recombinant bovine il17a protein  (Kingfisher Biotech)


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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
    Recombinant Bovine Il17a Protein, supplied by Kingfisher Biotech, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant bovine il17a protein/product/Kingfisher Biotech
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant bovine il17a protein - by Bioz Stars, 2022-07
    91/100 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 "Antigen-Specific Mammary Inflammation Depends on the Production of IL-17A and IFN-γ by Bovine CD4+ T Lymphocytes"

    Article Title: Antigen-Specific Mammary Inflammation Depends on the Production of IL-17A and IFN-γ by Bovine CD4+ T Lymphocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0137755

    Antigen-specific whole blood assay. ( A) Time-course of IL-17A and IFN-γ production by whole blood cultured with OVA. The blood of the 10 responder cows (taken 45 days after the first immunization) was cultured in the presence of OVA (10 μg/mL) for 1 to 4 days in 96-well microplates. For each cow triplicate wells were used for each incubation time. Results are median values and quartiles (Q1; Q3). (B) The effect of magnetic depletion of CD4+ cells on the production of IL-17A and IFN-γ in the antigen-specific whole blood assay. Results obtained by stimulating blood samples from responder cows 15 days after the booster immunization are expressed as the percentage of the production by CD4+-depleted PBMC relative to cytokine production by un processed PBMC (100%). The viability of cells was not altered by the magnetic cell separation.
    Figure Legend Snippet: Antigen-specific whole blood assay. ( A) Time-course of IL-17A and IFN-γ production by whole blood cultured with OVA. The blood of the 10 responder cows (taken 45 days after the first immunization) was cultured in the presence of OVA (10 μg/mL) for 1 to 4 days in 96-well microplates. For each cow triplicate wells were used for each incubation time. Results are median values and quartiles (Q1; Q3). (B) The effect of magnetic depletion of CD4+ cells on the production of IL-17A and IFN-γ in the antigen-specific whole blood assay. Results obtained by stimulating blood samples from responder cows 15 days after the booster immunization are expressed as the percentage of the production by CD4+-depleted PBMC relative to cytokine production by un processed PBMC (100%). The viability of cells was not altered by the magnetic cell separation.

    Techniques Used: Whole Blood Assay, Cell Culture, Incubation, Magnetic Cell Separation

    Persistence of reactivity to ovalbumin. Concentrations of IL-17A and IFN-γ yielded by the whole blood assay performed at different times after immunization with ovalbumin. Results are the median values (and interquartiles) from the 8 responder cows still available 10 months post-immunization.
    Figure Legend Snippet: Persistence of reactivity to ovalbumin. Concentrations of IL-17A and IFN-γ yielded by the whole blood assay performed at different times after immunization with ovalbumin. Results are the median values (and interquartiles) from the 8 responder cows still available 10 months post-immunization.

    Techniques Used: Whole Blood Assay

    Intracellular expression of IL-17A and IFN-γ by CD4+ T lymphocytes. PBMC were isolated one month after ovalbumin booster injection, stimulated in vitro with ovalbumin for 3 days, rested for 2 days and finally stimulated with PMA/ionomycin for 5 h with Brefeldin A for the last 3 hours. Cells were labeled for surface CD4 and intracellular IL-17A and IFN-γ. The numbers in the plots indicate the percentages of labeled cells in comparison to the isotype control. (A) Production of viable CD4+ T lymphoblasts after culture of PBMC with (OVA) or without (NS) ovalbumin. Left panels depict PBMC from a responder cow, right panels PBMC from a low-responder. (B) PBMC from two responder cows (R1 R2) were labeled for surface CD4 and intracellular IL-17A or IFN-γ, showing CD4+ and CD4- IL-17A- and IFN-γ-producing cells. (C) labeling of CD4+ cells with anti-IL-17A and anti-IFN-γ antibodies, showing single-producing and double-producing cells. D) Double labeling of CD4+ cells from two low-responders (R3 and R4). Percentages of labeled cells are indicated in the quadrants. Results are from a representative experiment.
    Figure Legend Snippet: Intracellular expression of IL-17A and IFN-γ by CD4+ T lymphocytes. PBMC were isolated one month after ovalbumin booster injection, stimulated in vitro with ovalbumin for 3 days, rested for 2 days and finally stimulated with PMA/ionomycin for 5 h with Brefeldin A for the last 3 hours. Cells were labeled for surface CD4 and intracellular IL-17A and IFN-γ. The numbers in the plots indicate the percentages of labeled cells in comparison to the isotype control. (A) Production of viable CD4+ T lymphoblasts after culture of PBMC with (OVA) or without (NS) ovalbumin. Left panels depict PBMC from a responder cow, right panels PBMC from a low-responder. (B) PBMC from two responder cows (R1 R2) were labeled for surface CD4 and intracellular IL-17A or IFN-γ, showing CD4+ and CD4- IL-17A- and IFN-γ-producing cells. (C) labeling of CD4+ cells with anti-IL-17A and anti-IFN-γ antibodies, showing single-producing and double-producing cells. D) Double labeling of CD4+ cells from two low-responders (R3 and R4). Percentages of labeled cells are indicated in the quadrants. Results are from a representative experiment.

    Techniques Used: Expressing, Isolation, Injection, In Vitro, Labeling

    Concentrations of cytokines in milk samples of the 10 responder cows. Time-course of concentration variation (median and interquartiles) of CXCL8 (A), IL-17A (B) and IFN- γ (C) in the milk of quarters infused with ovalbumin at 0 hpi.
    Figure Legend Snippet: Concentrations of cytokines in milk samples of the 10 responder cows. Time-course of concentration variation (median and interquartiles) of CXCL8 (A), IL-17A (B) and IFN- γ (C) in the milk of quarters infused with ovalbumin at 0 hpi.

    Techniques Used: Concentration Assay

    Time-course of the IL-17A and IFN-γ production in the antigen-specific whole blood assay following immunization and correlation with Peak SCC. Concentrations of IL-17A (A) or IFN-γ (B) after 3 days of culture with ovalbumin of blood samples taken before and after immunization at days 0 and 30 (median values and interquartiles) distinguishing the antigen-specific responses of responders and low-responder cows to the intramammary antigenic challenge. (B and C) Correlations (Spearman’s rank test) between peak SCC and IL-17A concentrations or IFN-γ concentrations yielded by the whole blood assay performed 45 days after the first immunization.
    Figure Legend Snippet: Time-course of the IL-17A and IFN-γ production in the antigen-specific whole blood assay following immunization and correlation with Peak SCC. Concentrations of IL-17A (A) or IFN-γ (B) after 3 days of culture with ovalbumin of blood samples taken before and after immunization at days 0 and 30 (median values and interquartiles) distinguishing the antigen-specific responses of responders and low-responder cows to the intramammary antigenic challenge. (B and C) Correlations (Spearman’s rank test) between peak SCC and IL-17A concentrations or IFN-γ concentrations yielded by the whole blood assay performed 45 days after the first immunization.

    Techniques Used: Whole Blood Assay

    3) Product Images from "Expansion, isolation and first characterization of bovine Th17 lymphocytes"

    Article Title: Expansion, isolation and first characterization of bovine Th17 lymphocytes

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-52562-2

    Choice of culture medium for the expansion of Th17 cells. ( a ) Cell growth after 3, 6, 8 or 13 days in RPMI (+FCS and 2 ng/mL TGF-β1) or IMDM (+10% KnockOut Serum Replacement) with different concentrations of recombinant human TGF-β1. ( b ) Proportions of IL-17A, IFN-γ and double positive cells after 6 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( c ) Proportions of IL-17A+ cells after 6 and 13 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( d ) Comparison of cell growth in RPMI and X-VIVO™ 15 media with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1), after 3 and 6 days of culture. ( e ) Proportions of cells IL-17A+ and IFN-γ+ (ICS) with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1) after 6 days of culture. Results are means from two cows.
    Figure Legend Snippet: Choice of culture medium for the expansion of Th17 cells. ( a ) Cell growth after 3, 6, 8 or 13 days in RPMI (+FCS and 2 ng/mL TGF-β1) or IMDM (+10% KnockOut Serum Replacement) with different concentrations of recombinant human TGF-β1. ( b ) Proportions of IL-17A, IFN-γ and double positive cells after 6 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( c ) Proportions of IL-17A+ cells after 6 and 13 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( d ) Comparison of cell growth in RPMI and X-VIVO™ 15 media with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1), after 3 and 6 days of culture. ( e ) Proportions of cells IL-17A+ and IFN-γ+ (ICS) with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1) after 6 days of culture. Results are means from two cows.

    Techniques Used: Knock-Out, Recombinant, Concentration Assay

    Identification of IL-17A and IFN-γ producing bovine lymphocytes. PBMC were stimulated with PMA/ionomycin, cytokine secretion blocked with Brefeldin A before flow cytometry analysis. Debris were excluded by gating according to FSC/SSC, and after gating on singlet cells, dead cells were excluded by gating on live cells. The CD4+ cells were gated by taking into account the isotype control, and the production of IL-17A and IFN-γ was measured by intracellular labelling with specific antibodies. Results shown are from a representative experiment. FSC: forward scatter: SSC: side scatter.
    Figure Legend Snippet: Identification of IL-17A and IFN-γ producing bovine lymphocytes. PBMC were stimulated with PMA/ionomycin, cytokine secretion blocked with Brefeldin A before flow cytometry analysis. Debris were excluded by gating according to FSC/SSC, and after gating on singlet cells, dead cells were excluded by gating on live cells. The CD4+ cells were gated by taking into account the isotype control, and the production of IL-17A and IFN-γ was measured by intracellular labelling with specific antibodies. Results shown are from a representative experiment. FSC: forward scatter: SSC: side scatter.

    Techniques Used: Flow Cytometry

    Unambiguous identification of bovine Th17. ( a ) Secretion of cytokines by sorted Th17 cells. After sorting, the cells were stimulated with α-CD3 and α-CD28 and cultured for 6 days. Supernatant contents were analysed by ELISA. Median values (10 to 90 percentiles) from five cell preparations from three cows are shown. ( b ) Comparison of Th17 signature gene expression between IL-17A+ and IL-17A- cells. Heatmap shows expression intensity expressed as log2(fold-change) normalized relative to three reference genes (ACTB, PPIA and GAPDH). Expression is relative to the CD4+ subset of the corresponding cow at day 6 after positive selection using MACS® beads. Cells were cultured in X-VIVO™ 15 medium without polarizing cytokines for 6 days after an initial TCR stimulation and a partial (half) renewal of medium with addition of IL-2 on the third day. Results are from three cows (C1, C2, C3).
    Figure Legend Snippet: Unambiguous identification of bovine Th17. ( a ) Secretion of cytokines by sorted Th17 cells. After sorting, the cells were stimulated with α-CD3 and α-CD28 and cultured for 6 days. Supernatant contents were analysed by ELISA. Median values (10 to 90 percentiles) from five cell preparations from three cows are shown. ( b ) Comparison of Th17 signature gene expression between IL-17A+ and IL-17A- cells. Heatmap shows expression intensity expressed as log2(fold-change) normalized relative to three reference genes (ACTB, PPIA and GAPDH). Expression is relative to the CD4+ subset of the corresponding cow at day 6 after positive selection using MACS® beads. Cells were cultured in X-VIVO™ 15 medium without polarizing cytokines for 6 days after an initial TCR stimulation and a partial (half) renewal of medium with addition of IL-2 on the third day. Results are from three cows (C1, C2, C3).

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Selection, Magnetic Cell Separation

    Comparison of the Cytokine Secretion Assay with the cytokine surface Staining. ( a ) Gating strategy. At the end of the culture expansion step, the polarized cell populations were split and analyzed side-by-side by flow cytometry with Cytokine Secretion (left) and surface staining (right) assays for IL-17A secretion. The squares indicate the IL-17A+ cells sorting windows, the circles the IL-17A- sorting windows. ( b ) Proportions of cytokine-positive cells 8 days after sorting. Median values from two sorting experiments with cells of two cows are shown. ( c ) Numbers of IL-17A+ and IL-17A- cells at different times after sorting. Stars indicate stimulations with anti-CD3/CD28 antibodies, arrows the sampling of cells for freezing or RNA preparation. Values are from one sorting experiment with two cows. ( d ) Proportions of sorted IL-17A+ cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. Values are from one sorting experiment with two cows. e) Proportions of sorted IL-17A- cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. CSA: cytokine secretion assay; CSS: cytokine surface staining.
    Figure Legend Snippet: Comparison of the Cytokine Secretion Assay with the cytokine surface Staining. ( a ) Gating strategy. At the end of the culture expansion step, the polarized cell populations were split and analyzed side-by-side by flow cytometry with Cytokine Secretion (left) and surface staining (right) assays for IL-17A secretion. The squares indicate the IL-17A+ cells sorting windows, the circles the IL-17A- sorting windows. ( b ) Proportions of cytokine-positive cells 8 days after sorting. Median values from two sorting experiments with cells of two cows are shown. ( c ) Numbers of IL-17A+ and IL-17A- cells at different times after sorting. Stars indicate stimulations with anti-CD3/CD28 antibodies, arrows the sampling of cells for freezing or RNA preparation. Values are from one sorting experiment with two cows. ( d ) Proportions of sorted IL-17A+ cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. Values are from one sorting experiment with two cows. e) Proportions of sorted IL-17A- cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. CSA: cytokine secretion assay; CSS: cytokine surface staining.

    Techniques Used: Staining, Flow Cytometry, Sampling

    Maintenance of the IL-17A and IFN-γ phenotype of sorted and frozen-thawed cells upon subculture. ( a ) Sorted IL-17A+ cells stimulated with α-CD3/α-CD28 were cultured with IL-2 with or without the polarizing cytokines TGF-β1 and IL-6 for 9 days and analyzed by flow cytometry (ICS). Percentages of IL-17A+/IFN-γ+ double positive cells and of IL-17A-/IFN-g+ cells are shown. ( b ) Thawed IL-17A+ cells were cultured for 22 days and analyzed by ICS at days 12 and 22. Percentages of IL-17A+ cells (all: both IL-17A+/IFN-γ- and double positive cells) and IL-17A-/IFN-γ+ cells are shown. Results from two cows (C1 C2) are shown.
    Figure Legend Snippet: Maintenance of the IL-17A and IFN-γ phenotype of sorted and frozen-thawed cells upon subculture. ( a ) Sorted IL-17A+ cells stimulated with α-CD3/α-CD28 were cultured with IL-2 with or without the polarizing cytokines TGF-β1 and IL-6 for 9 days and analyzed by flow cytometry (ICS). Percentages of IL-17A+/IFN-γ+ double positive cells and of IL-17A-/IFN-g+ cells are shown. ( b ) Thawed IL-17A+ cells were cultured for 22 days and analyzed by ICS at days 12 and 22. Percentages of IL-17A+ cells (all: both IL-17A+/IFN-γ- and double positive cells) and IL-17A-/IFN-γ+ cells are shown. Results from two cows (C1 C2) are shown.

    Techniques Used: Cell Culture, Flow Cytometry

    Culture conditions for expansion of Th17 cells. ( a ) Number of cells after 6, 9 and 13 days of culture as a function of the concentration (µg/mL) of the coating antibodies to CD3 with or without 10 ng/ml recombinant human IL-2. Results are from a representative experiment. ( b ) Concentrations of IL-17A and IFN-γ under these culture conditions. ( c ) Proportion of cells producing IL-17A or IFN-γ (ICS) at day 6 of culture. Results are means from the cells of two cows. Percentages of IL-17A positive cells differed as a function of treatment (p = 0.034, one-way ANOVA), but not percentages of IFN-γ positive cells (p = 0.24, one-way ANOVA). Results with α-CD3 1 µg/mL are not shown because too few cells were available for staining.
    Figure Legend Snippet: Culture conditions for expansion of Th17 cells. ( a ) Number of cells after 6, 9 and 13 days of culture as a function of the concentration (µg/mL) of the coating antibodies to CD3 with or without 10 ng/ml recombinant human IL-2. Results are from a representative experiment. ( b ) Concentrations of IL-17A and IFN-γ under these culture conditions. ( c ) Proportion of cells producing IL-17A or IFN-γ (ICS) at day 6 of culture. Results are means from the cells of two cows. Percentages of IL-17A positive cells differed as a function of treatment (p = 0.034, one-way ANOVA), but not percentages of IFN-γ positive cells (p = 0.24, one-way ANOVA). Results with α-CD3 1 µg/mL are not shown because too few cells were available for staining.

