A.1, red) or negative control primary anti-bovine CD34 pab (in-house) at an estimated 1 μg/mL equivalent (a, black) then detected with a secondary goat anti-rabbit alexafluor 488 at 1 μg/mL; B Directly conjugated mouse anti-human IL-17A eBio64DEC17-phycoerythrin (PE) mab (IgG1) at 2.5 μg/mL (B.1, red) and control IgG1 VPM21 mab (in-house) at an estimated 2.5 μg/mL equivalent (b, black) and detected with goat anti-mouse PE at 1 μg/mL; C Primary mouse anti-human IL-17A mabs MT44.6 (C.1, blue), MT241 (C.2, green), MT2770 (C.3, brown) and MT504 (C.4, red) [all IgG1] at 0.5 μg/mL and control IgG1 VPM21 mab (in-house) at an estimated 0.5 μg/mL equivalent (black), all detected with goat anti-mouse PE at 1 μg/mL; D Primary mouse anti-human IL-17A mabs #41809 (D.1, red) (IgG2b) and #41802 (D.2, blue) (IgG1) at 2.5 μg/mL and a mixture of control mabs VPM21 (IgG1) and VPM22 (IgG2b) at an estimated 2.5 μg/mL equivalent (d, black), all detected with goat anti-mouse PE at 1 μg/mL.
    Figure Legend Snippet: Evaluation of commercial antibodies for the intracellular detection of recombinant bovine and ovine IL-17A. The eight commercial antibodies listed in Table 1 were tested against fixed, permeabilised untransfected (UTF) CHO cells and CHO cells transfected with cDNA encoding bovIL-17A or ovIL-17A for their capacity to detect intracellular recombinant IL-17A by flow cytometry. Results are shown for one polyclonal antibody (pab) produced against bovIL-17A ( A ) and seven monoclonal antibodies (mabs) produced against human or mouse IL-17A ( B – D ). Profiles of the relevant control antibodies listed in Table 2 are included in the overlapping histograms. Events were acquired on the MacsQuant according to the gating strategy described previously (in brief) and shown in Additional file 2 . Line colours representing different antibody treatments are given in parentheses: A Primary rabbit anti-bovine IL-17A pab PB0274B-100 at 1 μg/mL (A.1, red) or negative control primary anti-bovine CD34 pab (in-house) at an estimated 1 μg/mL equivalent (a, black) then detected with a secondary goat anti-rabbit alexafluor 488 at 1 μg/mL; B Directly conjugated mouse anti-human IL-17A eBio64DEC17-phycoerythrin (PE) mab (IgG1) at 2.5 μg/mL (B.1, red) and control IgG1 VPM21 mab (in-house) at an estimated 2.5 μg/mL equivalent (b, black) and detected with goat anti-mouse PE at 1 μg/mL; C Primary mouse anti-human IL-17A mabs MT44.6 (C.1, blue), MT241 (C.2, green), MT2770 (C.3, brown) and MT504 (C.4, red) [all IgG1] at 0.5 μg/mL and control IgG1 VPM21 mab (in-house) at an estimated 0.5 μg/mL equivalent (black), all detected with goat anti-mouse PE at 1 μg/mL; D Primary mouse anti-human IL-17A mabs #41809 (D.1, red) (IgG2b) and #41802 (D.2, blue) (IgG1) at 2.5 μg/mL and a mixture of control mabs VPM21 (IgG1) and VPM22 (IgG2b) at an estimated 2.5 μg/mL equivalent (d, black), all detected with goat anti-mouse PE at 1 μg/mL.

    Techniques Used: Recombinant, Transfection, Flow Cytometry, Cytometry, Produced, Negative Control

    Measurement and biological function of recombinant bovine and ovine IL-17A and detection of native ovine IL-17A by ELISA. A Detection of rbov and rovIL-17A by ELISA. The supernatants from transfected CHO cells expressing rbovIL-17A or rovIL-17A, or control parent untransfected line (UTF) were serially diluted (Log 3 dilutions) and evaluated using the commercial bovIL-17A ELISA. Data presented are optical density (OD) values from the Spectrophotometer at 450 nm. The X-axis displays Dilution 1/X and the Y-axis gives the OD value. Readings from UTF supernatant were below the limit of detection. B Functional activity of rbov and rovIL-17A on bovine embryonic lung cells. Bovine embryonic lung (EBL) cells were stimulated with 100 ng/mL CHO-expressed rbovIL-17A or rovIL-17A or UTF CHO negative control supernatant. Following 24 h incubation, culture supernatants were collected from triplicate cultures then tested for CXCL8 by ELISA. The X-axis displays the bioassay treatments and the Y-axis shows CXCL8 production in pg/mL. Data are the arithmetic mean of three technical replicates with error bars representing the standard error from one of three experiments. CXCL8 expression between treatments was statistically assessed using Kruskal–Wallis test. C Functional activity of rbov and rovIL-17A on ovine ST-6 cells. Ovine ST-6 cells were stimulated with 100 ng/mL CHO-expressed rbovIL-17A or rovIL-17A or UTF CHO supernatant. Following 24 h incubation and culture supernatants collected, tested and analysed as described in Figure 2B. CXCL8 expression between treatments was statistically assessed using Kruskal–Wallis test. D Detection of native ovIL-17A by ELISA. Ovine PBMC were cultured at 2 × 10 6 cells/mL with or without 5 μg/mL ConA. Culture supernatants were analysed for IL-17A using the bovIL-17A ELISA. Data represent the arithmetic mean of PBMC from six ewes and error bars represent standard error. Data were analysed statistically for significance using the two-tailed Mann–Whitney test.
    Figure Legend Snippet: Measurement and biological function of recombinant bovine and ovine IL-17A and detection of native ovine IL-17A by ELISA. A Detection of rbov and rovIL-17A by ELISA. The supernatants from transfected CHO cells expressing rbovIL-17A or rovIL-17A, or control parent untransfected line (UTF) were serially diluted (Log 3 dilutions) and evaluated using the commercial bovIL-17A ELISA. Data presented are optical density (OD) values from the Spectrophotometer at 450 nm. The X-axis displays Dilution 1/X and the Y-axis gives the OD value. Readings from UTF supernatant were below the limit of detection. B Functional activity of rbov and rovIL-17A on bovine embryonic lung cells. Bovine embryonic lung (EBL) cells were stimulated with 100 ng/mL CHO-expressed rbovIL-17A or rovIL-17A or UTF CHO negative control supernatant. Following 24 h incubation, culture supernatants were collected from triplicate cultures then tested for CXCL8 by ELISA. The X-axis displays the bioassay treatments and the Y-axis shows CXCL8 production in pg/mL. Data are the arithmetic mean of three technical replicates with error bars representing the standard error from one of three experiments. CXCL8 expression between treatments was statistically assessed using Kruskal–Wallis test. C Functional activity of rbov and rovIL-17A on ovine ST-6 cells. Ovine ST-6 cells were stimulated with 100 ng/mL CHO-expressed rbovIL-17A or rovIL-17A or UTF CHO supernatant. Following 24 h incubation and culture supernatants collected, tested and analysed as described in Figure 2B. CXCL8 expression between treatments was statistically assessed using Kruskal–Wallis test. D Detection of native ovIL-17A by ELISA. Ovine PBMC were cultured at 2 × 10 6 cells/mL with or without 5 μg/mL ConA. Culture supernatants were analysed for IL-17A using the bovIL-17A ELISA. Data represent the arithmetic mean of PBMC from six ewes and error bars represent standard error. Data were analysed statistically for significance using the two-tailed Mann–Whitney test.