    Techniques Used: Concentration Assay, Recombinant, Staining

    Efficiency of the monoclonal antibodies in the cytokine secretion assay. ( a ) Percentages of CD4+ T cells labelled by the capture complex with either the α-Cter or α-Nter monoclonals, and comparison with the percentages of IL-17A+ cells identified by ICS. Data from two cows (C1 C2) are shown, one per row. ( b ) Concentrations of IL-17A in culture supernatant before capture, at the end of the 3.5 h of stimulation. ( c ) Concentrations of IL-17A in culture supernatant at the end of the secretion step (after capture) in the presence of the complex capture with either one of the two monoclonal antibodies to IL-17A, an isotype control or the polyclonal antiserum to IL-17A, showing the efficiency of the capture of IL-17A as it is secreted. PI: PMA/ionomycin, PIB: PMA/ionomycin/brefeldin A. ICS: intracellular staining.
    Figure Legend Snippet: Efficiency of the monoclonal antibodies in the cytokine secretion assay. ( a ) Percentages of CD4+ T cells labelled by the capture complex with either the α-Cter or α-Nter monoclonals, and comparison with the percentages of IL-17A+ cells identified by ICS. Data from two cows (C1 C2) are shown, one per row. ( b ) Concentrations of IL-17A in culture supernatant before capture, at the end of the 3.5 h of stimulation. ( c ) Concentrations of IL-17A in culture supernatant at the end of the secretion step (after capture) in the presence of the complex capture with either one of the two monoclonal antibodies to IL-17A, an isotype control or the polyclonal antiserum to IL-17A, showing the efficiency of the capture of IL-17A as it is secreted. PI: PMA/ionomycin, PIB: PMA/ionomycin/brefeldin A. ICS: intracellular staining.

    Techniques Used: Staining

    Schematic representation of the procedure used for the isolation and expansion of Th17 cells. CD4+ cells were isolated from PBMC by magnetic sorting, expanded in a culture medium without serum in the presence of polarizing cytokines and TCR-stimulation, activated, labelled for surface IL-17A and sorted by flow cytometry. The sorted cells were expanded and maintained in culture for several weeks. The indicated timeline is kept in all the figures to make it clear at which step cells were assayed. Concentrations of cytokines were 10 ng/mL (IL-2), 40 ng/mL (IL-6) and 2 ng/mL (TGF-β1). D: day; CSA: cytokine secretion assay; CSS: cytokine surface staining. α-: antibody anti-.
    Figure Legend Snippet: Schematic representation of the procedure used for the isolation and expansion of Th17 cells. CD4+ cells were isolated from PBMC by magnetic sorting, expanded in a culture medium without serum in the presence of polarizing cytokines and TCR-stimulation, activated, labelled for surface IL-17A and sorted by flow cytometry. The sorted cells were expanded and maintained in culture for several weeks. The indicated timeline is kept in all the figures to make it clear at which step cells were assayed. Concentrations of cytokines were 10 ng/mL (IL-2), 40 ng/mL (IL-6) and 2 ng/mL (TGF-β1). D: day; CSA: cytokine secretion assay; CSS: cytokine surface staining. α-: antibody anti-.

    Techniques Used: Isolation, Flow Cytometry, Staining

    Schematic representation of the assays used for surface labelling of IL-17A secreting cells. In the cytokine secretion assay (CSA), after stimulation with PMA/ionomycin, the cells start to secrete cytokines for 3.5 h. Then the cells are allowed to bind the capture complex (Capture complex antibody staining) for 15 min before dilution and incubation for 1.5 h under agitation (New cytokine secretion step). The secreted cytokine is captured by antibodies to IL-17A that are maintained at the surface of the cell by antibodies to CD45. The capture complex comprises the two types of biotinylated antibodies linked by a streptavidin molecule. After washing, rabbit anti-bovine IL-17A antibodies are added that bind to the captured IL-17A (IL-17A detection labelling and revelation). Then the cells are washed and the binding of rabbit antibodies revealed with a secondary antibody conjugated to phycoerythrin. Alternatively, in the cytokine surface staining assay (CSS), after washing at the end of the 3.5 h stimulation, cells are incubated with rabbit antibodies to IL-17A, washed, and the binding of rabbit antibodies to surface-associated IL-17A is revealed with a secondary antibody conjugated to phycoerythrin.
    Figure Legend Snippet: Schematic representation of the assays used for surface labelling of IL-17A secreting cells. In the cytokine secretion assay (CSA), after stimulation with PMA/ionomycin, the cells start to secrete cytokines for 3.5 h. Then the cells are allowed to bind the capture complex (Capture complex antibody staining) for 15 min before dilution and incubation for 1.5 h under agitation (New cytokine secretion step). The secreted cytokine is captured by antibodies to IL-17A that are maintained at the surface of the cell by antibodies to CD45. The capture complex comprises the two types of biotinylated antibodies linked by a streptavidin molecule. After washing, rabbit anti-bovine IL-17A antibodies are added that bind to the captured IL-17A (IL-17A detection labelling and revelation). Then the cells are washed and the binding of rabbit antibodies revealed with a secondary antibody conjugated to phycoerythrin. Alternatively, in the cytokine surface staining assay (CSS), after washing at the end of the 3.5 h stimulation, cells are incubated with rabbit antibodies to IL-17A, washed, and the binding of rabbit antibodies to surface-associated IL-17A is revealed with a secondary antibody conjugated to phycoerythrin.

    Techniques Used: Staining, Incubation, Binding Assay

    4) Product Images from "Expansion, isolation and first characterization of bovine Th17 lymphocytes"

    Article Title: Expansion, isolation and first characterization of bovine Th17 lymphocytes

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-52562-2

    Choice of culture medium for the expansion of Th17 cells. ( a ) Cell growth after 3, 6, 8 or 13 days in RPMI (+FCS and 2 ng/mL TGF-β1) or IMDM (+10% KnockOut Serum Replacement) with different concentrations of recombinant human TGF-β1. ( b ) Proportions of IL-17A, IFN-γ and double positive cells after 6 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( c ) Proportions of IL-17A+ cells after 6 and 13 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( d ) Comparison of cell growth in RPMI and X-VIVO™ 15 media with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1), after 3 and 6 days of culture. ( e ) Proportions of cells IL-17A+ and IFN-γ+ (ICS) with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1) after 6 days of culture. Results are means from two cows.
    Figure Legend Snippet: Choice of culture medium for the expansion of Th17 cells. ( a ) Cell growth after 3, 6, 8 or 13 days in RPMI (+FCS and 2 ng/mL TGF-β1) or IMDM (+10% KnockOut Serum Replacement) with different concentrations of recombinant human TGF-β1. ( b ) Proportions of IL-17A, IFN-γ and double positive cells after 6 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( c ) Proportions of IL-17A+ cells after 6 and 13 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( d ) Comparison of cell growth in RPMI and X-VIVO™ 15 media with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1), after 3 and 6 days of culture. ( e ) Proportions of cells IL-17A+ and IFN-γ+ (ICS) with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1) after 6 days of culture. Results are means from two cows.

    Techniques Used: Knock-Out, Recombinant, Concentration Assay

    Identification of IL-17A and IFN-γ producing bovine lymphocytes. PBMC were stimulated with PMA/ionomycin, cytokine secretion blocked with Brefeldin A before flow cytometry analysis. Debris were excluded by gating according to FSC/SSC, and after gating on singlet cells, dead cells were excluded by gating on live cells. The CD4+ cells were gated by taking into account the isotype control, and the production of IL-17A and IFN-γ was measured by intracellular labelling with specific antibodies. Results shown are from a representative experiment. FSC: forward scatter: SSC: side scatter.
    Figure Legend Snippet: Identification of IL-17A and IFN-γ producing bovine lymphocytes. PBMC were stimulated with PMA/ionomycin, cytokine secretion blocked with Brefeldin A before flow cytometry analysis. Debris were excluded by gating according to FSC/SSC, and after gating on singlet cells, dead cells were excluded by gating on live cells. The CD4+ cells were gated by taking into account the isotype control, and the production of IL-17A and IFN-γ was measured by intracellular labelling with specific antibodies. Results shown are from a representative experiment. FSC: forward scatter: SSC: side scatter.

    Techniques Used: Flow Cytometry

    Unambiguous identification of bovine Th17. ( a ) Secretion of cytokines by sorted Th17 cells. After sorting, the cells were stimulated with α-CD3 and α-CD28 and cultured for 6 days. Supernatant contents were analysed by ELISA. Median values (10 to 90 percentiles) from five cell preparations from three cows are shown. ( b ) Comparison of Th17 signature gene expression between IL-17A+ and IL-17A- cells. Heatmap shows expression intensity expressed as log2(fold-change) normalized relative to three reference genes (ACTB, PPIA and GAPDH). Expression is relative to the CD4+ subset of the corresponding cow at day 6 after positive selection using MACS® beads. Cells were cultured in X-VIVO™ 15 medium without polarizing cytokines for 6 days after an initial TCR stimulation and a partial (half) renewal of medium with addition of IL-2 on the third day. Results are from three cows (C1, C2, C3).
    Figure Legend Snippet: Unambiguous identification of bovine Th17. ( a ) Secretion of cytokines by sorted Th17 cells. After sorting, the cells were stimulated with α-CD3 and α-CD28 and cultured for 6 days. Supernatant contents were analysed by ELISA. Median values (10 to 90 percentiles) from five cell preparations from three cows are shown. ( b ) Comparison of Th17 signature gene expression between IL-17A+ and IL-17A- cells. Heatmap shows expression intensity expressed as log2(fold-change) normalized relative to three reference genes (ACTB, PPIA and GAPDH). Expression is relative to the CD4+ subset of the corresponding cow at day 6 after positive selection using MACS® beads. Cells were cultured in X-VIVO™ 15 medium without polarizing cytokines for 6 days after an initial TCR stimulation and a partial (half) renewal of medium with addition of IL-2 on the third day. Results are from three cows (C1, C2, C3).

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Selection, Magnetic Cell Separation

    Comparison of the Cytokine Secretion Assay with the cytokine surface Staining. ( a ) Gating strategy. At the end of the culture expansion step, the polarized cell populations were split and analyzed side-by-side by flow cytometry with Cytokine Secretion (left) and surface staining (right) assays for IL-17A secretion. The squares indicate the IL-17A+ cells sorting windows, the circles the IL-17A- sorting windows. ( b ) Proportions of cytokine-positive cells 8 days after sorting. Median values from two sorting experiments with cells of two cows are shown. ( c ) Numbers of IL-17A+ and IL-17A- cells at different times after sorting. Stars indicate stimulations with anti-CD3/CD28 antibodies, arrows the sampling of cells for freezing or RNA preparation. Values are from one sorting experiment with two cows. ( d ) Proportions of sorted IL-17A+ cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. Values are from one sorting experiment with two cows. e) Proportions of sorted IL-17A- cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. CSA: cytokine secretion assay; CSS: cytokine surface staining.
    Figure Legend Snippet: Comparison of the Cytokine Secretion Assay with the cytokine surface Staining. ( a ) Gating strategy. At the end of the culture expansion step, the polarized cell populations were split and analyzed side-by-side by flow cytometry with Cytokine Secretion (left) and surface staining (right) assays for IL-17A secretion. The squares indicate the IL-17A+ cells sorting windows, the circles the IL-17A- sorting windows. ( b ) Proportions of cytokine-positive cells 8 days after sorting. Median values from two sorting experiments with cells of two cows are shown. ( c ) Numbers of IL-17A+ and IL-17A- cells at different times after sorting. Stars indicate stimulations with anti-CD3/CD28 antibodies, arrows the sampling of cells for freezing or RNA preparation. Values are from one sorting experiment with two cows. ( d ) Proportions of sorted IL-17A+ cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. Values are from one sorting experiment with two cows. e) Proportions of sorted IL-17A- cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. CSA: cytokine secretion assay; CSS: cytokine surface staining.

    Techniques Used: Staining, Flow Cytometry, Sampling

    Maintenance of the IL-17A and IFN-γ phenotype of sorted and frozen-thawed cells upon subculture. ( a ) Sorted IL-17A+ cells stimulated with α-CD3/α-CD28 were cultured with IL-2 with or without the polarizing cytokines TGF-β1 and IL-6 for 9 days and analyzed by flow cytometry (ICS). Percentages of IL-17A+/IFN-γ+ double positive cells and of IL-17A-/IFN-g+ cells are shown. ( b ) Thawed IL-17A+ cells were cultured for 22 days and analyzed by ICS at days 12 and 22. Percentages of IL-17A+ cells (all: both IL-17A+/IFN-γ- and double positive cells) and IL-17A-/IFN-γ+ cells are shown. Results from two cows (C1 C2) are shown.
    Figure Legend Snippet: Maintenance of the IL-17A and IFN-γ phenotype of sorted and frozen-thawed cells upon subculture. ( a ) Sorted IL-17A+ cells stimulated with α-CD3/α-CD28 were cultured with IL-2 with or without the polarizing cytokines TGF-β1 and IL-6 for 9 days and analyzed by flow cytometry (ICS). Percentages of IL-17A+/IFN-γ+ double positive cells and of IL-17A-/IFN-g+ cells are shown. ( b ) Thawed IL-17A+ cells were cultured for 22 days and analyzed by ICS at days 12 and 22. Percentages of IL-17A+ cells (all: both IL-17A+/IFN-γ- and double positive cells) and IL-17A-/IFN-γ+ cells are shown. Results from two cows (C1 C2) are shown.

    Techniques Used: Cell Culture, Flow Cytometry

    Culture conditions for expansion of Th17 cells. ( a ) Number of cells after 6, 9 and 13 days of culture as a function of the concentration (µg/mL) of the coating antibodies to CD3 with or without 10 ng/ml recombinant human IL-2. Results are from a representative experiment. ( b ) Concentrations of IL-17A and IFN-γ under these culture conditions. ( c ) Proportion of cells producing IL-17A or IFN-γ (ICS) at day 6 of culture. Results are means from the cells of two cows. Percentages of IL-17A positive cells differed as a function of treatment (p = 0.034, one-way ANOVA), but not percentages of IFN-γ positive cells (p = 0.24, one-way ANOVA). Results with α-CD3 1 µg/mL are not shown because too few cells were available for staining.
    Figure Legend Snippet: Culture conditions for expansion of Th17 cells. ( a ) Number of cells after 6, 9 and 13 days of culture as a function of the concentration (µg/mL) of the coating antibodies to CD3 with or without 10 ng/ml recombinant human IL-2. Results are from a representative experiment. ( b ) Concentrations of IL-17A and IFN-γ under these culture conditions. ( c ) Proportion of cells producing IL-17A or IFN-γ (ICS) at day 6 of culture. Results are means from the cells of two cows. Percentages of IL-17A positive cells differed as a function of treatment (p = 0.034, one-way ANOVA), but not percentages of IFN-γ positive cells (p = 0.24, one-way ANOVA). Results with α-CD3 1 µg/mL are not shown because too few cells were available for staining.

    Techniques Used: Concentration Assay, Recombinant, Staining

    Efficiency of the monoclonal antibodies in the cytokine secretion assay. ( a ) Percentages of CD4+ T cells labelled by the capture complex with either the α-Cter or α-Nter monoclonals, and comparison with the percentages of IL-17A+ cells identified by ICS. Data from two cows (C1 C2) are shown, one per row. ( b ) Concentrations of IL-17A in culture supernatant before capture, at the end of the 3.5 h of stimulation. ( c ) Concentrations of IL-17A in culture supernatant at the end of the secretion step (after capture) in the presence of the complex capture with either one of the two monoclonal antibodies to IL-17A, an isotype control or the polyclonal antiserum to IL-17A, showing the efficiency of the capture of IL-17A as it is secreted. PI: PMA/ionomycin, PIB: PMA/ionomycin/brefeldin A. ICS: intracellular staining.
    Figure Legend Snippet: Efficiency of the monoclonal antibodies in the cytokine secretion assay. ( a ) Percentages of CD4+ T cells labelled by the capture complex with either the α-Cter or α-Nter monoclonals, and comparison with the percentages of IL-17A+ cells identified by ICS. Data from two cows (C1 C2) are shown, one per row. ( b ) Concentrations of IL-17A in culture supernatant before capture, at the end of the 3.5 h of stimulation. ( c ) Concentrations of IL-17A in culture supernatant at the end of the secretion step (after capture) in the presence of the complex capture with either one of the two monoclonal antibodies to IL-17A, an isotype control or the polyclonal antiserum to IL-17A, showing the efficiency of the capture of IL-17A as it is secreted. PI: PMA/ionomycin, PIB: PMA/ionomycin/brefeldin A. ICS: intracellular staining.

    Techniques Used: Staining

    Schematic representation of the procedure used for the isolation and expansion of Th17 cells. CD4+ cells were isolated from PBMC by magnetic sorting, expanded in a culture medium without serum in the presence of polarizing cytokines and TCR-stimulation, activated, labelled for surface IL-17A and sorted by flow cytometry. The sorted cells were expanded and maintained in culture for several weeks. The indicated timeline is kept in all the figures to make it clear at which step cells were assayed. Concentrations of cytokines were 10 ng/mL (IL-2), 40 ng/mL (IL-6) and 2 ng/mL (TGF-β1). D: day; CSA: cytokine secretion assay; CSS: cytokine surface staining. α-: antibody anti-.
    Figure Legend Snippet: Schematic representation of the procedure used for the isolation and expansion of Th17 cells. CD4+ cells were isolated from PBMC by magnetic sorting, expanded in a culture medium without serum in the presence of polarizing cytokines and TCR-stimulation, activated, labelled for surface IL-17A and sorted by flow cytometry. The sorted cells were expanded and maintained in culture for several weeks. The indicated timeline is kept in all the figures to make it clear at which step cells were assayed. Concentrations of cytokines were 10 ng/mL (IL-2), 40 ng/mL (IL-6) and 2 ng/mL (TGF-β1). D: day; CSA: cytokine secretion assay; CSS: cytokine surface staining. α-: antibody anti-.

    Techniques Used: Isolation, Flow Cytometry, Staining

    Schematic representation of the assays used for surface labelling of IL-17A secreting cells. In the cytokine secretion assay (CSA), after stimulation with PMA/ionomycin, the cells start to secrete cytokines for 3.5 h. Then the cells are allowed to bind the capture complex (Capture complex antibody staining) for 15 min before dilution and incubation for 1.5 h under agitation (New cytokine secretion step). The secreted cytokine is captured by antibodies to IL-17A that are maintained at the surface of the cell by antibodies to CD45. The capture complex comprises the two types of biotinylated antibodies linked by a streptavidin molecule. After washing, rabbit anti-bovine IL-17A antibodies are added that bind to the captured IL-17A (IL-17A detection labelling and revelation). Then the cells are washed and the binding of rabbit antibodies revealed with a secondary antibody conjugated to phycoerythrin. Alternatively, in the cytokine surface staining assay (CSS), after washing at the end of the 3.5 h stimulation, cells are incubated with rabbit antibodies to IL-17A, washed, and the binding of rabbit antibodies to surface-associated IL-17A is revealed with a secondary antibody conjugated to phycoerythrin.
    Figure Legend Snippet: Schematic representation of the assays used for surface labelling of IL-17A secreting cells. In the cytokine secretion assay (CSA), after stimulation with PMA/ionomycin, the cells start to secrete cytokines for 3.5 h. Then the cells are allowed to bind the capture complex (Capture complex antibody staining) for 15 min before dilution and incubation for 1.5 h under agitation (New cytokine secretion step). The secreted cytokine is captured by antibodies to IL-17A that are maintained at the surface of the cell by antibodies to CD45. The capture complex comprises the two types of biotinylated antibodies linked by a streptavidin molecule. After washing, rabbit anti-bovine IL-17A antibodies are added that bind to the captured IL-17A (IL-17A detection labelling and revelation). Then the cells are washed and the binding of rabbit antibodies revealed with a secondary antibody conjugated to phycoerythrin. Alternatively, in the cytokine surface staining assay (CSS), after washing at the end of the 3.5 h stimulation, cells are incubated with rabbit antibodies to IL-17A, washed, and the binding of rabbit antibodies to surface-associated IL-17A is revealed with a secondary antibody conjugated to phycoerythrin.

    Techniques Used: Staining, Incubation, Binding Assay

    5) 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

    6) Product Images from "Efficacy of mucosal polyanhydride nanovaccine against respiratory syncytial virus infection in the neonatal calf"

    Article Title: Efficacy of mucosal polyanhydride nanovaccine against respiratory syncytial virus infection in the neonatal calf

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-21292-2

    Enhanced BRSV-specific T cell responses in the peripheral blood and BAL of BRSV-F/G nanovaccine-administered calves. PBMC were collected on day 6 post-challenge, labeled with Cell Trace Violet and stimulated for 6 days with 5 μg/mL of the recombinant BRSV F and G proteins, or 0.01 MOI of BRSV strain 375. Pokeweed Mitogen was used at a concentration of 1 μg/mL as a positive control. Mock stimulated samples (negative control wells) were cultured with cRPMI and were used to correct for background proliferation. ( A ) After 6 days, antigen-specific CD4 T cell proliferation was assessed by flow cytometry, as measured by dilution of the Cell Trace Violet dye. Background levels of proliferation were subtracted and results are presented as change over mock. ( B ) Stimulated cell culture supernatants were collected after 6 days and concentrations of IFNγ (left panel) and IL-17A (right panel) were measured by commercial sandwich ELISAs. ( C ) BALs were performed on day 7 post-challenge. Cells were enumerated and stimulated for 6 days with recombinant BRSV F protein, G protein or BRSV as in A. After 6 days, cell culture supernatants were collected and concentrations of IFNγ (left panel) and IL-17A (right panel) were measured by commercial sandwich ELISAs. Results represent n = 6 animals/group. Data represent means ± SEM. *p
    Figure Legend Snippet: Enhanced BRSV-specific T cell responses in the peripheral blood and BAL of BRSV-F/G nanovaccine-administered calves. PBMC were collected on day 6 post-challenge, labeled with Cell Trace Violet and stimulated for 6 days with 5 μg/mL of the recombinant BRSV F and G proteins, or 0.01 MOI of BRSV strain 375. Pokeweed Mitogen was used at a concentration of 1 μg/mL as a positive control. Mock stimulated samples (negative control wells) were cultured with cRPMI and were used to correct for background proliferation. ( A ) After 6 days, antigen-specific CD4 T cell proliferation was assessed by flow cytometry, as measured by dilution of the Cell Trace Violet dye. Background levels of proliferation were subtracted and results are presented as change over mock. ( B ) Stimulated cell culture supernatants were collected after 6 days and concentrations of IFNγ (left panel) and IL-17A (right panel) were measured by commercial sandwich ELISAs. ( C ) BALs were performed on day 7 post-challenge. Cells were enumerated and stimulated for 6 days with recombinant BRSV F protein, G protein or BRSV as in A. After 6 days, cell culture supernatants were collected and concentrations of IFNγ (left panel) and IL-17A (right panel) were measured by commercial sandwich ELISAs. Results represent n = 6 animals/group. Data represent means ± SEM. *p

    Techniques Used: Labeling, Recombinant, Concentration Assay, Positive Control, Negative Control, Cell Culture, Flow Cytometry, Cytometry

    7) 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

    8) Product Images from "Expansion, isolation and first characterization of bovine Th17 lymphocytes"

    Article Title: Expansion, isolation and first characterization of bovine Th17 lymphocytes

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-52562-2

    Choice of culture medium for the expansion of Th17 cells. ( a ) Cell growth after 3, 6, 8 or 13 days in RPMI (+FCS and 2 ng/mL TGF-β1) or IMDM (+10% KnockOut Serum Replacement) with different concentrations of recombinant human TGF-β1. ( b ) Proportions of IL-17A, IFN-γ and double positive cells after 6 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( c ) Proportions of IL-17A+ cells after 6 and 13 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( d ) Comparison of cell growth in RPMI and X-VIVO™ 15 media with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1), after 3 and 6 days of culture. ( e ) Proportions of cells IL-17A+ and IFN-γ+ (ICS) with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1) after 6 days of culture. Results are means from two cows.
    Figure Legend Snippet: Choice of culture medium for the expansion of Th17 cells. ( a ) Cell growth after 3, 6, 8 or 13 days in RPMI (+FCS and 2 ng/mL TGF-β1) or IMDM (+10% KnockOut Serum Replacement) with different concentrations of recombinant human TGF-β1. ( b ) Proportions of IL-17A, IFN-γ and double positive cells after 6 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( c ) Proportions of IL-17A+ cells after 6 and 13 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( d ) Comparison of cell growth in RPMI and X-VIVO™ 15 media with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1), after 3 and 6 days of culture. ( e ) Proportions of cells IL-17A+ and IFN-γ+ (ICS) with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1) after 6 days of culture. Results are means from two cows.

    Techniques Used: Knock-Out, Recombinant, Concentration Assay

    Identification of IL-17A and IFN-γ producing bovine lymphocytes. PBMC were stimulated with PMA/ionomycin, cytokine secretion blocked with Brefeldin A before flow cytometry analysis. Debris were excluded by gating according to FSC/SSC, and after gating on singlet cells, dead cells were excluded by gating on live cells. The CD4+ cells were gated by taking into account the isotype control, and the production of IL-17A and IFN-γ was measured by intracellular labelling with specific antibodies. Results shown are from a representative experiment. FSC: forward scatter: SSC: side scatter.
    Figure Legend Snippet: Identification of IL-17A and IFN-γ producing bovine lymphocytes. PBMC were stimulated with PMA/ionomycin, cytokine secretion blocked with Brefeldin A before flow cytometry analysis. Debris were excluded by gating according to FSC/SSC, and after gating on singlet cells, dead cells were excluded by gating on live cells. The CD4+ cells were gated by taking into account the isotype control, and the production of IL-17A and IFN-γ was measured by intracellular labelling with specific antibodies. Results shown are from a representative experiment. FSC: forward scatter: SSC: side scatter.

    Techniques Used: Flow Cytometry

    Unambiguous identification of bovine Th17. ( a ) Secretion of cytokines by sorted Th17 cells. After sorting, the cells were stimulated with α-CD3 and α-CD28 and cultured for 6 days. Supernatant contents were analysed by ELISA. Median values (10 to 90 percentiles) from five cell preparations from three cows are shown. ( b ) Comparison of Th17 signature gene expression between IL-17A+ and IL-17A- cells. Heatmap shows expression intensity expressed as log2(fold-change) normalized relative to three reference genes (ACTB, PPIA and GAPDH). Expression is relative to the CD4+ subset of the corresponding cow at day 6 after positive selection using MACS® beads. Cells were cultured in X-VIVO™ 15 medium without polarizing cytokines for 6 days after an initial TCR stimulation and a partial (half) renewal of medium with addition of IL-2 on the third day. Results are from three cows (C1, C2, C3).
    Figure Legend Snippet: Unambiguous identification of bovine Th17. ( a ) Secretion of cytokines by sorted Th17 cells. After sorting, the cells were stimulated with α-CD3 and α-CD28 and cultured for 6 days. Supernatant contents were analysed by ELISA. Median values (10 to 90 percentiles) from five cell preparations from three cows are shown. ( b ) Comparison of Th17 signature gene expression between IL-17A+ and IL-17A- cells. Heatmap shows expression intensity expressed as log2(fold-change) normalized relative to three reference genes (ACTB, PPIA and GAPDH). Expression is relative to the CD4+ subset of the corresponding cow at day 6 after positive selection using MACS® beads. Cells were cultured in X-VIVO™ 15 medium without polarizing cytokines for 6 days after an initial TCR stimulation and a partial (half) renewal of medium with addition of IL-2 on the third day. Results are from three cows (C1, C2, C3).

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Selection, Magnetic Cell Separation

    Comparison of the Cytokine Secretion Assay with the cytokine surface Staining. ( a ) Gating strategy. At the end of the culture expansion step, the polarized cell populations were split and analyzed side-by-side by flow cytometry with Cytokine Secretion (left) and surface staining (right) assays for IL-17A secretion. The squares indicate the IL-17A+ cells sorting windows, the circles the IL-17A- sorting windows. ( b ) Proportions of cytokine-positive cells 8 days after sorting. Median values from two sorting experiments with cells of two cows are shown. ( c ) Numbers of IL-17A+ and IL-17A- cells at different times after sorting. Stars indicate stimulations with anti-CD3/CD28 antibodies, arrows the sampling of cells for freezing or RNA preparation. Values are from one sorting experiment with two cows. ( d ) Proportions of sorted IL-17A+ cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. Values are from one sorting experiment with two cows. e) Proportions of sorted IL-17A- cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. CSA: cytokine secretion assay; CSS: cytokine surface staining.
    Figure Legend Snippet: Comparison of the Cytokine Secretion Assay with the cytokine surface Staining. ( a ) Gating strategy. At the end of the culture expansion step, the polarized cell populations were split and analyzed side-by-side by flow cytometry with Cytokine Secretion (left) and surface staining (right) assays for IL-17A secretion. The squares indicate the IL-17A+ cells sorting windows, the circles the IL-17A- sorting windows. ( b ) Proportions of cytokine-positive cells 8 days after sorting. Median values from two sorting experiments with cells of two cows are shown. ( c ) Numbers of IL-17A+ and IL-17A- cells at different times after sorting. Stars indicate stimulations with anti-CD3/CD28 antibodies, arrows the sampling of cells for freezing or RNA preparation. Values are from one sorting experiment with two cows. ( d ) Proportions of sorted IL-17A+ cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. Values are from one sorting experiment with two cows. e) Proportions of sorted IL-17A- cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. CSA: cytokine secretion assay; CSS: cytokine surface staining.

    Techniques Used: Staining, Flow Cytometry, Sampling

    Maintenance of the IL-17A and IFN-γ phenotype of sorted and frozen-thawed cells upon subculture. ( a ) Sorted IL-17A+ cells stimulated with α-CD3/α-CD28 were cultured with IL-2 with or without the polarizing cytokines TGF-β1 and IL-6 for 9 days and analyzed by flow cytometry (ICS). Percentages of IL-17A+/IFN-γ+ double positive cells and of IL-17A-/IFN-g+ cells are shown. ( b ) Thawed IL-17A+ cells were cultured for 22 days and analyzed by ICS at days 12 and 22. Percentages of IL-17A+ cells (all: both IL-17A+/IFN-γ- and double positive cells) and IL-17A-/IFN-γ+ cells are shown. Results from two cows (C1 C2) are shown.
    Figure Legend Snippet: Maintenance of the IL-17A and IFN-γ phenotype of sorted and frozen-thawed cells upon subculture. ( a ) Sorted IL-17A+ cells stimulated with α-CD3/α-CD28 were cultured with IL-2 with or without the polarizing cytokines TGF-β1 and IL-6 for 9 days and analyzed by flow cytometry (ICS). Percentages of IL-17A+/IFN-γ+ double positive cells and of IL-17A-/IFN-g+ cells are shown. ( b ) Thawed IL-17A+ cells were cultured for 22 days and analyzed by ICS at days 12 and 22. Percentages of IL-17A+ cells (all: both IL-17A+/IFN-γ- and double positive cells) and IL-17A-/IFN-γ+ cells are shown. Results from two cows (C1 C2) are shown.

    Techniques Used: Cell Culture, Flow Cytometry

    Culture conditions for expansion of Th17 cells. ( a ) Number of cells after 6, 9 and 13 days of culture as a function of the concentration (µg/mL) of the coating antibodies to CD3 with or without 10 ng/ml recombinant human IL-2. Results are from a representative experiment. ( b ) Concentrations of IL-17A and IFN-γ under these culture conditions. ( c ) Proportion of cells producing IL-17A or IFN-γ (ICS) at day 6 of culture. Results are means from the cells of two cows. Percentages of IL-17A positive cells differed as a function of treatment (p = 0.034, one-way ANOVA), but not percentages of IFN-γ positive cells (p = 0.24, one-way ANOVA). Results with α-CD3 1 µg/mL are not shown because too few cells were available for staining.
    Figure Legend Snippet: Culture conditions for expansion of Th17 cells. ( a ) Number of cells after 6, 9 and 13 days of culture as a function of the concentration (µg/mL) of the coating antibodies to CD3 with or without 10 ng/ml recombinant human IL-2. Results are from a representative experiment. ( b ) Concentrations of IL-17A and IFN-γ under these culture conditions. ( c ) Proportion of cells producing IL-17A or IFN-γ (ICS) at day 6 of culture. Results are means from the cells of two cows. Percentages of IL-17A positive cells differed as a function of treatment (p = 0.034, one-way ANOVA), but not percentages of IFN-γ positive cells (p = 0.24, one-way ANOVA). Results with α-CD3 1 µg/mL are not shown because too few cells were available for staining.

    Techniques Used: Concentration Assay, Recombinant, Staining

    Efficiency of the monoclonal antibodies in the cytokine secretion assay. ( a ) Percentages of CD4+ T cells labelled by the capture complex with either the α-Cter or α-Nter monoclonals, and comparison with the percentages of IL-17A+ cells identified by ICS. Data from two cows (C1 C2) are shown, one per row. ( b ) Concentrations of IL-17A in culture supernatant before capture, at the end of the 3.5 h of stimulation. ( c ) Concentrations of IL-17A in culture supernatant at the end of the secretion step (after capture) in the presence of the complex capture with either one of the two monoclonal antibodies to IL-17A, an isotype control or the polyclonal antiserum to IL-17A, showing the efficiency of the capture of IL-17A as it is secreted. PI: PMA/ionomycin, PIB: PMA/ionomycin/brefeldin A. ICS: intracellular staining.
    Figure Legend Snippet: Efficiency of the monoclonal antibodies in the cytokine secretion assay. ( a ) Percentages of CD4+ T cells labelled by the capture complex with either the α-Cter or α-Nter monoclonals, and comparison with the percentages of IL-17A+ cells identified by ICS. Data from two cows (C1 C2) are shown, one per row. ( b ) Concentrations of IL-17A in culture supernatant before capture, at the end of the 3.5 h of stimulation. ( c ) Concentrations of IL-17A in culture supernatant at the end of the secretion step (after capture) in the presence of the complex capture with either one of the two monoclonal antibodies to IL-17A, an isotype control or the polyclonal antiserum to IL-17A, showing the efficiency of the capture of IL-17A as it is secreted. PI: PMA/ionomycin, PIB: PMA/ionomycin/brefeldin A. ICS: intracellular staining.

    Techniques Used: Staining

    Schematic representation of the procedure used for the isolation and expansion of Th17 cells. CD4+ cells were isolated from PBMC by magnetic sorting, expanded in a culture medium without serum in the presence of polarizing cytokines and TCR-stimulation, activated, labelled for surface IL-17A and sorted by flow cytometry. The sorted cells were expanded and maintained in culture for several weeks. The indicated timeline is kept in all the figures to make it clear at which step cells were assayed. Concentrations of cytokines were 10 ng/mL (IL-2), 40 ng/mL (IL-6) and 2 ng/mL (TGF-β1). D: day; CSA: cytokine secretion assay; CSS: cytokine surface staining. α-: antibody anti-.
    Figure Legend Snippet: Schematic representation of the procedure used for the isolation and expansion of Th17 cells. CD4+ cells were isolated from PBMC by magnetic sorting, expanded in a culture medium without serum in the presence of polarizing cytokines and TCR-stimulation, activated, labelled for surface IL-17A and sorted by flow cytometry. The sorted cells were expanded and maintained in culture for several weeks. The indicated timeline is kept in all the figures to make it clear at which step cells were assayed. Concentrations of cytokines were 10 ng/mL (IL-2), 40 ng/mL (IL-6) and 2 ng/mL (TGF-β1). D: day; CSA: cytokine secretion assay; CSS: cytokine surface staining. α-: antibody anti-.