    Techniques Used: Recombinant, Enzyme-linked Immunosorbent Assay, Transfection, Expressing, Spectrophotometry, Functional Assay, Activity Assay, Negative Control, Incubation, Cell Culture, Two Tailed Test, MANN-WHITNEY

    Relative intracellular expression of IL-17A and IFN-γ by activated bovine and ovine PBMC. The data sets described in “ Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section ” and presented in Figures 5 and 6 are summarised to compare overall intracellular expression of IL-17A ( A ) and IFN-γ ( B ) by PMA/ionomycin-stimulated bovine and ovine PBMC. Each bar represents the arithmetic mean of four cattle or four sheep and the error bars represent the standard error. The data for total percentage IFN-γ and IL-17A expression between species were assessed statistically using two-tailed Mann–Whitney tests allowing for ties.
    Figure Legend Snippet: Relative intracellular expression of IL-17A and IFN-γ by activated bovine and ovine PBMC. The data sets described in “ Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section ” and presented in Figures 5 and 6 are summarised to compare overall intracellular expression of IL-17A ( A ) and IFN-γ ( B ) by PMA/ionomycin-stimulated bovine and ovine PBMC. Each bar represents the arithmetic mean of four cattle or four sheep and the error bars represent the standard error. The data for total percentage IFN-γ and IL-17A expression between species were assessed statistically using two-tailed Mann–Whitney tests allowing for ties.

    Techniques Used: Expressing, Two Tailed Test, MANN-WHITNEY

    Detection of single-cell expression of ruminant IL-17A by ELISpot. Plates and PBMC were prepared and cultured as described in “ IL-17A ELISpot section ”. ELISpot images shown are representative of PBMC from one of three cattle ( A ) and one of three sheep ( B ) activated with ConA and PMA/ionomycin. The average number of spot-forming units (SFU) with standard errors are shown for 10 6 PBMC from all three cattle (grey bars) and sheep (black bars), stimulated under the different conditions ( C ). Data were modelled by fitting a Poisson generalised linear mixed model (GLMM) by maximum likelihood to the IL-17A SFU/10 6 values, using logarithmic link function and Laplace approximations to calculate log-likelihoods. The model included treatment (medium control, ConA and PMA/ionomycin), species (bovine, ovine) and their interaction as fixed effects and animal identification as a random effect in order to account for both within- and between-animal variability. An observation-level random effect term was specified to account for data over-dispersion. The statistical significance of the fixed effect terms was assessed using p values derived from type II Wald Chi square tests. Linear hypothesis tests were defined from the GLMM in order to conduct pair-wise comparisons of means between treatments and species. The associated p values were adjusted for false discovery rate (FDR) following Benjamini–Hochberg’s procedure.
    Figure Legend Snippet: Detection of single-cell expression of ruminant IL-17A by ELISpot. Plates and PBMC were prepared and cultured as described in “ IL-17A ELISpot section ”. ELISpot images shown are representative of PBMC from one of three cattle ( A ) and one of three sheep ( B ) activated with ConA and PMA/ionomycin. The average number of spot-forming units (SFU) with standard errors are shown for 10 6 PBMC from all three cattle (grey bars) and sheep (black bars), stimulated under the different conditions ( C ). Data were modelled by fitting a Poisson generalised linear mixed model (GLMM) by maximum likelihood to the IL-17A SFU/10 6 values, using logarithmic link function and Laplace approximations to calculate log-likelihoods. The model included treatment (medium control, ConA and PMA/ionomycin), species (bovine, ovine) and their interaction as fixed effects and animal identification as a random effect in order to account for both within- and between-animal variability. An observation-level random effect term was specified to account for data over-dispersion. The statistical significance of the fixed effect terms was assessed using p values derived from type II Wald Chi square tests. Linear hypothesis tests were defined from the GLMM in order to conduct pair-wise comparisons of means between treatments and species. The associated p values were adjusted for false discovery rate (FDR) following Benjamini–Hochberg’s procedure.

    Techniques Used: Expressing, Enzyme-linked Immunospot, Cell Culture, Derivative Assay

    Intracellular expression of IL-17A and IFN-γ by activated bovine T cell subsets. PBMC from four cattle were stimulated with phorbol 12-myristate 13 acetate, ionomycin and brefeldin A in RPMI culture medium for 4 h. Cells were harvested and stained for viability and with mabs specific for cell-surface phenotypic markers and intracellular cytokines as described in Table 3 and “ Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section ”. Cells were stained for CD4 with mab CC8-PE at 1:20 dilution ( A , D ), for CD8β with mab CC58-PE at 1:20 dilution ( B , D ) and for WC-1 (γδ T cells) with mab CC15-PE at 1:200 ( C , E ). Intracellular cytokine staining for IL-17A was conducted using mab eBioDEC17-APC at a 1:20 dilution ( A – C ) and for IFN-γ using mab CC302-Alexafluor 647 at a 1:200 dilution ( D – F ). Data are shown for PBMC from one representative animal of four.
    Figure Legend Snippet: Intracellular expression of IL-17A and IFN-γ by activated bovine T cell subsets. PBMC from four cattle were stimulated with phorbol 12-myristate 13 acetate, ionomycin and brefeldin A in RPMI culture medium for 4 h. Cells were harvested and stained for viability and with mabs specific for cell-surface phenotypic markers and intracellular cytokines as described in Table 3 and “ Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section ”. Cells were stained for CD4 with mab CC8-PE at 1:20 dilution ( A , D ), for CD8β with mab CC58-PE at 1:20 dilution ( B , D ) and for WC-1 (γδ T cells) with mab CC15-PE at 1:200 ( C , E ). Intracellular cytokine staining for IL-17A was conducted using mab eBioDEC17-APC at a 1:20 dilution ( A – C ) and for IFN-γ using mab CC302-Alexafluor 647 at a 1:200 dilution ( D – F ). Data are shown for PBMC from one representative animal of four.

    Techniques Used: Expressing, Staining

    Phylogenetic tree of mammalian IL-17A protein sequences. Evolutionary sequence comparisons were undertaken using 13 selected mammalian and other IL-17A sequences by initially conducting a multiple alignment using Clustal Omega (EMBL/EBI online, [ 21 ]). The evolutionary relationships between the sequences were inferred using Mr. Bayes launched from TOPALI v 2.5 using the Jones–Taylor–Thornton plus gamma (JTT + G) model with two runs each of 1 250 000 generations with a burn in period of 20% and sampling frequency of 1000. The horizontal lines are branches whose length represents the amount of genetic change over time. The scale bar shows the distance represented by 0.1 expected substitutions per site. The robustness of the clustering of sequences are shown by the Bayesian Posterior Probabilities at the nodes. Accession numbers of the sequences used for the comparison are: Human NP_002181.1; House mouse NP_034682.1; Cow NP_001008412.1; Sheep XP_004018936.1; Goat NP_001272654.1; Horse NP_001137264.1; Pig NP_001005729.1; Dog NP_001159350.1; Domestic guinea pig NP_001265697.1; Koala AHZ08738.1; Chicken NP_989791.1; EGW10039.1 Chinese hamster and European rabbit AMQ91106.1. The phylogenetic tree was annotated using Dendroscope.
    Figure Legend Snippet: Phylogenetic tree of mammalian IL-17A protein sequences. Evolutionary sequence comparisons were undertaken using 13 selected mammalian and other IL-17A sequences by initially conducting a multiple alignment using Clustal Omega (EMBL/EBI online, [ 21 ]). The evolutionary relationships between the sequences were inferred using Mr. Bayes launched from TOPALI v 2.5 using the Jones–Taylor–Thornton plus gamma (JTT + G) model with two runs each of 1 250 000 generations with a burn in period of 20% and sampling frequency of 1000. The horizontal lines are branches whose length represents the amount of genetic change over time. The scale bar shows the distance represented by 0.1 expected substitutions per site. The robustness of the clustering of sequences are shown by the Bayesian Posterior Probabilities at the nodes. Accession numbers of the sequences used for the comparison are: Human NP_002181.1; House mouse NP_034682.1; Cow NP_001008412.1; Sheep XP_004018936.1; Goat NP_001272654.1; Horse NP_001137264.1; Pig NP_001005729.1; Dog NP_001159350.1; Domestic guinea pig NP_001265697.1; Koala AHZ08738.1; Chicken NP_989791.1; EGW10039.1 Chinese hamster and European rabbit AMQ91106.1. The phylogenetic tree was annotated using Dendroscope.