    Techniques Used: Isolation, Flow Cytometry, Staining

    Schematic representation of the assays used for surface labelling of IL-17A secreting cells. In the cytokine secretion assay (CSA), after stimulation with PMA/ionomycin, the cells start to secrete cytokines for 3.5 h. Then the cells are allowed to bind the capture complex (Capture complex antibody staining) for 15 min before dilution and incubation for 1.5 h under agitation (New cytokine secretion step). The secreted cytokine is captured by antibodies to IL-17A that are maintained at the surface of the cell by antibodies to CD45. The capture complex comprises the two types of biotinylated antibodies linked by a streptavidin molecule. After washing, rabbit anti-bovine IL-17A antibodies are added that bind to the captured IL-17A (IL-17A detection labelling and revelation). Then the cells are washed and the binding of rabbit antibodies revealed with a secondary antibody conjugated to phycoerythrin. Alternatively, in the cytokine surface staining assay (CSS), after washing at the end of the 3.5 h stimulation, cells are incubated with rabbit antibodies to IL-17A, washed, and the binding of rabbit antibodies to surface-associated IL-17A is revealed with a secondary antibody conjugated to phycoerythrin.
    Figure Legend Snippet: Schematic representation of the assays used for surface labelling of IL-17A secreting cells. In the cytokine secretion assay (CSA), after stimulation with PMA/ionomycin, the cells start to secrete cytokines for 3.5 h. Then the cells are allowed to bind the capture complex (Capture complex antibody staining) for 15 min before dilution and incubation for 1.5 h under agitation (New cytokine secretion step). The secreted cytokine is captured by antibodies to IL-17A that are maintained at the surface of the cell by antibodies to CD45. The capture complex comprises the two types of biotinylated antibodies linked by a streptavidin molecule. After washing, rabbit anti-bovine IL-17A antibodies are added that bind to the captured IL-17A (IL-17A detection labelling and revelation). Then the cells are washed and the binding of rabbit antibodies revealed with a secondary antibody conjugated to phycoerythrin. Alternatively, in the cytokine surface staining assay (CSS), after washing at the end of the 3.5 h stimulation, cells are incubated with rabbit antibodies to IL-17A, washed, and the binding of rabbit antibodies to surface-associated IL-17A is revealed with a secondary antibody conjugated to phycoerythrin.

    Techniques Used: Staining, Incubation, Binding Assay

    9) Product Images from "A Recombinant BCG Vaccine Is Safe and Immunogenic in Neonatal Calves and Reduces the Clinical Disease Caused by the Respiratory Syncytial Virus"

    Article Title: A Recombinant BCG Vaccine Is Safe and Immunogenic in Neonatal Calves and Reduces the Clinical Disease Caused by the Respiratory Syncytial Virus

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2021.664212

    Vaccination with rBCG-N-hRSV increases virus and BCG-specific IFN-γ and IL-17 secretion in PBMC cultures. (A, C) Study 1 PBMCs and (B, D) Study 2 Tracheobronchial lymph node cells (TBLNs) were isolated on day 7 post-infection, labeled with Cell Trace Violet, and restimulated in vitro with PPD-B, Ag85A/TB10.4, N-hRSV or bRSV strain 375. Mock stimulated cultures were used as negative controls. ConA stimulated cultures were used as positive controls (not shown). Six days later, cell culture supernatants were analyzed for bovine IFN-γ and IL-17A secretion using commercial ELISA kits. *p
    Figure Legend Snippet: Vaccination with rBCG-N-hRSV increases virus and BCG-specific IFN-γ and IL-17 secretion in PBMC cultures. (A, C) Study 1 PBMCs and (B, D) Study 2 Tracheobronchial lymph node cells (TBLNs) were isolated on day 7 post-infection, labeled with Cell Trace Violet, and restimulated in vitro with PPD-B, Ag85A/TB10.4, N-hRSV or bRSV strain 375. Mock stimulated cultures were used as negative controls. ConA stimulated cultures were used as positive controls (not shown). Six days later, cell culture supernatants were analyzed for bovine IFN-γ and IL-17A secretion using commercial ELISA kits. *p

    Techniques Used: Isolation, Infection, Labeling, In Vitro, Cell Culture, Enzyme-linked Immunosorbent Assay

    10) 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

    11) Product Images from "Expansion, isolation and first characterization of bovine Th17 lymphocytes"

    Article Title: Expansion, isolation and first characterization of bovine Th17 lymphocytes

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-52562-2

    Choice of culture medium for the expansion of Th17 cells. ( a ) Cell growth after 3, 6, 8 or 13 days in RPMI (+FCS and 2 ng/mL TGF-β1) or IMDM (+10% KnockOut Serum Replacement) with different concentrations of recombinant human TGF-β1. ( b ) Proportions of IL-17A, IFN-γ and double positive cells after 6 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( c ) Proportions of IL-17A+ cells after 6 and 13 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( d ) Comparison of cell growth in RPMI and X-VIVO™ 15 media with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1), after 3 and 6 days of culture. ( e ) Proportions of cells IL-17A+ and IFN-γ+ (ICS) with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1) after 6 days of culture. Results are means from two cows.
    Figure Legend Snippet: Choice of culture medium for the expansion of Th17 cells. ( a ) Cell growth after 3, 6, 8 or 13 days in RPMI (+FCS and 2 ng/mL TGF-β1) or IMDM (+10% KnockOut Serum Replacement) with different concentrations of recombinant human TGF-β1. ( b ) Proportions of IL-17A, IFN-γ and double positive cells after 6 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( c ) Proportions of IL-17A+ cells after 6 and 13 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( d ) Comparison of cell growth in RPMI and X-VIVO™ 15 media with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1), after 3 and 6 days of culture. ( e ) Proportions of cells IL-17A+ and IFN-γ+ (ICS) with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1) after 6 days of culture. Results are means from two cows.

    Techniques Used: Knock-Out, Recombinant, Concentration Assay

    Identification of IL-17A and IFN-γ producing bovine lymphocytes. PBMC were stimulated with PMA/ionomycin, cytokine secretion blocked with Brefeldin A before flow cytometry analysis. Debris were excluded by gating according to FSC/SSC, and after gating on singlet cells, dead cells were excluded by gating on live cells. The CD4+ cells were gated by taking into account the isotype control, and the production of IL-17A and IFN-γ was measured by intracellular labelling with specific antibodies. Results shown are from a representative experiment. FSC: forward scatter: SSC: side scatter.
    Figure Legend Snippet: Identification of IL-17A and IFN-γ producing bovine lymphocytes. PBMC were stimulated with PMA/ionomycin, cytokine secretion blocked with Brefeldin A before flow cytometry analysis. Debris were excluded by gating according to FSC/SSC, and after gating on singlet cells, dead cells were excluded by gating on live cells. The CD4+ cells were gated by taking into account the isotype control, and the production of IL-17A and IFN-γ was measured by intracellular labelling with specific antibodies. Results shown are from a representative experiment. FSC: forward scatter: SSC: side scatter.

    Techniques Used: Flow Cytometry

    Unambiguous identification of bovine Th17. ( a ) Secretion of cytokines by sorted Th17 cells. After sorting, the cells were stimulated with α-CD3 and α-CD28 and cultured for 6 days. Supernatant contents were analysed by ELISA. Median values (10 to 90 percentiles) from five cell preparations from three cows are shown. ( b ) Comparison of Th17 signature gene expression between IL-17A+ and IL-17A- cells. Heatmap shows expression intensity expressed as log2(fold-change) normalized relative to three reference genes (ACTB, PPIA and GAPDH). Expression is relative to the CD4+ subset of the corresponding cow at day 6 after positive selection using MACS® beads. Cells were cultured in X-VIVO™ 15 medium without polarizing cytokines for 6 days after an initial TCR stimulation and a partial (half) renewal of medium with addition of IL-2 on the third day. Results are from three cows (C1, C2, C3).
    Figure Legend Snippet: Unambiguous identification of bovine Th17. ( a ) Secretion of cytokines by sorted Th17 cells. After sorting, the cells were stimulated with α-CD3 and α-CD28 and cultured for 6 days. Supernatant contents were analysed by ELISA. Median values (10 to 90 percentiles) from five cell preparations from three cows are shown. ( b ) Comparison of Th17 signature gene expression between IL-17A+ and IL-17A- cells. Heatmap shows expression intensity expressed as log2(fold-change) normalized relative to three reference genes (ACTB, PPIA and GAPDH). Expression is relative to the CD4+ subset of the corresponding cow at day 6 after positive selection using MACS® beads. Cells were cultured in X-VIVO™ 15 medium without polarizing cytokines for 6 days after an initial TCR stimulation and a partial (half) renewal of medium with addition of IL-2 on the third day. Results are from three cows (C1, C2, C3).

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Selection, Magnetic Cell Separation

    Comparison of the Cytokine Secretion Assay with the cytokine surface Staining. ( a ) Gating strategy. At the end of the culture expansion step, the polarized cell populations were split and analyzed side-by-side by flow cytometry with Cytokine Secretion (left) and surface staining (right) assays for IL-17A secretion. The squares indicate the IL-17A+ cells sorting windows, the circles the IL-17A- sorting windows. ( b ) Proportions of cytokine-positive cells 8 days after sorting. Median values from two sorting experiments with cells of two cows are shown. ( c ) Numbers of IL-17A+ and IL-17A- cells at different times after sorting. Stars indicate stimulations with anti-CD3/CD28 antibodies, arrows the sampling of cells for freezing or RNA preparation. Values are from one sorting experiment with two cows. ( d ) Proportions of sorted IL-17A+ cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. Values are from one sorting experiment with two cows. e) Proportions of sorted IL-17A- cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. CSA: cytokine secretion assay; CSS: cytokine surface staining.
    Figure Legend Snippet: Comparison of the Cytokine Secretion Assay with the cytokine surface Staining. ( a ) Gating strategy. At the end of the culture expansion step, the polarized cell populations were split and analyzed side-by-side by flow cytometry with Cytokine Secretion (left) and surface staining (right) assays for IL-17A secretion. The squares indicate the IL-17A+ cells sorting windows, the circles the IL-17A- sorting windows. ( b ) Proportions of cytokine-positive cells 8 days after sorting. Median values from two sorting experiments with cells of two cows are shown. ( c ) Numbers of IL-17A+ and IL-17A- cells at different times after sorting. Stars indicate stimulations with anti-CD3/CD28 antibodies, arrows the sampling of cells for freezing or RNA preparation. Values are from one sorting experiment with two cows. ( d ) Proportions of sorted IL-17A+ cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. Values are from one sorting experiment with two cows. e) Proportions of sorted IL-17A- cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. CSA: cytokine secretion assay; CSS: cytokine surface staining.

    Techniques Used: Staining, Flow Cytometry, Sampling

    Maintenance of the IL-17A and IFN-γ phenotype of sorted and frozen-thawed cells upon subculture. ( a ) Sorted IL-17A+ cells stimulated with α-CD3/α-CD28 were cultured with IL-2 with or without the polarizing cytokines TGF-β1 and IL-6 for 9 days and analyzed by flow cytometry (ICS). Percentages of IL-17A+/IFN-γ+ double positive cells and of IL-17A-/IFN-g+ cells are shown. ( b ) Thawed IL-17A+ cells were cultured for 22 days and analyzed by ICS at days 12 and 22. Percentages of IL-17A+ cells (all: both IL-17A+/IFN-γ- and double positive cells) and IL-17A-/IFN-γ+ cells are shown. Results from two cows (C1 C2) are shown.
    Figure Legend Snippet: Maintenance of the IL-17A and IFN-γ phenotype of sorted and frozen-thawed cells upon subculture. ( a ) Sorted IL-17A+ cells stimulated with α-CD3/α-CD28 were cultured with IL-2 with or without the polarizing cytokines TGF-β1 and IL-6 for 9 days and analyzed by flow cytometry (ICS). Percentages of IL-17A+/IFN-γ+ double positive cells and of IL-17A-/IFN-g+ cells are shown. ( b ) Thawed IL-17A+ cells were cultured for 22 days and analyzed by ICS at days 12 and 22. Percentages of IL-17A+ cells (all: both IL-17A+/IFN-γ- and double positive cells) and IL-17A-/IFN-γ+ cells are shown. Results from two cows (C1 C2) are shown.

    Techniques Used: Cell Culture, Flow Cytometry

    Culture conditions for expansion of Th17 cells. ( a ) Number of cells after 6, 9 and 13 days of culture as a function of the concentration (µg/mL) of the coating antibodies to CD3 with or without 10 ng/ml recombinant human IL-2. Results are from a representative experiment. ( b ) Concentrations of IL-17A and IFN-γ under these culture conditions. ( c ) Proportion of cells producing IL-17A or IFN-γ (ICS) at day 6 of culture. Results are means from the cells of two cows. Percentages of IL-17A positive cells differed as a function of treatment (p = 0.034, one-way ANOVA), but not percentages of IFN-γ positive cells (p = 0.24, one-way ANOVA). Results with α-CD3 1 µg/mL are not shown because too few cells were available for staining.
    Figure Legend Snippet: Culture conditions for expansion of Th17 cells. ( a ) Number of cells after 6, 9 and 13 days of culture as a function of the concentration (µg/mL) of the coating antibodies to CD3 with or without 10 ng/ml recombinant human IL-2. Results are from a representative experiment. ( b ) Concentrations of IL-17A and IFN-γ under these culture conditions. ( c ) Proportion of cells producing IL-17A or IFN-γ (ICS) at day 6 of culture. Results are means from the cells of two cows. Percentages of IL-17A positive cells differed as a function of treatment (p = 0.034, one-way ANOVA), but not percentages of IFN-γ positive cells (p = 0.24, one-way ANOVA). Results with α-CD3 1 µg/mL are not shown because too few cells were available for staining.

    Techniques Used: Concentration Assay, Recombinant, Staining

    Efficiency of the monoclonal antibodies in the cytokine secretion assay. ( a ) Percentages of CD4+ T cells labelled by the capture complex with either the α-Cter or α-Nter monoclonals, and comparison with the percentages of IL-17A+ cells identified by ICS. Data from two cows (C1 C2) are shown, one per row. ( b ) Concentrations of IL-17A in culture supernatant before capture, at the end of the 3.5 h of stimulation. ( c ) Concentrations of IL-17A in culture supernatant at the end of the secretion step (after capture) in the presence of the complex capture with either one of the two monoclonal antibodies to IL-17A, an isotype control or the polyclonal antiserum to IL-17A, showing the efficiency of the capture of IL-17A as it is secreted. PI: PMA/ionomycin, PIB: PMA/ionomycin/brefeldin A. ICS: intracellular staining.
    Figure Legend Snippet: Efficiency of the monoclonal antibodies in the cytokine secretion assay. ( a ) Percentages of CD4+ T cells labelled by the capture complex with either the α-Cter or α-Nter monoclonals, and comparison with the percentages of IL-17A+ cells identified by ICS. Data from two cows (C1 C2) are shown, one per row. ( b ) Concentrations of IL-17A in culture supernatant before capture, at the end of the 3.5 h of stimulation. ( c ) Concentrations of IL-17A in culture supernatant at the end of the secretion step (after capture) in the presence of the complex capture with either one of the two monoclonal antibodies to IL-17A, an isotype control or the polyclonal antiserum to IL-17A, showing the efficiency of the capture of IL-17A as it is secreted. PI: PMA/ionomycin, PIB: PMA/ionomycin/brefeldin A. ICS: intracellular staining.

    Techniques Used: Staining

    Schematic representation of the procedure used for the isolation and expansion of Th17 cells. CD4+ cells were isolated from PBMC by magnetic sorting, expanded in a culture medium without serum in the presence of polarizing cytokines and TCR-stimulation, activated, labelled for surface IL-17A and sorted by flow cytometry. The sorted cells were expanded and maintained in culture for several weeks. The indicated timeline is kept in all the figures to make it clear at which step cells were assayed. Concentrations of cytokines were 10 ng/mL (IL-2), 40 ng/mL (IL-6) and 2 ng/mL (TGF-β1). D: day; CSA: cytokine secretion assay; CSS: cytokine surface staining. α-: antibody anti-.
    Figure Legend Snippet: Schematic representation of the procedure used for the isolation and expansion of Th17 cells. CD4+ cells were isolated from PBMC by magnetic sorting, expanded in a culture medium without serum in the presence of polarizing cytokines and TCR-stimulation, activated, labelled for surface IL-17A and sorted by flow cytometry. The sorted cells were expanded and maintained in culture for several weeks. The indicated timeline is kept in all the figures to make it clear at which step cells were assayed. Concentrations of cytokines were 10 ng/mL (IL-2), 40 ng/mL (IL-6) and 2 ng/mL (TGF-β1). D: day; CSA: cytokine secretion assay; CSS: cytokine surface staining. α-: antibody anti-.

    Techniques Used: Isolation, Flow Cytometry, Staining

    Schematic representation of the assays used for surface labelling of IL-17A secreting cells. In the cytokine secretion assay (CSA), after stimulation with PMA/ionomycin, the cells start to secrete cytokines for 3.5 h. Then the cells are allowed to bind the capture complex (Capture complex antibody staining) for 15 min before dilution and incubation for 1.5 h under agitation (New cytokine secretion step). The secreted cytokine is captured by antibodies to IL-17A that are maintained at the surface of the cell by antibodies to CD45. The capture complex comprises the two types of biotinylated antibodies linked by a streptavidin molecule. After washing, rabbit anti-bovine IL-17A antibodies are added that bind to the captured IL-17A (IL-17A detection labelling and revelation). Then the cells are washed and the binding of rabbit antibodies revealed with a secondary antibody conjugated to phycoerythrin. Alternatively, in the cytokine surface staining assay (CSS), after washing at the end of the 3.5 h stimulation, cells are incubated with rabbit antibodies to IL-17A, washed, and the binding of rabbit antibodies to surface-associated IL-17A is revealed with a secondary antibody conjugated to phycoerythrin.
    Figure Legend Snippet: Schematic representation of the assays used for surface labelling of IL-17A secreting cells. In the cytokine secretion assay (CSA), after stimulation with PMA/ionomycin, the cells start to secrete cytokines for 3.5 h. Then the cells are allowed to bind the capture complex (Capture complex antibody staining) for 15 min before dilution and incubation for 1.5 h under agitation (New cytokine secretion step). The secreted cytokine is captured by antibodies to IL-17A that are maintained at the surface of the cell by antibodies to CD45. The capture complex comprises the two types of biotinylated antibodies linked by a streptavidin molecule. After washing, rabbit anti-bovine IL-17A antibodies are added that bind to the captured IL-17A (IL-17A detection labelling and revelation). Then the cells are washed and the binding of rabbit antibodies revealed with a secondary antibody conjugated to phycoerythrin. Alternatively, in the cytokine surface staining assay (CSS), after washing at the end of the 3.5 h stimulation, cells are incubated with rabbit antibodies to IL-17A, washed, and the binding of rabbit antibodies to surface-associated IL-17A is revealed with a secondary antibody conjugated to phycoerythrin.

    Techniques Used: Staining, Incubation, Binding Assay

    12) Product Images from "Expansion, isolation and first characterization of bovine Th17 lymphocytes"

    Article Title: Expansion, isolation and first characterization of bovine Th17 lymphocytes

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-52562-2

    Choice of culture medium for the expansion of Th17 cells. ( a ) Cell growth after 3, 6, 8 or 13 days in RPMI (+FCS and 2 ng/mL TGF-β1) or IMDM (+10% KnockOut Serum Replacement) with different concentrations of recombinant human TGF-β1. ( b ) Proportions of IL-17A, IFN-γ and double positive cells after 6 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( c ) Proportions of IL-17A+ cells after 6 and 13 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( d ) Comparison of cell growth in RPMI and X-VIVO™ 15 media with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1), after 3 and 6 days of culture. ( e ) Proportions of cells IL-17A+ and IFN-γ+ (ICS) with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1) after 6 days of culture. Results are means from two cows.
    Figure Legend Snippet: Choice of culture medium for the expansion of Th17 cells. ( a ) Cell growth after 3, 6, 8 or 13 days in RPMI (+FCS and 2 ng/mL TGF-β1) or IMDM (+10% KnockOut Serum Replacement) with different concentrations of recombinant human TGF-β1. ( b ) Proportions of IL-17A, IFN-γ and double positive cells after 6 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( c ) Proportions of IL-17A+ cells after 6 and 13 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( d ) Comparison of cell growth in RPMI and X-VIVO™ 15 media with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1), after 3 and 6 days of culture. ( e ) Proportions of cells IL-17A+ and IFN-γ+ (ICS) with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1) after 6 days of culture. Results are means from two cows.

    Techniques Used: Knock-Out, Recombinant, Concentration Assay

    Identification of IL-17A and IFN-γ producing bovine lymphocytes. PBMC were stimulated with PMA/ionomycin, cytokine secretion blocked with Brefeldin A before flow cytometry analysis. Debris were excluded by gating according to FSC/SSC, and after gating on singlet cells, dead cells were excluded by gating on live cells. The CD4+ cells were gated by taking into account the isotype control, and the production of IL-17A and IFN-γ was measured by intracellular labelling with specific antibodies. Results shown are from a representative experiment. FSC: forward scatter: SSC: side scatter.
    Figure Legend Snippet: Identification of IL-17A and IFN-γ producing bovine lymphocytes. PBMC were stimulated with PMA/ionomycin, cytokine secretion blocked with Brefeldin A before flow cytometry analysis. Debris were excluded by gating according to FSC/SSC, and after gating on singlet cells, dead cells were excluded by gating on live cells. The CD4+ cells were gated by taking into account the isotype control, and the production of IL-17A and IFN-γ was measured by intracellular labelling with specific antibodies. Results shown are from a representative experiment. FSC: forward scatter: SSC: side scatter.

    Techniques Used: Flow Cytometry

    Unambiguous identification of bovine Th17. ( a ) Secretion of cytokines by sorted Th17 cells. After sorting, the cells were stimulated with α-CD3 and α-CD28 and cultured for 6 days. Supernatant contents were analysed by ELISA. Median values (10 to 90 percentiles) from five cell preparations from three cows are shown. ( b ) Comparison of Th17 signature gene expression between IL-17A+ and IL-17A- cells. Heatmap shows expression intensity expressed as log2(fold-change) normalized relative to three reference genes (ACTB, PPIA and GAPDH). Expression is relative to the CD4+ subset of the corresponding cow at day 6 after positive selection using MACS® beads. Cells were cultured in X-VIVO™ 15 medium without polarizing cytokines for 6 days after an initial TCR stimulation and a partial (half) renewal of medium with addition of IL-2 on the third day. Results are from three cows (C1, C2, C3).
    Figure Legend Snippet: Unambiguous identification of bovine Th17. ( a ) Secretion of cytokines by sorted Th17 cells. After sorting, the cells were stimulated with α-CD3 and α-CD28 and cultured for 6 days. Supernatant contents were analysed by ELISA. Median values (10 to 90 percentiles) from five cell preparations from three cows are shown. ( b ) Comparison of Th17 signature gene expression between IL-17A+ and IL-17A- cells. Heatmap shows expression intensity expressed as log2(fold-change) normalized relative to three reference genes (ACTB, PPIA and GAPDH). Expression is relative to the CD4+ subset of the corresponding cow at day 6 after positive selection using MACS® beads. Cells were cultured in X-VIVO™ 15 medium without polarizing cytokines for 6 days after an initial TCR stimulation and a partial (half) renewal of medium with addition of IL-2 on the third day. Results are from three cows (C1, C2, C3).

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Selection, Magnetic Cell Separation

    Comparison of the Cytokine Secretion Assay with the cytokine surface Staining. ( a ) Gating strategy. At the end of the culture expansion step, the polarized cell populations were split and analyzed side-by-side by flow cytometry with Cytokine Secretion (left) and surface staining (right) assays for IL-17A secretion. The squares indicate the IL-17A+ cells sorting windows, the circles the IL-17A- sorting windows. ( b ) Proportions of cytokine-positive cells 8 days after sorting. Median values from two sorting experiments with cells of two cows are shown. ( c ) Numbers of IL-17A+ and IL-17A- cells at different times after sorting. Stars indicate stimulations with anti-CD3/CD28 antibodies, arrows the sampling of cells for freezing or RNA preparation. Values are from one sorting experiment with two cows. ( d ) Proportions of sorted IL-17A+ cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. Values are from one sorting experiment with two cows. e) Proportions of sorted IL-17A- cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. CSA: cytokine secretion assay; CSS: cytokine surface staining.
    Figure Legend Snippet: Comparison of the Cytokine Secretion Assay with the cytokine surface Staining. ( a ) Gating strategy. At the end of the culture expansion step, the polarized cell populations were split and analyzed side-by-side by flow cytometry with Cytokine Secretion (left) and surface staining (right) assays for IL-17A secretion. The squares indicate the IL-17A+ cells sorting windows, the circles the IL-17A- sorting windows. ( b ) Proportions of cytokine-positive cells 8 days after sorting. Median values from two sorting experiments with cells of two cows are shown. ( c ) Numbers of IL-17A+ and IL-17A- cells at different times after sorting. Stars indicate stimulations with anti-CD3/CD28 antibodies, arrows the sampling of cells for freezing or RNA preparation. Values are from one sorting experiment with two cows. ( d ) Proportions of sorted IL-17A+ cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. Values are from one sorting experiment with two cows. e) Proportions of sorted IL-17A- cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. CSA: cytokine secretion assay; CSS: cytokine surface staining.

    Techniques Used: Staining, Flow Cytometry, Sampling

    Maintenance of the IL-17A and IFN-γ phenotype of sorted and frozen-thawed cells upon subculture. ( a ) Sorted IL-17A+ cells stimulated with α-CD3/α-CD28 were cultured with IL-2 with or without the polarizing cytokines TGF-β1 and IL-6 for 9 days and analyzed by flow cytometry (ICS). Percentages of IL-17A+/IFN-γ+ double positive cells and of IL-17A-/IFN-g+ cells are shown. ( b ) Thawed IL-17A+ cells were cultured for 22 days and analyzed by ICS at days 12 and 22. Percentages of IL-17A+ cells (all: both IL-17A+/IFN-γ- and double positive cells) and IL-17A-/IFN-γ+ cells are shown. Results from two cows (C1 C2) are shown.
    Figure Legend Snippet: Maintenance of the IL-17A and IFN-γ phenotype of sorted and frozen-thawed cells upon subculture. ( a ) Sorted IL-17A+ cells stimulated with α-CD3/α-CD28 were cultured with IL-2 with or without the polarizing cytokines TGF-β1 and IL-6 for 9 days and analyzed by flow cytometry (ICS). Percentages of IL-17A+/IFN-γ+ double positive cells and of IL-17A-/IFN-g+ cells are shown. ( b ) Thawed IL-17A+ cells were cultured for 22 days and analyzed by ICS at days 12 and 22. Percentages of IL-17A+ cells (all: both IL-17A+/IFN-γ- and double positive cells) and IL-17A-/IFN-γ+ cells are shown. Results from two cows (C1 C2) are shown.

    Techniques Used: Cell Culture, Flow Cytometry

    Culture conditions for expansion of Th17 cells. ( a ) Number of cells after 6, 9 and 13 days of culture as a function of the concentration (µg/mL) of the coating antibodies to CD3 with or without 10 ng/ml recombinant human IL-2. Results are from a representative experiment. ( b ) Concentrations of IL-17A and IFN-γ under these culture conditions. ( c ) Proportion of cells producing IL-17A or IFN-γ (ICS) at day 6 of culture. Results are means from the cells of two cows. Percentages of IL-17A positive cells differed as a function of treatment (p = 0.034, one-way ANOVA), but not percentages of IFN-γ positive cells (p = 0.24, one-way ANOVA). Results with α-CD3 1 µg/mL are not shown because too few cells were available for staining.
    Figure Legend Snippet: Culture conditions for expansion of Th17 cells. ( a ) Number of cells after 6, 9 and 13 days of culture as a function of the concentration (µg/mL) of the coating antibodies to CD3 with or without 10 ng/ml recombinant human IL-2. Results are from a representative experiment. ( b ) Concentrations of IL-17A and IFN-γ under these culture conditions. ( c ) Proportion of cells producing IL-17A or IFN-γ (ICS) at day 6 of culture. Results are means from the cells of two cows. Percentages of IL-17A positive cells differed as a function of treatment (p = 0.034, one-way ANOVA), but not percentages of IFN-γ positive cells (p = 0.24, one-way ANOVA). Results with α-CD3 1 µg/mL are not shown because too few cells were available for staining.

    Techniques Used: Concentration Assay, Recombinant, Staining

    Efficiency of the monoclonal antibodies in the cytokine secretion assay. ( a ) Percentages of CD4+ T cells labelled by the capture complex with either the α-Cter or α-Nter monoclonals, and comparison with the percentages of IL-17A+ cells identified by ICS. Data from two cows (C1 C2) are shown, one per row. ( b ) Concentrations of IL-17A in culture supernatant before capture, at the end of the 3.5 h of stimulation. ( c ) Concentrations of IL-17A in culture supernatant at the end of the secretion step (after capture) in the presence of the complex capture with either one of the two monoclonal antibodies to IL-17A, an isotype control or the polyclonal antiserum to IL-17A, showing the efficiency of the capture of IL-17A as it is secreted. PI: PMA/ionomycin, PIB: PMA/ionomycin/brefeldin A. ICS: intracellular staining.
    Figure Legend Snippet: Efficiency of the monoclonal antibodies in the cytokine secretion assay. ( a ) Percentages of CD4+ T cells labelled by the capture complex with either the α-Cter or α-Nter monoclonals, and comparison with the percentages of IL-17A+ cells identified by ICS. Data from two cows (C1 C2) are shown, one per row. ( b ) Concentrations of IL-17A in culture supernatant before capture, at the end of the 3.5 h of stimulation. ( c ) Concentrations of IL-17A in culture supernatant at the end of the secretion step (after capture) in the presence of the complex capture with either one of the two monoclonal antibodies to IL-17A, an isotype control or the polyclonal antiserum to IL-17A, showing the efficiency of the capture of IL-17A as it is secreted. PI: PMA/ionomycin, PIB: PMA/ionomycin/brefeldin A. ICS: intracellular staining.

    Techniques Used: Staining

    Schematic representation of the procedure used for the isolation and expansion of Th17 cells. CD4+ cells were isolated from PBMC by magnetic sorting, expanded in a culture medium without serum in the presence of polarizing cytokines and TCR-stimulation, activated, labelled for surface IL-17A and sorted by flow cytometry. The sorted cells were expanded and maintained in culture for several weeks. The indicated timeline is kept in all the figures to make it clear at which step cells were assayed. Concentrations of cytokines were 10 ng/mL (IL-2), 40 ng/mL (IL-6) and 2 ng/mL (TGF-β1). D: day; CSA: cytokine secretion assay; CSS: cytokine surface staining. α-: antibody anti-.
    Figure Legend Snippet: Schematic representation of the procedure used for the isolation and expansion of Th17 cells. CD4+ cells were isolated from PBMC by magnetic sorting, expanded in a culture medium without serum in the presence of polarizing cytokines and TCR-stimulation, activated, labelled for surface IL-17A and sorted by flow cytometry. The sorted cells were expanded and maintained in culture for several weeks. The indicated timeline is kept in all the figures to make it clear at which step cells were assayed. Concentrations of cytokines were 10 ng/mL (IL-2), 40 ng/mL (IL-6) and 2 ng/mL (TGF-β1). D: day; CSA: cytokine secretion assay; CSS: cytokine surface staining. α-: antibody anti-.

    Techniques Used: Isolation, Flow Cytometry, Staining

    Schematic representation of the assays used for surface labelling of IL-17A secreting cells. In the cytokine secretion assay (CSA), after stimulation with PMA/ionomycin, the cells start to secrete cytokines for 3.5 h. Then the cells are allowed to bind the capture complex (Capture complex antibody staining) for 15 min before dilution and incubation for 1.5 h under agitation (New cytokine secretion step). The secreted cytokine is captured by antibodies to IL-17A that are maintained at the surface of the cell by antibodies to CD45. The capture complex comprises the two types of biotinylated antibodies linked by a streptavidin molecule. After washing, rabbit anti-bovine IL-17A antibodies are added that bind to the captured IL-17A (IL-17A detection labelling and revelation). Then the cells are washed and the binding of rabbit antibodies revealed with a secondary antibody conjugated to phycoerythrin. Alternatively, in the cytokine surface staining assay (CSS), after washing at the end of the 3.5 h stimulation, cells are incubated with rabbit antibodies to IL-17A, washed, and the binding of rabbit antibodies to surface-associated IL-17A is revealed with a secondary antibody conjugated to phycoerythrin.
    Figure Legend Snippet: Schematic representation of the assays used for surface labelling of IL-17A secreting cells. In the cytokine secretion assay (CSA), after stimulation with PMA/ionomycin, the cells start to secrete cytokines for 3.5 h. Then the cells are allowed to bind the capture complex (Capture complex antibody staining) for 15 min before dilution and incubation for 1.5 h under agitation (New cytokine secretion step). The secreted cytokine is captured by antibodies to IL-17A that are maintained at the surface of the cell by antibodies to CD45. The capture complex comprises the two types of biotinylated antibodies linked by a streptavidin molecule. After washing, rabbit anti-bovine IL-17A antibodies are added that bind to the captured IL-17A (IL-17A detection labelling and revelation). Then the cells are washed and the binding of rabbit antibodies revealed with a secondary antibody conjugated to phycoerythrin. Alternatively, in the cytokine surface staining assay (CSS), after washing at the end of the 3.5 h stimulation, cells are incubated with rabbit antibodies to IL-17A, washed, and the binding of rabbit antibodies to surface-associated IL-17A is revealed with a secondary antibody conjugated to phycoerythrin.