    Techniques Used: Sequencing, Sampling

    Intracellular expression of IL-17A and IFN-γ by activated ovine T cell subsets. PBMC from four sheep were stimulated with phorbol 12-myristate 13 acetate, ionomycin and brefeldin A in RPMI culture medium for 4 h. Cells were harvested and stained for viability and with mabs specific for cell-surface phenotypic markers and intracellular cytokines as described in Table 3 and “ Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section ”. Cells were then stained for CD4 with mab 44.38-PE at 1:20 dilution ( A , D ), CD8β with mab CC58-PE at 1:20 dilution ( B , D ) and WC-1 (γδ) with mab CC15-PE at 1:200 ( C , E ). Intracellular cytokine staining for IL-17A was conducted using mab eBio64DEC17-APC a 1:20 dilution ( A – C ) and for IFN-γ using mab CC302-alexafluor 647 at a 1:200 dilution ( D – F ). Data shown is for one representative animal out of four.
    Figure Legend Snippet: Intracellular expression of IL-17A and IFN-γ by activated ovine T cell subsets. PBMC from four sheep were stimulated with phorbol 12-myristate 13 acetate, ionomycin and brefeldin A in RPMI culture medium for 4 h. Cells were harvested and stained for viability and with mabs specific for cell-surface phenotypic markers and intracellular cytokines as described in Table 3 and “ Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section ”. Cells were then stained for CD4 with mab 44.38-PE at 1:20 dilution ( A , D ), CD8β with mab CC58-PE at 1:20 dilution ( B , D ) and WC-1 (γδ) with mab CC15-PE at 1:200 ( C , E ). Intracellular cytokine staining for IL-17A was conducted using mab eBio64DEC17-APC a 1:20 dilution ( A – C ) and for IFN-γ using mab CC302-alexafluor 647 at a 1:200 dilution ( D – F ). Data shown is for one representative animal out of four.

    Techniques Used: Expressing, Staining

    7) Product Images from "Regulation of tracheal antimicrobial peptide gene expression in airway epithelial cells of cattle"

    Article Title: Regulation of tracheal antimicrobial peptide gene expression in airway epithelial cells of cattle

    Journal: Veterinary Research

    doi: 10.1186/s13567-016-0329-x

    Effect of dexamethasone on LPS-, Pam3CSK4- and IL-17A-induced up-regulation of TAP mRNA. Bovine tracheal epithelial cells were grown in the presence or absence of 10 −6 M dexamethasone for 24 h. LPS (0.1 μg/mL), Pam3CSK4 (1 μg/mL) or IL-17A (316 ng/mL) were added for 16 h. Each of these agonists significantly upregulated TAP gene expression relative to the reference gene GAPDH ( P
    Figure Legend Snippet: Effect of dexamethasone on LPS-, Pam3CSK4- and IL-17A-induced up-regulation of TAP mRNA. Bovine tracheal epithelial cells were grown in the presence or absence of 10 −6 M dexamethasone for 24 h. LPS (0.1 μg/mL), Pam3CSK4 (1 μg/mL) or IL-17A (316 ng/mL) were added for 16 h. Each of these agonists significantly upregulated TAP gene expression relative to the reference gene GAPDH ( P

    Techniques Used: Expressing

    Effect of the NF-κB inhibitor caffeic acid phenylester (CAPE) on induction of TAP gene expression by LPS, Pam3CSK4 and IL-17A. Stimulation of bovine tracheal epithelial cells (bTEC) with 0.1 µg/mL LPS (panel A ), 1 µg/mL Pam3CSK4 (panel B ), or 316 ng/mL IL-17A (panel C ) induced significantly greater TAP gene expression than in non-stimulated cells (NS). Pre-treatment with CAPE for 2 h before stimulation inhibited each of these responses. Asterisks indicate significant effects of CAPE treatment compared to no CAPE, P
    Figure Legend Snippet: Effect of the NF-κB inhibitor caffeic acid phenylester (CAPE) on induction of TAP gene expression by LPS, Pam3CSK4 and IL-17A. Stimulation of bovine tracheal epithelial cells (bTEC) with 0.1 µg/mL LPS (panel A ), 1 µg/mL Pam3CSK4 (panel B ), or 316 ng/mL IL-17A (panel C ) induced significantly greater TAP gene expression than in non-stimulated cells (NS). Pre-treatment with CAPE for 2 h before stimulation inhibited each of these responses. Asterisks indicate significant effects of CAPE treatment compared to no CAPE, P

    Techniques Used: Expressing

    Western analysis of NF-kB p65 in nuclear and cytoplasmic extracts of bTEC stimulated with LPS, Pam3CSK4 or IL-17A. NF-kB p65 was localized to the cytoplasm of non-stimulated cells (NS), but showed translocation to the nucleus following treatment of the cells with either LPS (panel A ), Pam3CSK4 (Pam, panel B ) or IL-17A (panel C ). However, pre-treating the cells with the NF-κB inhibitor CAPE (10 μM) prior to exposure to agonists fully abrogated nuclear translocation of NF-kB p65 in the studies involving LPS and Pam3CSK4 (panels A and B ), and partially in the studies involving IL-17A (panel C ). Pos control: positive control, NF-kB p65. The findings were similar when the experiments were repeated using cells from different animals.
    Figure Legend Snippet: Western analysis of NF-kB p65 in nuclear and cytoplasmic extracts of bTEC stimulated with LPS, Pam3CSK4 or IL-17A. NF-kB p65 was localized to the cytoplasm of non-stimulated cells (NS), but showed translocation to the nucleus following treatment of the cells with either LPS (panel A ), Pam3CSK4 (Pam, panel B ) or IL-17A (panel C ). However, pre-treating the cells with the NF-κB inhibitor CAPE (10 μM) prior to exposure to agonists fully abrogated nuclear translocation of NF-kB p65 in the studies involving LPS and Pam3CSK4 (panels A and B ), and partially in the studies involving IL-17A (panel C ). Pos control: positive control, NF-kB p65. The findings were similar when the experiments were repeated using cells from different animals.

    Techniques Used: Western Blot, Translocation Assay, Positive Control

    Related Articles

    Enzyme-linked Immunosorbent Assay:

    Article Title: Enhancing the toolbox to study IL-17A in cattle and sheep
    Article Snippet: .. Measurement of IL-17 family members at the protein level in ruminant species has been limited by the paucity of species-specific reagents with the exception of one commercially-available ELISA kit to detect bovine IL-17A (Kingfisher Biotech). ..

    Article Title: Prophylactic digoxin treatment reduces IL-17 production in vivo in the neonatal calf and moderates RSV-associated disease
    Article Snippet: .. ELISAs Bovine IL-17A and IFNγ VetSet ELISA Development kits were purchased from Kingfisher Biotech, Inc and performed according to manufacturer’s instructions. ..

    Article Title: Enhancing the toolbox to study IL-17A in cattle and sheep
    Article Snippet: .. Expression, detection and biological function of recombinant ruminant IL-17A The Kingfisher Biotech bovine IL-17A VetSet is a pab-based ELISA for detection of bovine IL-17A and based on the homology between the bovine and ovine orthologues, we predicted that it would detect both of the expressed recombinant proteins. ..

    Blocking Assay:

    Article Title: Immunization of young heifers with staphylococcal immune evasion proteins before natural exposure to Staphylococcus aureus induces a humoral immune response in serum and milk
    Article Snippet: .. Plates were blocked using Blocking buffer and supernatant samples diluted 1:2 in Blocking buffer were added in triplicate and incubated at room temperature for 2 h. Standard curves of recombinant bovine IL17a protein (Kingfisher Biotech, Inc.), ranging from 15.6–1000 pg/mL, were included in triplicate on each plate. ..