    Techniques Used: Staining, Incubation, Binding Assay

    13) Product Images from "Efficacy of mucosal polyanhydride nanovaccine against respiratory syncytial virus infection in the neonatal calf"

    Article Title: Efficacy of mucosal polyanhydride nanovaccine against respiratory syncytial virus infection in the neonatal calf

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-21292-2

    Enhanced BRSV-specific T cell responses in the peripheral blood and BAL of BRSV-F/G nanovaccine-administered calves. PBMC were collected on day 6 post-challenge, labeled with Cell Trace Violet and stimulated for 6 days with 5 μg/mL of the recombinant BRSV F and G proteins, or 0.01 MOI of BRSV strain 375. Pokeweed Mitogen was used at a concentration of 1 μg/mL as a positive control. Mock stimulated samples (negative control wells) were cultured with cRPMI and were used to correct for background proliferation. ( A ) After 6 days, antigen-specific CD4 T cell proliferation was assessed by flow cytometry, as measured by dilution of the Cell Trace Violet dye. Background levels of proliferation were subtracted and results are presented as change over mock. ( B ) Stimulated cell culture supernatants were collected after 6 days and concentrations of IFNγ (left panel) and IL-17A (right panel) were measured by commercial sandwich ELISAs. ( C ) BALs were performed on day 7 post-challenge. Cells were enumerated and stimulated for 6 days with recombinant BRSV F protein, G protein or BRSV as in A. After 6 days, cell culture supernatants were collected and concentrations of IFNγ (left panel) and IL-17A (right panel) were measured by commercial sandwich ELISAs. Results represent n = 6 animals/group. Data represent means ± SEM. *p
    Figure Legend Snippet: Enhanced BRSV-specific T cell responses in the peripheral blood and BAL of BRSV-F/G nanovaccine-administered calves. PBMC were collected on day 6 post-challenge, labeled with Cell Trace Violet and stimulated for 6 days with 5 μg/mL of the recombinant BRSV F and G proteins, or 0.01 MOI of BRSV strain 375. Pokeweed Mitogen was used at a concentration of 1 μg/mL as a positive control. Mock stimulated samples (negative control wells) were cultured with cRPMI and were used to correct for background proliferation. ( A ) After 6 days, antigen-specific CD4 T cell proliferation was assessed by flow cytometry, as measured by dilution of the Cell Trace Violet dye. Background levels of proliferation were subtracted and results are presented as change over mock. ( B ) Stimulated cell culture supernatants were collected after 6 days and concentrations of IFNγ (left panel) and IL-17A (right panel) were measured by commercial sandwich ELISAs. ( C ) BALs were performed on day 7 post-challenge. Cells were enumerated and stimulated for 6 days with recombinant BRSV F protein, G protein or BRSV as in A. After 6 days, cell culture supernatants were collected and concentrations of IFNγ (left panel) and IL-17A (right panel) were measured by commercial sandwich ELISAs. Results represent n = 6 animals/group. Data represent means ± SEM. *p

    Techniques Used: Labeling, Recombinant, Concentration Assay, Positive Control, Negative Control, Cell Culture, Flow Cytometry, Cytometry

    14) 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 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:

    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:

    15) Product Images from "Antigen-Specific Mammary Inflammation Depends on the Production of IL-17A and IFN-γ by Bovine CD4+ T Lymphocytes"

    Article Title: Antigen-Specific Mammary Inflammation Depends on the Production of IL-17A and IFN-γ by Bovine CD4+ T Lymphocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0137755

    Antigen-specific whole blood assay. ( A) Time-course of IL-17A and IFN-γ production by whole blood cultured with OVA. The blood of the 10 responder cows (taken 45 days after the first immunization) was cultured in the presence of OVA (10 μg/mL) for 1 to 4 days in 96-well microplates. For each cow triplicate wells were used for each incubation time. Results are median values and quartiles (Q1; Q3). (B) The effect of magnetic depletion of CD4+ cells on the production of IL-17A and IFN-γ in the antigen-specific whole blood assay. Results obtained by stimulating blood samples from responder cows 15 days after the booster immunization are expressed as the percentage of the production by CD4+-depleted PBMC relative to cytokine production by un processed PBMC (100%). The viability of cells was not altered by the magnetic cell separation.
    Figure Legend Snippet: Antigen-specific whole blood assay. ( A) Time-course of IL-17A and IFN-γ production by whole blood cultured with OVA. The blood of the 10 responder cows (taken 45 days after the first immunization) was cultured in the presence of OVA (10 μg/mL) for 1 to 4 days in 96-well microplates. For each cow triplicate wells were used for each incubation time. Results are median values and quartiles (Q1; Q3). (B) The effect of magnetic depletion of CD4+ cells on the production of IL-17A and IFN-γ in the antigen-specific whole blood assay. Results obtained by stimulating blood samples from responder cows 15 days after the booster immunization are expressed as the percentage of the production by CD4+-depleted PBMC relative to cytokine production by un processed PBMC (100%). The viability of cells was not altered by the magnetic cell separation.

    Techniques Used: Whole Blood Assay, Cell Culture, Incubation, Magnetic Cell Separation

    Persistence of reactivity to ovalbumin. Concentrations of IL-17A and IFN-γ yielded by the whole blood assay performed at different times after immunization with ovalbumin. Results are the median values (and interquartiles) from the 8 responder cows still available 10 months post-immunization.
    Figure Legend Snippet: Persistence of reactivity to ovalbumin. Concentrations of IL-17A and IFN-γ yielded by the whole blood assay performed at different times after immunization with ovalbumin. Results are the median values (and interquartiles) from the 8 responder cows still available 10 months post-immunization.

    Techniques Used: Whole Blood Assay

    Intracellular expression of IL-17A and IFN-γ by CD4+ T lymphocytes. PBMC were isolated one month after ovalbumin booster injection, stimulated in vitro with ovalbumin for 3 days, rested for 2 days and finally stimulated with PMA/ionomycin for 5 h with Brefeldin A for the last 3 hours. Cells were labeled for surface CD4 and intracellular IL-17A and IFN-γ. The numbers in the plots indicate the percentages of labeled cells in comparison to the isotype control. (A) Production of viable CD4+ T lymphoblasts after culture of PBMC with (OVA) or without (NS) ovalbumin. Left panels depict PBMC from a responder cow, right panels PBMC from a low-responder. (B) PBMC from two responder cows (R1 R2) were labeled for surface CD4 and intracellular IL-17A or IFN-γ, showing CD4+ and CD4- IL-17A- and IFN-γ-producing cells. (C) labeling of CD4+ cells with anti-IL-17A and anti-IFN-γ antibodies, showing single-producing and double-producing cells. D) Double labeling of CD4+ cells from two low-responders (R3 and R4). Percentages of labeled cells are indicated in the quadrants. Results are from a representative experiment.
    Figure Legend Snippet: Intracellular expression of IL-17A and IFN-γ by CD4+ T lymphocytes. PBMC were isolated one month after ovalbumin booster injection, stimulated in vitro with ovalbumin for 3 days, rested for 2 days and finally stimulated with PMA/ionomycin for 5 h with Brefeldin A for the last 3 hours. Cells were labeled for surface CD4 and intracellular IL-17A and IFN-γ. The numbers in the plots indicate the percentages of labeled cells in comparison to the isotype control. (A) Production of viable CD4+ T lymphoblasts after culture of PBMC with (OVA) or without (NS) ovalbumin. Left panels depict PBMC from a responder cow, right panels PBMC from a low-responder. (B) PBMC from two responder cows (R1 R2) were labeled for surface CD4 and intracellular IL-17A or IFN-γ, showing CD4+ and CD4- IL-17A- and IFN-γ-producing cells. (C) labeling of CD4+ cells with anti-IL-17A and anti-IFN-γ antibodies, showing single-producing and double-producing cells. D) Double labeling of CD4+ cells from two low-responders (R3 and R4). Percentages of labeled cells are indicated in the quadrants. Results are from a representative experiment.

    Techniques Used: Expressing, Isolation, Injection, In Vitro, Labeling

    Concentrations of cytokines in milk samples of the 10 responder cows. Time-course of concentration variation (median and interquartiles) of CXCL8 (A), IL-17A (B) and IFN- γ (C) in the milk of quarters infused with ovalbumin at 0 hpi.
    Figure Legend Snippet: Concentrations of cytokines in milk samples of the 10 responder cows. Time-course of concentration variation (median and interquartiles) of CXCL8 (A), IL-17A (B) and IFN- γ (C) in the milk of quarters infused with ovalbumin at 0 hpi.

    Techniques Used: Concentration Assay

    Time-course of the IL-17A and IFN-γ production in the antigen-specific whole blood assay following immunization and correlation with Peak SCC. Concentrations of IL-17A (A) or IFN-γ (B) after 3 days of culture with ovalbumin of blood samples taken before and after immunization at days 0 and 30 (median values and interquartiles) distinguishing the antigen-specific responses of responders and low-responder cows to the intramammary antigenic challenge. (B and C) Correlations (Spearman’s rank test) between peak SCC and IL-17A concentrations or IFN-γ concentrations yielded by the whole blood assay performed 45 days after the first immunization.
    Figure Legend Snippet: Time-course of the IL-17A and IFN-γ production in the antigen-specific whole blood assay following immunization and correlation with Peak SCC. Concentrations of IL-17A (A) or IFN-γ (B) after 3 days of culture with ovalbumin of blood samples taken before and after immunization at days 0 and 30 (median values and interquartiles) distinguishing the antigen-specific responses of responders and low-responder cows to the intramammary antigenic challenge. (B and C) Correlations (Spearman’s rank test) between peak SCC and IL-17A concentrations or IFN-γ concentrations yielded by the whole blood assay performed 45 days after the first immunization.

    Techniques Used: Whole Blood Assay

    16) 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

    17) 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

    18) 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

    19) 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

    20) Product Images from "Expansion, isolation and first characterization of bovine Th17 lymphocytes"

    Article Title: Expansion, isolation and first characterization of bovine Th17 lymphocytes

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-52562-2

    Choice of culture medium for the expansion of Th17 cells. ( a ) Cell growth after 3, 6, 8 or 13 days in RPMI (+FCS and 2 ng/mL TGF-β1) or IMDM (+10% KnockOut Serum Replacement) with different concentrations of recombinant human TGF-β1. ( b ) Proportions of IL-17A, IFN-γ and double positive cells after 6 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( c ) Proportions of IL-17A+ cells after 6 and 13 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( d ) Comparison of cell growth in RPMI and X-VIVO™ 15 media with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1), after 3 and 6 days of culture. ( e ) Proportions of cells IL-17A+ and IFN-γ+ (ICS) with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1) after 6 days of culture. Results are means from two cows.
    Figure Legend Snippet: Choice of culture medium for the expansion of Th17 cells. ( a ) Cell growth after 3, 6, 8 or 13 days in RPMI (+FCS and 2 ng/mL TGF-β1) or IMDM (+10% KnockOut Serum Replacement) with different concentrations of recombinant human TGF-β1. ( b ) Proportions of IL-17A, IFN-γ and double positive cells after 6 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( c ) Proportions of IL-17A+ cells after 6 and 13 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( d ) Comparison of cell growth in RPMI and X-VIVO™ 15 media with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1), after 3 and 6 days of culture. ( e ) Proportions of cells IL-17A+ and IFN-γ+ (ICS) with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1) after 6 days of culture. Results are means from two cows.

    Techniques Used: Knock-Out, Recombinant, Concentration Assay

    Identification of IL-17A and IFN-γ producing bovine lymphocytes. PBMC were stimulated with PMA/ionomycin, cytokine secretion blocked with Brefeldin A before flow cytometry analysis. Debris were excluded by gating according to FSC/SSC, and after gating on singlet cells, dead cells were excluded by gating on live cells. The CD4+ cells were gated by taking into account the isotype control, and the production of IL-17A and IFN-γ was measured by intracellular labelling with specific antibodies. Results shown are from a representative experiment. FSC: forward scatter: SSC: side scatter.
    Figure Legend Snippet: Identification of IL-17A and IFN-γ producing bovine lymphocytes. PBMC were stimulated with PMA/ionomycin, cytokine secretion blocked with Brefeldin A before flow cytometry analysis. Debris were excluded by gating according to FSC/SSC, and after gating on singlet cells, dead cells were excluded by gating on live cells. The CD4+ cells were gated by taking into account the isotype control, and the production of IL-17A and IFN-γ was measured by intracellular labelling with specific antibodies. Results shown are from a representative experiment. FSC: forward scatter: SSC: side scatter.

    Techniques Used: Flow Cytometry

    Unambiguous identification of bovine Th17. ( a ) Secretion of cytokines by sorted Th17 cells. After sorting, the cells were stimulated with α-CD3 and α-CD28 and cultured for 6 days. Supernatant contents were analysed by ELISA. Median values (10 to 90 percentiles) from five cell preparations from three cows are shown. ( b ) Comparison of Th17 signature gene expression between IL-17A+ and IL-17A- cells. Heatmap shows expression intensity expressed as log2(fold-change) normalized relative to three reference genes (ACTB, PPIA and GAPDH). Expression is relative to the CD4+ subset of the corresponding cow at day 6 after positive selection using MACS® beads. Cells were cultured in X-VIVO™ 15 medium without polarizing cytokines for 6 days after an initial TCR stimulation and a partial (half) renewal of medium with addition of IL-2 on the third day. Results are from three cows (C1, C2, C3).
    Figure Legend Snippet: Unambiguous identification of bovine Th17. ( a ) Secretion of cytokines by sorted Th17 cells. After sorting, the cells were stimulated with α-CD3 and α-CD28 and cultured for 6 days. Supernatant contents were analysed by ELISA. Median values (10 to 90 percentiles) from five cell preparations from three cows are shown. ( b ) Comparison of Th17 signature gene expression between IL-17A+ and IL-17A- cells. Heatmap shows expression intensity expressed as log2(fold-change) normalized relative to three reference genes (ACTB, PPIA and GAPDH). Expression is relative to the CD4+ subset of the corresponding cow at day 6 after positive selection using MACS® beads. Cells were cultured in X-VIVO™ 15 medium without polarizing cytokines for 6 days after an initial TCR stimulation and a partial (half) renewal of medium with addition of IL-2 on the third day. Results are from three cows (C1, C2, C3).

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Selection, Magnetic Cell Separation

    Comparison of the Cytokine Secretion Assay with the cytokine surface Staining. ( a ) Gating strategy. At the end of the culture expansion step, the polarized cell populations were split and analyzed side-by-side by flow cytometry with Cytokine Secretion (left) and surface staining (right) assays for IL-17A secretion. The squares indicate the IL-17A+ cells sorting windows, the circles the IL-17A- sorting windows. ( b ) Proportions of cytokine-positive cells 8 days after sorting. Median values from two sorting experiments with cells of two cows are shown. ( c ) Numbers of IL-17A+ and IL-17A- cells at different times after sorting. Stars indicate stimulations with anti-CD3/CD28 antibodies, arrows the sampling of cells for freezing or RNA preparation. Values are from one sorting experiment with two cows. ( d ) Proportions of sorted IL-17A+ cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. Values are from one sorting experiment with two cows. e) Proportions of sorted IL-17A- cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. CSA: cytokine secretion assay; CSS: cytokine surface staining.
    Figure Legend Snippet: Comparison of the Cytokine Secretion Assay with the cytokine surface Staining. ( a ) Gating strategy. At the end of the culture expansion step, the polarized cell populations were split and analyzed side-by-side by flow cytometry with Cytokine Secretion (left) and surface staining (right) assays for IL-17A secretion. The squares indicate the IL-17A+ cells sorting windows, the circles the IL-17A- sorting windows. ( b ) Proportions of cytokine-positive cells 8 days after sorting. Median values from two sorting experiments with cells of two cows are shown. ( c ) Numbers of IL-17A+ and IL-17A- cells at different times after sorting. Stars indicate stimulations with anti-CD3/CD28 antibodies, arrows the sampling of cells for freezing or RNA preparation. Values are from one sorting experiment with two cows. ( d ) Proportions of sorted IL-17A+ cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. Values are from one sorting experiment with two cows. e) Proportions of sorted IL-17A- cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. CSA: cytokine secretion assay; CSS: cytokine surface staining.

    Techniques Used: Staining, Flow Cytometry, Sampling

    Maintenance of the IL-17A and IFN-γ phenotype of sorted and frozen-thawed cells upon subculture. ( a ) Sorted IL-17A+ cells stimulated with α-CD3/α-CD28 were cultured with IL-2 with or without the polarizing cytokines TGF-β1 and IL-6 for 9 days and analyzed by flow cytometry (ICS). Percentages of IL-17A+/IFN-γ+ double positive cells and of IL-17A-/IFN-g+ cells are shown. ( b ) Thawed IL-17A+ cells were cultured for 22 days and analyzed by ICS at days 12 and 22. Percentages of IL-17A+ cells (all: both IL-17A+/IFN-γ- and double positive cells) and IL-17A-/IFN-γ+ cells are shown. Results from two cows (C1 C2) are shown.
    Figure Legend Snippet: Maintenance of the IL-17A and IFN-γ phenotype of sorted and frozen-thawed cells upon subculture. ( a ) Sorted IL-17A+ cells stimulated with α-CD3/α-CD28 were cultured with IL-2 with or without the polarizing cytokines TGF-β1 and IL-6 for 9 days and analyzed by flow cytometry (ICS). Percentages of IL-17A+/IFN-γ+ double positive cells and of IL-17A-/IFN-g+ cells are shown. ( b ) Thawed IL-17A+ cells were cultured for 22 days and analyzed by ICS at days 12 and 22. Percentages of IL-17A+ cells (all: both IL-17A+/IFN-γ- and double positive cells) and IL-17A-/IFN-γ+ cells are shown. Results from two cows (C1 C2) are shown.

    Techniques Used: Cell Culture, Flow Cytometry

    Culture conditions for expansion of Th17 cells. ( a ) Number of cells after 6, 9 and 13 days of culture as a function of the concentration (µg/mL) of the coating antibodies to CD3 with or without 10 ng/ml recombinant human IL-2. Results are from a representative experiment. ( b ) Concentrations of IL-17A and IFN-γ under these culture conditions. ( c ) Proportion of cells producing IL-17A or IFN-γ (ICS) at day 6 of culture. Results are means from the cells of two cows. Percentages of IL-17A positive cells differed as a function of treatment (p = 0.034, one-way ANOVA), but not percentages of IFN-γ positive cells (p = 0.24, one-way ANOVA). Results with α-CD3 1 µg/mL are not shown because too few cells were available for staining.
    Figure Legend Snippet: Culture conditions for expansion of Th17 cells. ( a ) Number of cells after 6, 9 and 13 days of culture as a function of the concentration (µg/mL) of the coating antibodies to CD3 with or without 10 ng/ml recombinant human IL-2. Results are from a representative experiment. ( b ) Concentrations of IL-17A and IFN-γ under these culture conditions. ( c ) Proportion of cells producing IL-17A or IFN-γ (ICS) at day 6 of culture. Results are means from the cells of two cows. Percentages of IL-17A positive cells differed as a function of treatment (p = 0.034, one-way ANOVA), but not percentages of IFN-γ positive cells (p = 0.24, one-way ANOVA). Results with α-CD3 1 µg/mL are not shown because too few cells were available for staining.

    Techniques Used: Concentration Assay, Recombinant, Staining

    Efficiency of the monoclonal antibodies in the cytokine secretion assay. ( a ) Percentages of CD4+ T cells labelled by the capture complex with either the α-Cter or α-Nter monoclonals, and comparison with the percentages of IL-17A+ cells identified by ICS. Data from two cows (C1 C2) are shown, one per row. ( b ) Concentrations of IL-17A in culture supernatant before capture, at the end of the 3.5 h of stimulation. ( c ) Concentrations of IL-17A in culture supernatant at the end of the secretion step (after capture) in the presence of the complex capture with either one of the two monoclonal antibodies to IL-17A, an isotype control or the polyclonal antiserum to IL-17A, showing the efficiency of the capture of IL-17A as it is secreted. PI: PMA/ionomycin, PIB: PMA/ionomycin/brefeldin A. ICS: intracellular staining.
    Figure Legend Snippet: Efficiency of the monoclonal antibodies in the cytokine secretion assay. ( a ) Percentages of CD4+ T cells labelled by the capture complex with either the α-Cter or α-Nter monoclonals, and comparison with the percentages of IL-17A+ cells identified by ICS. Data from two cows (C1 C2) are shown, one per row. ( b ) Concentrations of IL-17A in culture supernatant before capture, at the end of the 3.5 h of stimulation. ( c ) Concentrations of IL-17A in culture supernatant at the end of the secretion step (after capture) in the presence of the complex capture with either one of the two monoclonal antibodies to IL-17A, an isotype control or the polyclonal antiserum to IL-17A, showing the efficiency of the capture of IL-17A as it is secreted. PI: PMA/ionomycin, PIB: PMA/ionomycin/brefeldin A. ICS: intracellular staining.

    Techniques Used: Staining

    Schematic representation of the procedure used for the isolation and expansion of Th17 cells. CD4+ cells were isolated from PBMC by magnetic sorting, expanded in a culture medium without serum in the presence of polarizing cytokines and TCR-stimulation, activated, labelled for surface IL-17A and sorted by flow cytometry. The sorted cells were expanded and maintained in culture for several weeks. The indicated timeline is kept in all the figures to make it clear at which step cells were assayed. Concentrations of cytokines were 10 ng/mL (IL-2), 40 ng/mL (IL-6) and 2 ng/mL (TGF-β1). D: day; CSA: cytokine secretion assay; CSS: cytokine surface staining. α-: antibody anti-.
    Figure Legend Snippet: Schematic representation of the procedure used for the isolation and expansion of Th17 cells. CD4+ cells were isolated from PBMC by magnetic sorting, expanded in a culture medium without serum in the presence of polarizing cytokines and TCR-stimulation, activated, labelled for surface IL-17A and sorted by flow cytometry. The sorted cells were expanded and maintained in culture for several weeks. The indicated timeline is kept in all the figures to make it clear at which step cells were assayed. Concentrations of cytokines were 10 ng/mL (IL-2), 40 ng/mL (IL-6) and 2 ng/mL (TGF-β1). D: day; CSA: cytokine secretion assay; CSS: cytokine surface staining. α-: antibody anti-.

    Techniques Used: Isolation, Flow Cytometry, Staining

    Schematic representation of the assays used for surface labelling of IL-17A secreting cells. In the cytokine secretion assay (CSA), after stimulation with PMA/ionomycin, the cells start to secrete cytokines for 3.5 h. Then the cells are allowed to bind the capture complex (Capture complex antibody staining) for 15 min before dilution and incubation for 1.5 h under agitation (New cytokine secretion step). The secreted cytokine is captured by antibodies to IL-17A that are maintained at the surface of the cell by antibodies to CD45. The capture complex comprises the two types of biotinylated antibodies linked by a streptavidin molecule. After washing, rabbit anti-bovine IL-17A antibodies are added that bind to the captured IL-17A (IL-17A detection labelling and revelation). Then the cells are washed and the binding of rabbit antibodies revealed with a secondary antibody conjugated to phycoerythrin. Alternatively, in the cytokine surface staining assay (CSS), after washing at the end of the 3.5 h stimulation, cells are incubated with rabbit antibodies to IL-17A, washed, and the binding of rabbit antibodies to surface-associated IL-17A is revealed with a secondary antibody conjugated to phycoerythrin.
    Figure Legend Snippet: Schematic representation of the assays used for surface labelling of IL-17A secreting cells. In the cytokine secretion assay (CSA), after stimulation with PMA/ionomycin, the cells start to secrete cytokines for 3.5 h. Then the cells are allowed to bind the capture complex (Capture complex antibody staining) for 15 min before dilution and incubation for 1.5 h under agitation (New cytokine secretion step). The secreted cytokine is captured by antibodies to IL-17A that are maintained at the surface of the cell by antibodies to CD45. The capture complex comprises the two types of biotinylated antibodies linked by a streptavidin molecule. After washing, rabbit anti-bovine IL-17A antibodies are added that bind to the captured IL-17A (IL-17A detection labelling and revelation). Then the cells are washed and the binding of rabbit antibodies revealed with a secondary antibody conjugated to phycoerythrin. Alternatively, in the cytokine surface staining assay (CSS), after washing at the end of the 3.5 h stimulation, cells are incubated with rabbit antibodies to IL-17A, washed, and the binding of rabbit antibodies to surface-associated IL-17A is revealed with a secondary antibody conjugated to phycoerythrin.

    Techniques Used: Staining, Incubation, Binding Assay

    21) 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

    22) 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

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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: 91/100, 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:

    Antigen-specific whole blood assay. ( A) Time-course of IL-17A and IFN-γ production by whole blood cultured with OVA. The blood of the 10 responder cows (taken 45 days after the first immunization) was cultured in the presence of OVA (10 μg/mL) for 1 to 4 days in 96-well microplates. For each cow triplicate wells were used for each incubation time. Results are median values and quartiles (Q1; Q3). (B) The effect of magnetic depletion of CD4+ cells on the production of IL-17A and IFN-γ in the antigen-specific whole blood assay. Results obtained by stimulating blood samples from responder cows 15 days after the booster immunization are expressed as the percentage of the production by CD4+-depleted PBMC relative to cytokine production by un processed PBMC (100%). The viability of cells was not altered by the magnetic cell separation.

    Journal: PLoS ONE

    Article Title: Antigen-Specific Mammary Inflammation Depends on the Production of IL-17A and IFN-γ by Bovine CD4+ T Lymphocytes

    doi: 10.1371/journal.pone.0137755

    Figure Lengend Snippet: Antigen-specific whole blood assay. ( A) Time-course of IL-17A and IFN-γ production by whole blood cultured with OVA. The blood of the 10 responder cows (taken 45 days after the first immunization) was cultured in the presence of OVA (10 μg/mL) for 1 to 4 days in 96-well microplates. For each cow triplicate wells were used for each incubation time. Results are median values and quartiles (Q1; Q3). (B) The effect of magnetic depletion of CD4+ cells on the production of IL-17A and IFN-γ in the antigen-specific whole blood assay. Results obtained by stimulating blood samples from responder cows 15 days after the booster immunization are expressed as the percentage of the production by CD4+-depleted PBMC relative to cytokine production by un processed PBMC (100%). The viability of cells was not altered by the magnetic cell separation.

    Article Snippet: Cells were then labeled to reveal intracellular IL-17A with either rabbit antiserum to bovine IL-17A (Kingfisher Biotech) followed by RPE-conjugated anti-rabbit antibody, or PE-conjugated mouse monoclonal antibody to human IL-17A (eBioscience).

    Techniques: Whole Blood Assay, Cell Culture, Incubation, Magnetic Cell Separation

    Persistence of reactivity to ovalbumin. Concentrations of IL-17A and IFN-γ yielded by the whole blood assay performed at different times after immunization with ovalbumin. Results are the median values (and interquartiles) from the 8 responder cows still available 10 months post-immunization.

    Journal: PLoS ONE

    Article Title: Antigen-Specific Mammary Inflammation Depends on the Production of IL-17A and IFN-γ by Bovine CD4+ T Lymphocytes

    doi: 10.1371/journal.pone.0137755

    Figure Lengend Snippet: Persistence of reactivity to ovalbumin. Concentrations of IL-17A and IFN-γ yielded by the whole blood assay performed at different times after immunization with ovalbumin. Results are the median values (and interquartiles) from the 8 responder cows still available 10 months post-immunization.

    Article Snippet: Cells were then labeled to reveal intracellular IL-17A with either rabbit antiserum to bovine IL-17A (Kingfisher Biotech) followed by RPE-conjugated anti-rabbit antibody, or PE-conjugated mouse monoclonal antibody to human IL-17A (eBioscience).

    Techniques: Whole Blood Assay

    Intracellular expression of IL-17A and IFN-γ by CD4+ T lymphocytes. PBMC were isolated one month after ovalbumin booster injection, stimulated in vitro with ovalbumin for 3 days, rested for 2 days and finally stimulated with PMA/ionomycin for 5 h with Brefeldin A for the last 3 hours. Cells were labeled for surface CD4 and intracellular IL-17A and IFN-γ. The numbers in the plots indicate the percentages of labeled cells in comparison to the isotype control. (A) Production of viable CD4+ T lymphoblasts after culture of PBMC with (OVA) or without (NS) ovalbumin. Left panels depict PBMC from a responder cow, right panels PBMC from a low-responder. (B) PBMC from two responder cows (R1 R2) were labeled for surface CD4 and intracellular IL-17A or IFN-γ, showing CD4+ and CD4- IL-17A- and IFN-γ-producing cells. (C) labeling of CD4+ cells with anti-IL-17A and anti-IFN-γ antibodies, showing single-producing and double-producing cells. D) Double labeling of CD4+ cells from two low-responders (R3 and R4). Percentages of labeled cells are indicated in the quadrants. Results are from a representative experiment.

    Journal: PLoS ONE

    Article Title: Antigen-Specific Mammary Inflammation Depends on the Production of IL-17A and IFN-γ by Bovine CD4+ T Lymphocytes

    doi: 10.1371/journal.pone.0137755

    Figure Lengend Snippet: Intracellular expression of IL-17A and IFN-γ by CD4+ T lymphocytes. PBMC were isolated one month after ovalbumin booster injection, stimulated in vitro with ovalbumin for 3 days, rested for 2 days and finally stimulated with PMA/ionomycin for 5 h with Brefeldin A for the last 3 hours. Cells were labeled for surface CD4 and intracellular IL-17A and IFN-γ. The numbers in the plots indicate the percentages of labeled cells in comparison to the isotype control. (A) Production of viable CD4+ T lymphoblasts after culture of PBMC with (OVA) or without (NS) ovalbumin. Left panels depict PBMC from a responder cow, right panels PBMC from a low-responder. (B) PBMC from two responder cows (R1 R2) were labeled for surface CD4 and intracellular IL-17A or IFN-γ, showing CD4+ and CD4- IL-17A- and IFN-γ-producing cells. (C) labeling of CD4+ cells with anti-IL-17A and anti-IFN-γ antibodies, showing single-producing and double-producing cells. D) Double labeling of CD4+ cells from two low-responders (R3 and R4). Percentages of labeled cells are indicated in the quadrants. Results are from a representative experiment.

    Article Snippet: Cells were then labeled to reveal intracellular IL-17A with either rabbit antiserum to bovine IL-17A (Kingfisher Biotech) followed by RPE-conjugated anti-rabbit antibody, or PE-conjugated mouse monoclonal antibody to human IL-17A (eBioscience).

    Techniques: Expressing, Isolation, Injection, In Vitro, Labeling

    Concentrations of cytokines in milk samples of the 10 responder cows. Time-course of concentration variation (median and interquartiles) of CXCL8 (A), IL-17A (B) and IFN- γ (C) in the milk of quarters infused with ovalbumin at 0 hpi.

    Journal: PLoS ONE

    Article Title: Antigen-Specific Mammary Inflammation Depends on the Production of IL-17A and IFN-γ by Bovine CD4+ T Lymphocytes

    doi: 10.1371/journal.pone.0137755

    Figure Lengend Snippet: Concentrations of cytokines in milk samples of the 10 responder cows. Time-course of concentration variation (median and interquartiles) of CXCL8 (A), IL-17A (B) and IFN- γ (C) in the milk of quarters infused with ovalbumin at 0 hpi.

    Article Snippet: Cells were then labeled to reveal intracellular IL-17A with either rabbit antiserum to bovine IL-17A (Kingfisher Biotech) followed by RPE-conjugated anti-rabbit antibody, or PE-conjugated mouse monoclonal antibody to human IL-17A (eBioscience).

    Techniques: Concentration Assay

    Time-course of the IL-17A and IFN-γ production in the antigen-specific whole blood assay following immunization and correlation with Peak SCC. Concentrations of IL-17A (A) or IFN-γ (B) after 3 days of culture with ovalbumin of blood samples taken before and after immunization at days 0 and 30 (median values and interquartiles) distinguishing the antigen-specific responses of responders and low-responder cows to the intramammary antigenic challenge. (B and C) Correlations (Spearman’s rank test) between peak SCC and IL-17A concentrations or IFN-γ concentrations yielded by the whole blood assay performed 45 days after the first immunization.

    Journal: PLoS ONE

    Article Title: Antigen-Specific Mammary Inflammation Depends on the Production of IL-17A and IFN-γ by Bovine CD4+ T Lymphocytes

    doi: 10.1371/journal.pone.0137755

    Figure Lengend Snippet: Time-course of the IL-17A and IFN-γ production in the antigen-specific whole blood assay following immunization and correlation with Peak SCC. Concentrations of IL-17A (A) or IFN-γ (B) after 3 days of culture with ovalbumin of blood samples taken before and after immunization at days 0 and 30 (median values and interquartiles) distinguishing the antigen-specific responses of responders and low-responder cows to the intramammary antigenic challenge. (B and C) Correlations (Spearman’s rank test) between peak SCC and IL-17A concentrations or IFN-γ concentrations yielded by the whole blood assay performed 45 days after the first immunization.

    Article Snippet: Cells were then labeled to reveal intracellular IL-17A with either rabbit antiserum to bovine IL-17A (Kingfisher Biotech) followed by RPE-conjugated anti-rabbit antibody, or PE-conjugated mouse monoclonal antibody to human IL-17A (eBioscience).

    Techniques: Whole Blood Assay

    Choice of culture medium for the expansion of Th17 cells. ( a ) Cell growth after 3, 6, 8 or 13 days in RPMI (+FCS and 2 ng/mL TGF-β1) or IMDM (+10% KnockOut Serum Replacement) with different concentrations of recombinant human TGF-β1. ( b ) Proportions of IL-17A, IFN-γ and double positive cells after 6 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( c ) Proportions of IL-17A+ cells after 6 and 13 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( d ) Comparison of cell growth in RPMI and X-VIVO™ 15 media with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1), after 3 and 6 days of culture. ( e ) Proportions of cells IL-17A+ and IFN-γ+ (ICS) with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1) after 6 days of culture. Results are means from two cows.