    Incubation:

    Article Title: Immunization of young heifers with staphylococcal immune evasion proteins before natural exposure to Staphylococcus aureus induces a humoral immune response in serum and milk
    Article Snippet: .. Plates were blocked using Blocking buffer and supernatant samples diluted 1:2 in Blocking buffer were added in triplicate and incubated at room temperature for 2 h. Standard curves of recombinant bovine IL17a protein (Kingfisher Biotech, Inc.), ranging from 15.6–1000 pg/mL, were included in triplicate on each plate. ..

    Recombinant:

    Article Title: Immunization of young heifers with staphylococcal immune evasion proteins before natural exposure to Staphylococcus aureus induces a humoral immune response in serum and milk
    Article Snippet: .. Plates were blocked using Blocking buffer and supernatant samples diluted 1:2 in Blocking buffer were added in triplicate and incubated at room temperature for 2 h. Standard curves of recombinant bovine IL17a protein (Kingfisher Biotech, Inc.), ranging from 15.6–1000 pg/mL, were included in triplicate on each plate. ..

    Article Title: Functionally distinct T-helper cell phenotypes predict resistance to different types of parasites in a wild mammal
    Article Snippet: .. For the quantification of IL-17A, polyclonal rabbit anti-bovine IL-17A antibodies were used alongside bovine recombinant protein (Kingfisher Biotech, Inc., St. Paul, MN). ..

    Article Title: Cellular and humoral immune responses associated with protection in sheep vaccinated against Teladorsagia circumcincta
    Article Snippet: .. For the quantification of IL-17A, polyclonal rabbit anti-bovine IL-17A antibodies were used alongside bovine recombinant protein (all from Kingfisher Biotech, Inc., St. Paul, MN). ..

    Article Title: Enhancing the toolbox to study IL-17A in cattle and sheep
    Article Snippet: .. Bougarn et al. [ ] described the cloning of bovine IL-17A and IL-17F and the expression of biologically-active recombinant proteins in insect cells that could induce expression of a range of cytokines and chemokines (including CXCL8) in primary bovine mammary epithelial cells. ..

    Article Title: Enhancing the toolbox to study IL-17A in cattle and sheep
    Article Snippet: .. Expression, detection and biological function of recombinant ruminant IL-17A The Kingfisher Biotech bovine IL-17A VetSet is a pab-based ELISA for detection of bovine IL-17A and based on the homology between the bovine and ovine orthologues, we predicted that it would detect both of the expressed recombinant proteins. ..

    Clone Assay:

    Article Title: Enhancing the toolbox to study IL-17A in cattle and sheep
    Article Snippet: .. Bougarn et al. [ ] described the cloning of bovine IL-17A and IL-17F and the expression of biologically-active recombinant proteins in insect cells that could induce expression of a range of cytokines and chemokines (including CXCL8) in primary bovine mammary epithelial cells. ..

    Expressing:

    Article Title: Enhancing the toolbox to study IL-17A in cattle and sheep
    Article Snippet: .. Bougarn et al. [ ] described the cloning of bovine IL-17A and IL-17F and the expression of biologically-active recombinant proteins in insect cells that could induce expression of a range of cytokines and chemokines (including CXCL8) in primary bovine mammary epithelial cells. ..

    Article Title: Enhancing the toolbox to study IL-17A in cattle and sheep
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    Kingfisher Biotech recombinant bovine il17a protein
    Cellular immune responses following stimulation with LukM. IFNg ( a ) and <t>IL17a</t> ( c ) production following stimulation of whole blood with LukM for 48 h and 72 h, respectively. Proliferation measured as the percentage of CD4 ( b ) and CD8 ( d ) T-cells with diluted CFSE signal following 96 h stimulation with LukM. S/P = Sample to positive ratio. + = P
    Recombinant Bovine Il17a Protein, supplied by Kingfisher Biotech, 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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    Cellular immune responses following stimulation with LukM. IFNg ( a ) and IL17a ( c ) production following stimulation of whole blood with LukM for 48 h and 72 h, respectively. Proliferation measured as the percentage of CD4 ( b ) and CD8 ( d ) T-cells with diluted CFSE signal following 96 h stimulation with LukM. S/P = Sample to positive ratio. + = P

    Journal: BMC Veterinary Research

    Article Title: Immunization of young heifers with staphylococcal immune evasion proteins before natural exposure to Staphylococcus aureus induces a humoral immune response in serum and milk

    doi: 10.1186/s12917-018-1765-9

    Figure Lengend Snippet: Cellular immune responses following stimulation with LukM. IFNg ( a ) and IL17a ( c ) production following stimulation of whole blood with LukM for 48 h and 72 h, respectively. Proliferation measured as the percentage of CD4 ( b ) and CD8 ( d ) T-cells with diluted CFSE signal following 96 h stimulation with LukM. S/P = Sample to positive ratio. + = P

    Article Snippet: Plates were blocked using Blocking buffer and supernatant samples diluted 1:2 in Blocking buffer were added in triplicate and incubated at room temperature for 2 h. Standard curves of recombinant bovine IL17a protein (Kingfisher Biotech, Inc.), ranging from 15.6–1000 pg/mL, were included in triplicate on each plate.

    Techniques:

    Cellular immune responses following stimulation with EfB. IFNg ( a ) and IL17a ( c ) production following stimulation of whole blood with EfB for 48 h and 72 h, respectively. Proliferation measured as the percentage of CD4 ( b ) and CD8 ( d ) T-cells with diluted CFSE signal following 96 h stimulation with EfB. S/P = Sample to positive ratio. + = P

    Journal: BMC Veterinary Research

    Article Title: Immunization of young heifers with staphylococcal immune evasion proteins before natural exposure to Staphylococcus aureus induces a humoral immune response in serum and milk

    doi: 10.1186/s12917-018-1765-9

    Figure Lengend Snippet: Cellular immune responses following stimulation with EfB. IFNg ( a ) and IL17a ( c ) production following stimulation of whole blood with EfB for 48 h and 72 h, respectively. Proliferation measured as the percentage of CD4 ( b ) and CD8 ( d ) T-cells with diluted CFSE signal following 96 h stimulation with EfB. S/P = Sample to positive ratio. + = P

    Article Snippet: Plates were blocked using Blocking buffer and supernatant samples diluted 1:2 in Blocking buffer were added in triplicate and incubated at room temperature for 2 h. Standard curves of recombinant bovine IL17a protein (Kingfisher Biotech, Inc.), ranging from 15.6–1000 pg/mL, were included in triplicate on each plate.

    Techniques:

    Evaluation of commercial antibodies for the intracellular detection of recombinant bovine and ovine IL-17A. The eight commercial antibodies listed in Table 1 were tested against fixed, permeabilised untransfected (UTF) CHO cells and CHO cells transfected with cDNA encoding bovIL-17A or ovIL-17A for their capacity to detect intracellular recombinant IL-17A by flow cytometry. Results are shown for one polyclonal antibody (pab) produced against bovIL-17A ( A ) and seven monoclonal antibodies (mabs) produced against human or mouse IL-17A ( B – D ). Profiles of the relevant control antibodies listed in Table 2 are included in the overlapping histograms. Events were acquired on the MacsQuant according to the gating strategy described previously (in brief) and shown in Additional file 2 . Line colours representing different antibody treatments are given in parentheses: A Primary rabbit anti-bovine IL-17A pab PB0274B-100 at 1 μg/mL (A.1, red) or negative control primary anti-bovine CD34 pab (in-house) at an estimated 1 μg/mL equivalent (a, black) then detected with a secondary goat anti-rabbit alexafluor 488 at 1 μg/mL; B Directly conjugated mouse anti-human IL-17A eBio64DEC17-phycoerythrin (PE) mab (IgG1) at 2.5 μg/mL (B.1, red) and control IgG1 VPM21 mab (in-house) at an estimated 2.5 μg/mL equivalent (b, black) and detected with goat anti-mouse PE at 1 μg/mL; C Primary mouse anti-human IL-17A mabs MT44.6 (C.1, blue), MT241 (C.2, green), MT2770 (C.3, brown) and MT504 (C.4, red) [all IgG1] at 0.5 μg/mL and control IgG1 VPM21 mab (in-house) at an estimated 0.5 μg/mL equivalent (black), all detected with goat anti-mouse PE at 1 μg/mL; D Primary mouse anti-human IL-17A mabs #41809 (D.1, red) (IgG2b) and #41802 (D.2, blue) (IgG1) at 2.5 μg/mL and a mixture of control mabs VPM21 (IgG1) and VPM22 (IgG2b) at an estimated 2.5 μg/mL equivalent (d, black), all detected with goat anti-mouse PE at 1 μg/mL.

    Journal: Veterinary Research

    Article Title: Enhancing the toolbox to study IL-17A in cattle and sheep

    doi: 10.1186/s13567-017-0426-5

    Figure Lengend Snippet: Evaluation of commercial antibodies for the intracellular detection of recombinant bovine and ovine IL-17A. The eight commercial antibodies listed in Table 1 were tested against fixed, permeabilised untransfected (UTF) CHO cells and CHO cells transfected with cDNA encoding bovIL-17A or ovIL-17A for their capacity to detect intracellular recombinant IL-17A by flow cytometry. Results are shown for one polyclonal antibody (pab) produced against bovIL-17A ( A ) and seven monoclonal antibodies (mabs) produced against human or mouse IL-17A ( B – D ). Profiles of the relevant control antibodies listed in Table 2 are included in the overlapping histograms. Events were acquired on the MacsQuant according to the gating strategy described previously (in brief) and shown in Additional file 2 . Line colours representing different antibody treatments are given in parentheses: A Primary rabbit anti-bovine IL-17A pab PB0274B-100 at 1 μg/mL (A.1, red) or negative control primary anti-bovine CD34 pab (in-house) at an estimated 1 μg/mL equivalent (a, black) then detected with a secondary goat anti-rabbit alexafluor 488 at 1 μg/mL; B Directly conjugated mouse anti-human IL-17A eBio64DEC17-phycoerythrin (PE) mab (IgG1) at 2.5 μg/mL (B.1, red) and control IgG1 VPM21 mab (in-house) at an estimated 2.5 μg/mL equivalent (b, black) and detected with goat anti-mouse PE at 1 μg/mL; C Primary mouse anti-human IL-17A mabs MT44.6 (C.1, blue), MT241 (C.2, green), MT2770 (C.3, brown) and MT504 (C.4, red) [all IgG1] at 0.5 μg/mL and control IgG1 VPM21 mab (in-house) at an estimated 0.5 μg/mL equivalent (black), all detected with goat anti-mouse PE at 1 μg/mL; D Primary mouse anti-human IL-17A mabs #41809 (D.1, red) (IgG2b) and #41802 (D.2, blue) (IgG1) at 2.5 μg/mL and a mixture of control mabs VPM21 (IgG1) and VPM22 (IgG2b) at an estimated 2.5 μg/mL equivalent (d, black), all detected with goat anti-mouse PE at 1 μg/mL.

    Article Snippet: Expression, detection and biological function of recombinant ruminant IL-17A The Kingfisher Biotech bovine IL-17A VetSet is a pab-based ELISA for detection of bovine IL-17A and based on the homology between the bovine and ovine orthologues, we predicted that it would detect both of the expressed recombinant proteins.

    Techniques: Recombinant, Transfection, Flow Cytometry, Cytometry, Produced, Negative Control

    Measurement and biological function of recombinant bovine and ovine IL-17A and detection of native ovine IL-17A by ELISA. A Detection of rbov and rovIL-17A by ELISA. The supernatants from transfected CHO cells expressing rbovIL-17A or rovIL-17A, or control parent untransfected line (UTF) were serially diluted (Log 3 dilutions) and evaluated using the commercial bovIL-17A ELISA. Data presented are optical density (OD) values from the Spectrophotometer at 450 nm. The X-axis displays Dilution 1/X and the Y-axis gives the OD value. Readings from UTF supernatant were below the limit of detection. B Functional activity of rbov and rovIL-17A on bovine embryonic lung cells. Bovine embryonic lung (EBL) cells were stimulated with 100 ng/mL CHO-expressed rbovIL-17A or rovIL-17A or UTF CHO negative control supernatant. Following 24 h incubation, culture supernatants were collected from triplicate cultures then tested for CXCL8 by ELISA. The X-axis displays the bioassay treatments and the Y-axis shows CXCL8 production in pg/mL. Data are the arithmetic mean of three technical replicates with error bars representing the standard error from one of three experiments. CXCL8 expression between treatments was statistically assessed using Kruskal–Wallis test. C Functional activity of rbov and rovIL-17A on ovine ST-6 cells. Ovine ST-6 cells were stimulated with 100 ng/mL CHO-expressed rbovIL-17A or rovIL-17A or UTF CHO supernatant. Following 24 h incubation and culture supernatants collected, tested and analysed as described in Figure 2B. CXCL8 expression between treatments was statistically assessed using Kruskal–Wallis test. D Detection of native ovIL-17A by ELISA. Ovine PBMC were cultured at 2 × 10 6 cells/mL with or without 5 μg/mL ConA. Culture supernatants were analysed for IL-17A using the bovIL-17A ELISA. Data represent the arithmetic mean of PBMC from six ewes and error bars represent standard error. Data were analysed statistically for significance using the two-tailed Mann–Whitney test.

    Journal: Veterinary Research

    Article Title: Enhancing the toolbox to study IL-17A in cattle and sheep

    doi: 10.1186/s13567-017-0426-5

    Figure Lengend Snippet: Measurement and biological function of recombinant bovine and ovine IL-17A and detection of native ovine IL-17A by ELISA. A Detection of rbov and rovIL-17A by ELISA. The supernatants from transfected CHO cells expressing rbovIL-17A or rovIL-17A, or control parent untransfected line (UTF) were serially diluted (Log 3 dilutions) and evaluated using the commercial bovIL-17A ELISA. Data presented are optical density (OD) values from the Spectrophotometer at 450 nm. The X-axis displays Dilution 1/X and the Y-axis gives the OD value. Readings from UTF supernatant were below the limit of detection. B Functional activity of rbov and rovIL-17A on bovine embryonic lung cells. Bovine embryonic lung (EBL) cells were stimulated with 100 ng/mL CHO-expressed rbovIL-17A or rovIL-17A or UTF CHO negative control supernatant. Following 24 h incubation, culture supernatants were collected from triplicate cultures then tested for CXCL8 by ELISA. The X-axis displays the bioassay treatments and the Y-axis shows CXCL8 production in pg/mL. Data are the arithmetic mean of three technical replicates with error bars representing the standard error from one of three experiments. CXCL8 expression between treatments was statistically assessed using Kruskal–Wallis test. C Functional activity of rbov and rovIL-17A on ovine ST-6 cells. Ovine ST-6 cells were stimulated with 100 ng/mL CHO-expressed rbovIL-17A or rovIL-17A or UTF CHO supernatant. Following 24 h incubation and culture supernatants collected, tested and analysed as described in Figure 2B. CXCL8 expression between treatments was statistically assessed using Kruskal–Wallis test. D Detection of native ovIL-17A by ELISA. Ovine PBMC were cultured at 2 × 10 6 cells/mL with or without 5 μg/mL ConA. Culture supernatants were analysed for IL-17A using the bovIL-17A ELISA. Data represent the arithmetic mean of PBMC from six ewes and error bars represent standard error. Data were analysed statistically for significance using the two-tailed Mann–Whitney test.

    Article Snippet: Expression, detection and biological function of recombinant ruminant IL-17A The Kingfisher Biotech bovine IL-17A VetSet is a pab-based ELISA for detection of bovine IL-17A and based on the homology between the bovine and ovine orthologues, we predicted that it would detect both of the expressed recombinant proteins.

    Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Transfection, Expressing, Spectrophotometry, Functional Assay, Activity Assay, Negative Control, Incubation, Cell Culture, Two Tailed Test, MANN-WHITNEY

    Relative intracellular expression of IL-17A and IFN-γ by activated bovine and ovine PBMC. The data sets described in “ Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section ” and presented in Figures 5 and 6 are summarised to compare overall intracellular expression of IL-17A ( A ) and IFN-γ ( B ) by PMA/ionomycin-stimulated bovine and ovine PBMC. Each bar represents the arithmetic mean of four cattle or four sheep and the error bars represent the standard error. The data for total percentage IFN-γ and IL-17A expression between species were assessed statistically using two-tailed Mann–Whitney tests allowing for ties.

    Journal: Veterinary Research

    Article Title: Enhancing the toolbox to study IL-17A in cattle and sheep

    doi: 10.1186/s13567-017-0426-5

    Figure Lengend Snippet: Relative intracellular expression of IL-17A and IFN-γ by activated bovine and ovine PBMC. The data sets described in “ Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section ” and presented in Figures 5 and 6 are summarised to compare overall intracellular expression of IL-17A ( A ) and IFN-γ ( B ) by PMA/ionomycin-stimulated bovine and ovine PBMC. Each bar represents the arithmetic mean of four cattle or four sheep and the error bars represent the standard error. The data for total percentage IFN-γ and IL-17A expression between species were assessed statistically using two-tailed Mann–Whitney tests allowing for ties.

    Article Snippet: Expression, detection and biological function of recombinant ruminant IL-17A The Kingfisher Biotech bovine IL-17A VetSet is a pab-based ELISA for detection of bovine IL-17A and based on the homology between the bovine and ovine orthologues, we predicted that it would detect both of the expressed recombinant proteins.

    Techniques: Expressing, Two Tailed Test, MANN-WHITNEY

    Detection of single-cell expression of ruminant IL-17A by ELISpot. Plates and PBMC were prepared and cultured as described in “ IL-17A ELISpot section ”. ELISpot images shown are representative of PBMC from one of three cattle ( A ) and one of three sheep ( B ) activated with ConA and PMA/ionomycin. The average number of spot-forming units (SFU) with standard errors are shown for 10 6 PBMC from all three cattle (grey bars) and sheep (black bars), stimulated under the different conditions ( C ). Data were modelled by fitting a Poisson generalised linear mixed model (GLMM) by maximum likelihood to the IL-17A SFU/10 6 values, using logarithmic link function and Laplace approximations to calculate log-likelihoods. The model included treatment (medium control, ConA and PMA/ionomycin), species (bovine, ovine) and their interaction as fixed effects and animal identification as a random effect in order to account for both within- and between-animal variability. An observation-level random effect term was specified to account for data over-dispersion. The statistical significance of the fixed effect terms was assessed using p values derived from type II Wald Chi square tests. Linear hypothesis tests were defined from the GLMM in order to conduct pair-wise comparisons of means between treatments and species. The associated p values were adjusted for false discovery rate (FDR) following Benjamini–Hochberg’s procedure.

    Journal: Veterinary Research

    Article Title: Enhancing the toolbox to study IL-17A in cattle and sheep

    doi: 10.1186/s13567-017-0426-5

    Figure Lengend Snippet: Detection of single-cell expression of ruminant IL-17A by ELISpot. Plates and PBMC were prepared and cultured as described in “ IL-17A ELISpot section ”. ELISpot images shown are representative of PBMC from one of three cattle ( A ) and one of three sheep ( B ) activated with ConA and PMA/ionomycin. The average number of spot-forming units (SFU) with standard errors are shown for 10 6 PBMC from all three cattle (grey bars) and sheep (black bars), stimulated under the different conditions ( C ). Data were modelled by fitting a Poisson generalised linear mixed model (GLMM) by maximum likelihood to the IL-17A SFU/10 6 values, using logarithmic link function and Laplace approximations to calculate log-likelihoods. The model included treatment (medium control, ConA and PMA/ionomycin), species (bovine, ovine) and their interaction as fixed effects and animal identification as a random effect in order to account for both within- and between-animal variability. An observation-level random effect term was specified to account for data over-dispersion. The statistical significance of the fixed effect terms was assessed using p values derived from type II Wald Chi square tests. Linear hypothesis tests were defined from the GLMM in order to conduct pair-wise comparisons of means between treatments and species. The associated p values were adjusted for false discovery rate (FDR) following Benjamini–Hochberg’s procedure.

    Article Snippet: Expression, detection and biological function of recombinant ruminant IL-17A The Kingfisher Biotech bovine IL-17A VetSet is a pab-based ELISA for detection of bovine IL-17A and based on the homology between the bovine and ovine orthologues, we predicted that it would detect both of the expressed recombinant proteins.

    Techniques: Expressing, Enzyme-linked Immunospot, Cell Culture, Derivative Assay

    Intracellular expression of IL-17A and IFN-γ by activated bovine T cell subsets. PBMC from four cattle were stimulated with phorbol 12-myristate 13 acetate, ionomycin and brefeldin A in RPMI culture medium for 4 h. Cells were harvested and stained for viability and with mabs specific for cell-surface phenotypic markers and intracellular cytokines as described in Table 3 and “ Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section ”. Cells were stained for CD4 with mab CC8-PE at 1:20 dilution ( A , D ), for CD8β with mab CC58-PE at 1:20 dilution ( B , D ) and for WC-1 (γδ T cells) with mab CC15-PE at 1:200 ( C , E ). Intracellular cytokine staining for IL-17A was conducted using mab eBioDEC17-APC at a 1:20 dilution ( A – C ) and for IFN-γ using mab CC302-Alexafluor 647 at a 1:200 dilution ( D – F ). Data are shown for PBMC from one representative animal of four.

    Journal: Veterinary Research

    Article Title: Enhancing the toolbox to study IL-17A in cattle and sheep

    doi: 10.1186/s13567-017-0426-5

    Figure Lengend Snippet: Intracellular expression of IL-17A and IFN-γ by activated bovine T cell subsets. PBMC from four cattle were stimulated with phorbol 12-myristate 13 acetate, ionomycin and brefeldin A in RPMI culture medium for 4 h. Cells were harvested and stained for viability and with mabs specific for cell-surface phenotypic markers and intracellular cytokines as described in Table 3 and “ Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section ”. Cells were stained for CD4 with mab CC8-PE at 1:20 dilution ( A , D ), for CD8β with mab CC58-PE at 1:20 dilution ( B , D ) and for WC-1 (γδ T cells) with mab CC15-PE at 1:200 ( C , E ). Intracellular cytokine staining for IL-17A was conducted using mab eBioDEC17-APC at a 1:20 dilution ( A – C ) and for IFN-γ using mab CC302-Alexafluor 647 at a 1:200 dilution ( D – F ). Data are shown for PBMC from one representative animal of four.

    Article Snippet: Expression, detection and biological function of recombinant ruminant IL-17A The Kingfisher Biotech bovine IL-17A VetSet is a pab-based ELISA for detection of bovine IL-17A and based on the homology between the bovine and ovine orthologues, we predicted that it would detect both of the expressed recombinant proteins.

    Techniques: Expressing, Staining

    Phylogenetic tree of mammalian IL-17A protein sequences. Evolutionary sequence comparisons were undertaken using 13 selected mammalian and other IL-17A sequences by initially conducting a multiple alignment using Clustal Omega (EMBL/EBI online, [ 21 ]). The evolutionary relationships between the sequences were inferred using Mr. Bayes launched from TOPALI v 2.5 using the Jones–Taylor–Thornton plus gamma (JTT + G) model with two runs each of 1 250 000 generations with a burn in period of 20% and sampling frequency of 1000. The horizontal lines are branches whose length represents the amount of genetic change over time. The scale bar shows the distance represented by 0.1 expected substitutions per site. The robustness of the clustering of sequences are shown by the Bayesian Posterior Probabilities at the nodes. Accession numbers of the sequences used for the comparison are: Human NP_002181.1; House mouse NP_034682.1; Cow NP_001008412.1; Sheep XP_004018936.1; Goat NP_001272654.1; Horse NP_001137264.1; Pig NP_001005729.1; Dog NP_001159350.1; Domestic guinea pig NP_001265697.1; Koala AHZ08738.1; Chicken NP_989791.1; EGW10039.1 Chinese hamster and European rabbit AMQ91106.1. The phylogenetic tree was annotated using Dendroscope.