    Journal: Scientific Reports

    Article Title: Expansion, isolation and first characterization of bovine Th17 lymphocytes

    doi: 10.1038/s41598-019-52562-2

    Figure Lengend Snippet: Choice of culture medium for the expansion of Th17 cells. ( a ) Cell growth after 3, 6, 8 or 13 days in RPMI (+FCS and 2 ng/mL TGF-β1) or IMDM (+10% KnockOut Serum Replacement) with different concentrations of recombinant human TGF-β1. ( b ) Proportions of IL-17A, IFN-γ and double positive cells after 6 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( c ) Proportions of IL-17A+ cells after 6 and 13 days of culture as a function of TGF-β1 concentration (0 to 5 ng/mL). Results are means from the cells of two cows. ( d ) Comparison of cell growth in RPMI and X-VIVO™ 15 media with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1), after 3 and 6 days of culture. ( e ) Proportions of cells IL-17A+ and IFN-γ+ (ICS) with or without polarizing cytokines (40 ng/mL IL-6 and 2 ng/mL TGF-β1) after 6 days of culture. Results are means from two cows.

    Article Snippet: Rabbit antibodies to bovine IL-17A (Kingfisher) were then used as capture antibody with monoclonal antibodies to bovine IL-17A N- or C-terminal amino acid sequences as detection antibodies.

    Techniques: Knock-Out, Recombinant, Concentration Assay

    Identification of IL-17A and IFN-γ producing bovine lymphocytes. PBMC were stimulated with PMA/ionomycin, cytokine secretion blocked with Brefeldin A before flow cytometry analysis. Debris were excluded by gating according to FSC/SSC, and after gating on singlet cells, dead cells were excluded by gating on live cells. The CD4+ cells were gated by taking into account the isotype control, and the production of IL-17A and IFN-γ was measured by intracellular labelling with specific antibodies. Results shown are from a representative experiment. FSC: forward scatter: SSC: side scatter.

    Journal: Scientific Reports

    Article Title: Expansion, isolation and first characterization of bovine Th17 lymphocytes

    doi: 10.1038/s41598-019-52562-2

    Figure Lengend Snippet: Identification of IL-17A and IFN-γ producing bovine lymphocytes. PBMC were stimulated with PMA/ionomycin, cytokine secretion blocked with Brefeldin A before flow cytometry analysis. Debris were excluded by gating according to FSC/SSC, and after gating on singlet cells, dead cells were excluded by gating on live cells. The CD4+ cells were gated by taking into account the isotype control, and the production of IL-17A and IFN-γ was measured by intracellular labelling with specific antibodies. Results shown are from a representative experiment. FSC: forward scatter: SSC: side scatter.

    Article Snippet: Rabbit antibodies to bovine IL-17A (Kingfisher) were then used as capture antibody with monoclonal antibodies to bovine IL-17A N- or C-terminal amino acid sequences as detection antibodies.

    Techniques: Flow Cytometry

    Unambiguous identification of bovine Th17. ( a ) Secretion of cytokines by sorted Th17 cells. After sorting, the cells were stimulated with α-CD3 and α-CD28 and cultured for 6 days. Supernatant contents were analysed by ELISA. Median values (10 to 90 percentiles) from five cell preparations from three cows are shown. ( b ) Comparison of Th17 signature gene expression between IL-17A+ and IL-17A- cells. Heatmap shows expression intensity expressed as log2(fold-change) normalized relative to three reference genes (ACTB, PPIA and GAPDH). Expression is relative to the CD4+ subset of the corresponding cow at day 6 after positive selection using MACS® beads. Cells were cultured in X-VIVO™ 15 medium without polarizing cytokines for 6 days after an initial TCR stimulation and a partial (half) renewal of medium with addition of IL-2 on the third day. Results are from three cows (C1, C2, C3).

    Journal: Scientific Reports

    Article Title: Expansion, isolation and first characterization of bovine Th17 lymphocytes

    doi: 10.1038/s41598-019-52562-2

    Figure Lengend Snippet: Unambiguous identification of bovine Th17. ( a ) Secretion of cytokines by sorted Th17 cells. After sorting, the cells were stimulated with α-CD3 and α-CD28 and cultured for 6 days. Supernatant contents were analysed by ELISA. Median values (10 to 90 percentiles) from five cell preparations from three cows are shown. ( b ) Comparison of Th17 signature gene expression between IL-17A+ and IL-17A- cells. Heatmap shows expression intensity expressed as log2(fold-change) normalized relative to three reference genes (ACTB, PPIA and GAPDH). Expression is relative to the CD4+ subset of the corresponding cow at day 6 after positive selection using MACS® beads. Cells were cultured in X-VIVO™ 15 medium without polarizing cytokines for 6 days after an initial TCR stimulation and a partial (half) renewal of medium with addition of IL-2 on the third day. Results are from three cows (C1, C2, C3).

    Article Snippet: Rabbit antibodies to bovine IL-17A (Kingfisher) were then used as capture antibody with monoclonal antibodies to bovine IL-17A N- or C-terminal amino acid sequences as detection antibodies.

    Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Selection, Magnetic Cell Separation

    Comparison of the Cytokine Secretion Assay with the cytokine surface Staining. ( a ) Gating strategy. At the end of the culture expansion step, the polarized cell populations were split and analyzed side-by-side by flow cytometry with Cytokine Secretion (left) and surface staining (right) assays for IL-17A secretion. The squares indicate the IL-17A+ cells sorting windows, the circles the IL-17A- sorting windows. ( b ) Proportions of cytokine-positive cells 8 days after sorting. Median values from two sorting experiments with cells of two cows are shown. ( c ) Numbers of IL-17A+ and IL-17A- cells at different times after sorting. Stars indicate stimulations with anti-CD3/CD28 antibodies, arrows the sampling of cells for freezing or RNA preparation. Values are from one sorting experiment with two cows. ( d ) Proportions of sorted IL-17A+ cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. Values are from one sorting experiment with two cows. e) Proportions of sorted IL-17A- cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. CSA: cytokine secretion assay; CSS: cytokine surface staining.

    Journal: Scientific Reports

    Article Title: Expansion, isolation and first characterization of bovine Th17 lymphocytes

    doi: 10.1038/s41598-019-52562-2

    Figure Lengend Snippet: Comparison of the Cytokine Secretion Assay with the cytokine surface Staining. ( a ) Gating strategy. At the end of the culture expansion step, the polarized cell populations were split and analyzed side-by-side by flow cytometry with Cytokine Secretion (left) and surface staining (right) assays for IL-17A secretion. The squares indicate the IL-17A+ cells sorting windows, the circles the IL-17A- sorting windows. ( b ) Proportions of cytokine-positive cells 8 days after sorting. Median values from two sorting experiments with cells of two cows are shown. ( c ) Numbers of IL-17A+ and IL-17A- cells at different times after sorting. Stars indicate stimulations with anti-CD3/CD28 antibodies, arrows the sampling of cells for freezing or RNA preparation. Values are from one sorting experiment with two cows. ( d ) Proportions of sorted IL-17A+ cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. Values are from one sorting experiment with two cows. e) Proportions of sorted IL-17A- cells producing (ICS) IL-17A, IFN-γ or IL-22 at different times after sorting. CSA: cytokine secretion assay; CSS: cytokine surface staining.

    Article Snippet: Rabbit antibodies to bovine IL-17A (Kingfisher) were then used as capture antibody with monoclonal antibodies to bovine IL-17A N- or C-terminal amino acid sequences as detection antibodies.

    Techniques: Staining, Flow Cytometry, Sampling

    Maintenance of the IL-17A and IFN-γ phenotype of sorted and frozen-thawed cells upon subculture. ( a ) Sorted IL-17A+ cells stimulated with α-CD3/α-CD28 were cultured with IL-2 with or without the polarizing cytokines TGF-β1 and IL-6 for 9 days and analyzed by flow cytometry (ICS). Percentages of IL-17A+/IFN-γ+ double positive cells and of IL-17A-/IFN-g+ cells are shown. ( b ) Thawed IL-17A+ cells were cultured for 22 days and analyzed by ICS at days 12 and 22. Percentages of IL-17A+ cells (all: both IL-17A+/IFN-γ- and double positive cells) and IL-17A-/IFN-γ+ cells are shown. Results from two cows (C1 C2) are shown.

    Journal: Scientific Reports

    Article Title: Expansion, isolation and first characterization of bovine Th17 lymphocytes

    doi: 10.1038/s41598-019-52562-2

    Figure Lengend Snippet: Maintenance of the IL-17A and IFN-γ phenotype of sorted and frozen-thawed cells upon subculture. ( a ) Sorted IL-17A+ cells stimulated with α-CD3/α-CD28 were cultured with IL-2 with or without the polarizing cytokines TGF-β1 and IL-6 for 9 days and analyzed by flow cytometry (ICS). Percentages of IL-17A+/IFN-γ+ double positive cells and of IL-17A-/IFN-g+ cells are shown. ( b ) Thawed IL-17A+ cells were cultured for 22 days and analyzed by ICS at days 12 and 22. Percentages of IL-17A+ cells (all: both IL-17A+/IFN-γ- and double positive cells) and IL-17A-/IFN-γ+ cells are shown. Results from two cows (C1 C2) are shown.

    Article Snippet: Rabbit antibodies to bovine IL-17A (Kingfisher) were then used as capture antibody with monoclonal antibodies to bovine IL-17A N- or C-terminal amino acid sequences as detection antibodies.

    Techniques: Cell Culture, Flow Cytometry

    Culture conditions for expansion of Th17 cells. ( a ) Number of cells after 6, 9 and 13 days of culture as a function of the concentration (µg/mL) of the coating antibodies to CD3 with or without 10 ng/ml recombinant human IL-2. Results are from a representative experiment. ( b ) Concentrations of IL-17A and IFN-γ under these culture conditions. ( c ) Proportion of cells producing IL-17A or IFN-γ (ICS) at day 6 of culture. Results are means from the cells of two cows. Percentages of IL-17A positive cells differed as a function of treatment (p = 0.034, one-way ANOVA), but not percentages of IFN-γ positive cells (p = 0.24, one-way ANOVA). Results with α-CD3 1 µg/mL are not shown because too few cells were available for staining.

    Journal: Scientific Reports

    Article Title: Expansion, isolation and first characterization of bovine Th17 lymphocytes

    doi: 10.1038/s41598-019-52562-2

    Figure Lengend Snippet: Culture conditions for expansion of Th17 cells. ( a ) Number of cells after 6, 9 and 13 days of culture as a function of the concentration (µg/mL) of the coating antibodies to CD3 with or without 10 ng/ml recombinant human IL-2. Results are from a representative experiment. ( b ) Concentrations of IL-17A and IFN-γ under these culture conditions. ( c ) Proportion of cells producing IL-17A or IFN-γ (ICS) at day 6 of culture. Results are means from the cells of two cows. Percentages of IL-17A positive cells differed as a function of treatment (p = 0.034, one-way ANOVA), but not percentages of IFN-γ positive cells (p = 0.24, one-way ANOVA). Results with α-CD3 1 µg/mL are not shown because too few cells were available for staining.

    Article Snippet: Rabbit antibodies to bovine IL-17A (Kingfisher) were then used as capture antibody with monoclonal antibodies to bovine IL-17A N- or C-terminal amino acid sequences as detection antibodies.

    Techniques: Concentration Assay, Recombinant, Staining

    Efficiency of the monoclonal antibodies in the cytokine secretion assay. ( a ) Percentages of CD4+ T cells labelled by the capture complex with either the α-Cter or α-Nter monoclonals, and comparison with the percentages of IL-17A+ cells identified by ICS. Data from two cows (C1 C2) are shown, one per row. ( b ) Concentrations of IL-17A in culture supernatant before capture, at the end of the 3.5 h of stimulation. ( c ) Concentrations of IL-17A in culture supernatant at the end of the secretion step (after capture) in the presence of the complex capture with either one of the two monoclonal antibodies to IL-17A, an isotype control or the polyclonal antiserum to IL-17A, showing the efficiency of the capture of IL-17A as it is secreted. PI: PMA/ionomycin, PIB: PMA/ionomycin/brefeldin A. ICS: intracellular staining.

    Journal: Scientific Reports

    Article Title: Expansion, isolation and first characterization of bovine Th17 lymphocytes

    doi: 10.1038/s41598-019-52562-2

    Figure Lengend Snippet: Efficiency of the monoclonal antibodies in the cytokine secretion assay. ( a ) Percentages of CD4+ T cells labelled by the capture complex with either the α-Cter or α-Nter monoclonals, and comparison with the percentages of IL-17A+ cells identified by ICS. Data from two cows (C1 C2) are shown, one per row. ( b ) Concentrations of IL-17A in culture supernatant before capture, at the end of the 3.5 h of stimulation. ( c ) Concentrations of IL-17A in culture supernatant at the end of the secretion step (after capture) in the presence of the complex capture with either one of the two monoclonal antibodies to IL-17A, an isotype control or the polyclonal antiserum to IL-17A, showing the efficiency of the capture of IL-17A as it is secreted. PI: PMA/ionomycin, PIB: PMA/ionomycin/brefeldin A. ICS: intracellular staining.

    Article Snippet: Rabbit antibodies to bovine IL-17A (Kingfisher) were then used as capture antibody with monoclonal antibodies to bovine IL-17A N- or C-terminal amino acid sequences as detection antibodies.

    Techniques: Staining

    Schematic representation of the procedure used for the isolation and expansion of Th17 cells. CD4+ cells were isolated from PBMC by magnetic sorting, expanded in a culture medium without serum in the presence of polarizing cytokines and TCR-stimulation, activated, labelled for surface IL-17A and sorted by flow cytometry. The sorted cells were expanded and maintained in culture for several weeks. The indicated timeline is kept in all the figures to make it clear at which step cells were assayed. Concentrations of cytokines were 10 ng/mL (IL-2), 40 ng/mL (IL-6) and 2 ng/mL (TGF-β1). D: day; CSA: cytokine secretion assay; CSS: cytokine surface staining. α-: antibody anti-.

    Journal: Scientific Reports

    Article Title: Expansion, isolation and first characterization of bovine Th17 lymphocytes

    doi: 10.1038/s41598-019-52562-2

    Figure Lengend Snippet: Schematic representation of the procedure used for the isolation and expansion of Th17 cells. CD4+ cells were isolated from PBMC by magnetic sorting, expanded in a culture medium without serum in the presence of polarizing cytokines and TCR-stimulation, activated, labelled for surface IL-17A and sorted by flow cytometry. The sorted cells were expanded and maintained in culture for several weeks. The indicated timeline is kept in all the figures to make it clear at which step cells were assayed. Concentrations of cytokines were 10 ng/mL (IL-2), 40 ng/mL (IL-6) and 2 ng/mL (TGF-β1). D: day; CSA: cytokine secretion assay; CSS: cytokine surface staining. α-: antibody anti-.

    Article Snippet: Rabbit antibodies to bovine IL-17A (Kingfisher) were then used as capture antibody with monoclonal antibodies to bovine IL-17A N- or C-terminal amino acid sequences as detection antibodies.

    Techniques: Isolation, Flow Cytometry, Staining

    Schematic representation of the assays used for surface labelling of IL-17A secreting cells. In the cytokine secretion assay (CSA), after stimulation with PMA/ionomycin, the cells start to secrete cytokines for 3.5 h. Then the cells are allowed to bind the capture complex (Capture complex antibody staining) for 15 min before dilution and incubation for 1.5 h under agitation (New cytokine secretion step). The secreted cytokine is captured by antibodies to IL-17A that are maintained at the surface of the cell by antibodies to CD45. The capture complex comprises the two types of biotinylated antibodies linked by a streptavidin molecule. After washing, rabbit anti-bovine IL-17A antibodies are added that bind to the captured IL-17A (IL-17A detection labelling and revelation). Then the cells are washed and the binding of rabbit antibodies revealed with a secondary antibody conjugated to phycoerythrin. Alternatively, in the cytokine surface staining assay (CSS), after washing at the end of the 3.5 h stimulation, cells are incubated with rabbit antibodies to IL-17A, washed, and the binding of rabbit antibodies to surface-associated IL-17A is revealed with a secondary antibody conjugated to phycoerythrin.

    Journal: Scientific Reports

    Article Title: Expansion, isolation and first characterization of bovine Th17 lymphocytes

    doi: 10.1038/s41598-019-52562-2

    Figure Lengend Snippet: Schematic representation of the assays used for surface labelling of IL-17A secreting cells. In the cytokine secretion assay (CSA), after stimulation with PMA/ionomycin, the cells start to secrete cytokines for 3.5 h. Then the cells are allowed to bind the capture complex (Capture complex antibody staining) for 15 min before dilution and incubation for 1.5 h under agitation (New cytokine secretion step). The secreted cytokine is captured by antibodies to IL-17A that are maintained at the surface of the cell by antibodies to CD45. The capture complex comprises the two types of biotinylated antibodies linked by a streptavidin molecule. After washing, rabbit anti-bovine IL-17A antibodies are added that bind to the captured IL-17A (IL-17A detection labelling and revelation). Then the cells are washed and the binding of rabbit antibodies revealed with a secondary antibody conjugated to phycoerythrin. Alternatively, in the cytokine surface staining assay (CSS), after washing at the end of the 3.5 h stimulation, cells are incubated with rabbit antibodies to IL-17A, washed, and the binding of rabbit antibodies to surface-associated IL-17A is revealed with a secondary antibody conjugated to phycoerythrin.

    Article Snippet: Rabbit antibodies to bovine IL-17A (Kingfisher) were then used as capture antibody with monoclonal antibodies to bovine IL-17A N- or C-terminal amino acid sequences as detection antibodies.

    Techniques: Staining, Incubation, Binding Assay