    Journal: Veterinary Research

    Article Title: Enhancing the toolbox to study IL-17A in cattle and sheep

    doi: 10.1186/s13567-017-0426-5

    Figure Lengend Snippet: Phylogenetic tree of mammalian IL-17A protein sequences. Evolutionary sequence comparisons were undertaken using 13 selected mammalian and other IL-17A sequences by initially conducting a multiple alignment using Clustal Omega (EMBL/EBI online, [ 21 ]). The evolutionary relationships between the sequences were inferred using Mr. Bayes launched from TOPALI v 2.5 using the Jones–Taylor–Thornton plus gamma (JTT + G) model with two runs each of 1 250 000 generations with a burn in period of 20% and sampling frequency of 1000. The horizontal lines are branches whose length represents the amount of genetic change over time. The scale bar shows the distance represented by 0.1 expected substitutions per site. The robustness of the clustering of sequences are shown by the Bayesian Posterior Probabilities at the nodes. Accession numbers of the sequences used for the comparison are: Human NP_002181.1; House mouse NP_034682.1; Cow NP_001008412.1; Sheep XP_004018936.1; Goat NP_001272654.1; Horse NP_001137264.1; Pig NP_001005729.1; Dog NP_001159350.1; Domestic guinea pig NP_001265697.1; Koala AHZ08738.1; Chicken NP_989791.1; EGW10039.1 Chinese hamster and European rabbit AMQ91106.1. The phylogenetic tree was annotated using Dendroscope.

    Article Snippet: Expression, detection and biological function of recombinant ruminant IL-17A The Kingfisher Biotech bovine IL-17A VetSet is a pab-based ELISA for detection of bovine IL-17A and based on the homology between the bovine and ovine orthologues, we predicted that it would detect both of the expressed recombinant proteins.

    Techniques: Sequencing, Sampling

    Intracellular expression of IL-17A and IFN-γ by activated ovine T cell subsets. PBMC from four sheep were stimulated with phorbol 12-myristate 13 acetate, ionomycin and brefeldin A in RPMI culture medium for 4 h. Cells were harvested and stained for viability and with mabs specific for cell-surface phenotypic markers and intracellular cytokines as described in Table 3 and “ Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section ”. Cells were then stained for CD4 with mab 44.38-PE at 1:20 dilution ( A , D ), CD8β with mab CC58-PE at 1:20 dilution ( B , D ) and WC-1 (γδ) with mab CC15-PE at 1:200 ( C , E ). Intracellular cytokine staining for IL-17A was conducted using mab eBio64DEC17-APC a 1:20 dilution ( A – C ) and for IFN-γ using mab CC302-alexafluor 647 at a 1:200 dilution ( D – F ). Data shown is for one representative animal out of four.

    Journal: Veterinary Research

    Article Title: Enhancing the toolbox to study IL-17A in cattle and sheep

    doi: 10.1186/s13567-017-0426-5

    Figure Lengend Snippet: Intracellular expression of IL-17A and IFN-γ by activated ovine T cell subsets. PBMC from four sheep were stimulated with phorbol 12-myristate 13 acetate, ionomycin and brefeldin A in RPMI culture medium for 4 h. Cells were harvested and stained for viability and with mabs specific for cell-surface phenotypic markers and intracellular cytokines as described in Table 3 and “ Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section ”. Cells were then stained for CD4 with mab 44.38-PE at 1:20 dilution ( A , D ), CD8β with mab CC58-PE at 1:20 dilution ( B , D ) and WC-1 (γδ) with mab CC15-PE at 1:200 ( C , E ). Intracellular cytokine staining for IL-17A was conducted using mab eBio64DEC17-APC a 1:20 dilution ( A – C ) and for IFN-γ using mab CC302-alexafluor 647 at a 1:200 dilution ( D – F ). Data shown is for one representative animal out of four.

    Article Snippet: Expression, detection and biological function of recombinant ruminant IL-17A The Kingfisher Biotech bovine IL-17A VetSet is a pab-based ELISA for detection of bovine IL-17A and based on the homology between the bovine and ovine orthologues, we predicted that it would detect both of the expressed recombinant proteins.

    Techniques: Expressing, Staining

    Analysis by immunohistochemistry of representative sections of mammary tissue of ovalbumin-infused glands. A) Immunoreactivity of the epithelial lining the alveoli and of cells in the connective tissue (cow #4019) to antibody against N-terminal peptide of bovine IL-17A; B) Immunoreactivity of the mammary tissue of another cow (#1039) to the N-term antibody; C) Inhibition of labeling by Ab to N-terminal IL-17A peptide with the peptide antigen (cow #4019); D) Negative control with Ab to ovalbumin and second antibody conjugated to horseradish peroxidase; E, F) Immunoreactivity of the epithelial lining the alveoli and of cells in the connective tissue of cows #4019 and 1039 to the abcam antibody; G-H) Immunoreactivity of mammary tissue of cows #4019 and 1039 to the to the C-term and antibody. Scale bars indicate 25 µm.

    Journal: PLoS ONE

    Article Title: T Helper 17-Associated Cytokines Are Produced during Antigen-Specific Inflammation in the Mammary Gland

    doi: 10.1371/journal.pone.0063471

    Figure Lengend Snippet: Analysis by immunohistochemistry of representative sections of mammary tissue of ovalbumin-infused glands. A) Immunoreactivity of the epithelial lining the alveoli and of cells in the connective tissue (cow #4019) to antibody against N-terminal peptide of bovine IL-17A; B) Immunoreactivity of the mammary tissue of another cow (#1039) to the N-term antibody; C) Inhibition of labeling by Ab to N-terminal IL-17A peptide with the peptide antigen (cow #4019); D) Negative control with Ab to ovalbumin and second antibody conjugated to horseradish peroxidase; E, F) Immunoreactivity of the epithelial lining the alveoli and of cells in the connective tissue of cows #4019 and 1039 to the abcam antibody; G-H) Immunoreactivity of mammary tissue of cows #4019 and 1039 to the to the C-term and antibody. Scale bars indicate 25 µm.

    Article Snippet: Commercial affinity-purified antibodies to whole recombinant bovine IL-17A were from Kingfisher Biotech (St Paul, MN, USA), and rabbit antibody (ab79056) to human IL-17A, raised against a synthetic 19 amino acid-peptide from near the center of human IL-17A and affinity-purified with the immunogen, was from Abcam.

    Techniques: Immunohistochemistry, Inhibition, Labeling, Negative Control

    Analysis by immunohistochemistry of representative tissue sections of uninfused, healthy mammary glands. A–B) Immunoreactivity of the apical side of the epithelial cells lining the alveoli to the N-term antibody to IL-17A of cows #1018 and 2014, respectively; C) Inhibition of labeling by Ab to N-terminal IL-17A peptide with the peptide antigen (cow #1018); D) Negative control with Ab to ovalbumin and second antibody conjugated to horseradish peroxidase (cow #1018); E–F) Immunoreactivity of the epithelial lining the alveoli to abcam antibody (cows #1018 and 2014, respectively); G–H) Immunoreactivity of mammary tissue of healthy uninfused quarters of cows 1018 and 2014 to the C-term antibody, respectively. Scale bars indicate 25 µm.

    Journal: PLoS ONE

    Article Title: T Helper 17-Associated Cytokines Are Produced during Antigen-Specific Inflammation in the Mammary Gland

    doi: 10.1371/journal.pone.0063471

    Figure Lengend Snippet: Analysis by immunohistochemistry of representative tissue sections of uninfused, healthy mammary glands. A–B) Immunoreactivity of the apical side of the epithelial cells lining the alveoli to the N-term antibody to IL-17A of cows #1018 and 2014, respectively; C) Inhibition of labeling by Ab to N-terminal IL-17A peptide with the peptide antigen (cow #1018); D) Negative control with Ab to ovalbumin and second antibody conjugated to horseradish peroxidase (cow #1018); E–F) Immunoreactivity of the epithelial lining the alveoli to abcam antibody (cows #1018 and 2014, respectively); G–H) Immunoreactivity of mammary tissue of healthy uninfused quarters of cows 1018 and 2014 to the C-term antibody, respectively. Scale bars indicate 25 µm.

    Article Snippet: Commercial affinity-purified antibodies to whole recombinant bovine IL-17A were from Kingfisher Biotech (St Paul, MN, USA), and rabbit antibody (ab79056) to human IL-17A, raised against a synthetic 19 amino acid-peptide from near the center of human IL-17A and affinity-purified with the immunogen, was from Abcam.

    Techniques: Immunohistochemistry, Inhibition, Labeling, Negative Control

    Concentrations of chemoattractants and cytokines in milk samples of the 9 responsive cows. Concentrations were measured by ELISA in the milk samples of the 9 responder cows. Quarters were infused with 25 µg ovalbumin at time 0 and milk samples taken at indicated times. Median values (Q1, Q3) are shown. Concentrations varied significantly (Friedman test) as a function of hpi for C5a, CXCL8, IL-1β, IL-6, IFN-γ, IL-17A (p

    Journal: PLoS ONE

    Article Title: T Helper 17-Associated Cytokines Are Produced during Antigen-Specific Inflammation in the Mammary Gland

    doi: 10.1371/journal.pone.0063471

    Figure Lengend Snippet: Concentrations of chemoattractants and cytokines in milk samples of the 9 responsive cows. Concentrations were measured by ELISA in the milk samples of the 9 responder cows. Quarters were infused with 25 µg ovalbumin at time 0 and milk samples taken at indicated times. Median values (Q1, Q3) are shown. Concentrations varied significantly (Friedman test) as a function of hpi for C5a, CXCL8, IL-1β, IL-6, IFN-γ, IL-17A (p

    Article Snippet: Commercial affinity-purified antibodies to whole recombinant bovine IL-17A were from Kingfisher Biotech (St Paul, MN, USA), and rabbit antibody (ab79056) to human IL-17A, raised against a synthetic 19 amino acid-peptide from near the center of human IL-17A and affinity-purified with the immunogen, was from Abcam.

    Techniques: Enzyme-linked Immunosorbent Assay

    Effects of CpG oligodinucleotide, interleukin-17A and interferon-α on tracheal antimicrobial peptide gene expression. Dose- and time-dependent effects were measured for CpG oligodinucleotide (A,B) , a TLR9 agonist; interleukin-17A (C,D) ; and interferon-α (E,F) . For the dose–response studies (A,C,E) , confluent bTEC were stimulated in triplicate with various concentrations of agonist for 16 h. For the time-course studies (B,D,F) , confluent bTEC were stimulated in triplicate for 4, 8, 16 or 24 h with the doses of agonist shown. Gene expression of TAP relative to that of GAPDH was measured using real-time RT-qPCR. Lipopolysaccharide (LPS, 0.1 μg/mL) was used in all assays as a positive control and standard. *, significantly different from unstimulated cells ( P

    Journal: Veterinary Research

    Article Title: Comparison of innate immune agonists for induction of tracheal antimicrobial peptide gene expression in tracheal epithelial cells of cattle

    doi: 10.1186/s13567-014-0105-8

    Figure Lengend Snippet: Effects of CpG oligodinucleotide, interleukin-17A and interferon-α on tracheal antimicrobial peptide gene expression. Dose- and time-dependent effects were measured for CpG oligodinucleotide (A,B) , a TLR9 agonist; interleukin-17A (C,D) ; and interferon-α (E,F) . For the dose–response studies (A,C,E) , confluent bTEC were stimulated in triplicate with various concentrations of agonist for 16 h. For the time-course studies (B,D,F) , confluent bTEC were stimulated in triplicate for 4, 8, 16 or 24 h with the doses of agonist shown. Gene expression of TAP relative to that of GAPDH was measured using real-time RT-qPCR. Lipopolysaccharide (LPS, 0.1 μg/mL) was used in all assays as a positive control and standard. *, significantly different from unstimulated cells ( P

    Article Snippet: The cytokines included bovine recombinant IL-17A (Kingfisher Biotech, St. Paul, MN, USA, item number RP0056B-005) and bovine recombinant IFN-α (Kingfisher Biotech, item RP0008B-025).

    Techniques: Expressing, Quantitative RT-PCR, Positive Control

    Comparison of the effects of IL-17A, Pam3CSK4 and LPS on tracheal antimicrobial peptide gene expression. Confluent cultures of tracheal epithelial cells from 4 different calves were non-stimulated (NS) or stimulated with 1 μg/mL Pam3CSK4, 316 ng/mL IL-17A, or 0.1 μg/mL LPS for 8 h (A) and 16 h (B) in triplicate. Gene expression was assessed using real-time RT-qPCR. Pam3CSK4 induced significantly higher tracheal antimicrobial peptide gene expression than IL-17A and LPS at both 8 and 16 h ( P

    Journal: Veterinary Research

    Article Title: Comparison of innate immune agonists for induction of tracheal antimicrobial peptide gene expression in tracheal epithelial cells of cattle

    doi: 10.1186/s13567-014-0105-8

    Figure Lengend Snippet: Comparison of the effects of IL-17A, Pam3CSK4 and LPS on tracheal antimicrobial peptide gene expression. Confluent cultures of tracheal epithelial cells from 4 different calves were non-stimulated (NS) or stimulated with 1 μg/mL Pam3CSK4, 316 ng/mL IL-17A, or 0.1 μg/mL LPS for 8 h (A) and 16 h (B) in triplicate. Gene expression was assessed using real-time RT-qPCR. Pam3CSK4 induced significantly higher tracheal antimicrobial peptide gene expression than IL-17A and LPS at both 8 and 16 h ( P

    Article Snippet: The cytokines included bovine recombinant IL-17A (Kingfisher Biotech, St. Paul, MN, USA, item number RP0056B-005) and bovine recombinant IFN-α (Kingfisher Biotech, item RP0008B-025).

    Techniques: Expressing, Quantitative RT-PCR

    Effect of stimulation with single agonists compared to combined agonists. Cultured bovine tracheal epithelial cells were stimulated for 16 h in triplicate with various combinations of 1 μg/mL Pam3CSK4, 316 ng/mL IL-17A, or 0.1 μg/mL LPS. Tracheal antimicrobial peptide gene expression was measured as above. The effects of combined agonists were greater than that of interleukin-17A (IL-17A) alone, but minimally or not different than that of lipopolysaccharide (LPS) or Pam3CSK4 alone. The data shown (panels A, B and C) represent 3 studies conducted on different days using cells from different calves.

    Journal: Veterinary Research

    Article Title: Comparison of innate immune agonists for induction of tracheal antimicrobial peptide gene expression in tracheal epithelial cells of cattle

    doi: 10.1186/s13567-014-0105-8

    Figure Lengend Snippet: Effect of stimulation with single agonists compared to combined agonists. Cultured bovine tracheal epithelial cells were stimulated for 16 h in triplicate with various combinations of 1 μg/mL Pam3CSK4, 316 ng/mL IL-17A, or 0.1 μg/mL LPS. Tracheal antimicrobial peptide gene expression was measured as above. The effects of combined agonists were greater than that of interleukin-17A (IL-17A) alone, but minimally or not different than that of lipopolysaccharide (LPS) or Pam3CSK4 alone. The data shown (panels A, B and C) represent 3 studies conducted on different days using cells from different calves.

    Article Snippet: The cytokines included bovine recombinant IL-17A (Kingfisher Biotech, St. Paul, MN, USA, item number RP0056B-005) and bovine recombinant IFN-α (Kingfisher Biotech, item RP0008B-025).

    Techniques: Cell Culture, Expressing