bovine recombinant il 17a  (Kingfisher Biotech)


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    Kingfisher Biotech bovine recombinant il 17a
    Effects of CpG oligodinucleotide, <t>interleukin-17A</t> and interferon-α on tracheal antimicrobial peptide gene expression. Dose- and time-dependent effects were measured for CpG oligodinucleotide (A,B) , a TLR9 agonist; interleukin-17A (C,D) ; and interferon-α (E,F) . For the dose–response studies (A,C,E) , confluent bTEC were stimulated in triplicate with various concentrations of agonist for 16 h. For the time-course studies (B,D,F) , confluent bTEC were stimulated in triplicate for 4, 8, 16 or 24 h with the doses of agonist shown. Gene expression of TAP relative to that of GAPDH was measured using real-time RT-qPCR. Lipopolysaccharide (LPS, 0.1 μg/mL) was used in all assays as a positive control and standard. *, significantly different from unstimulated cells ( P
    Bovine Recombinant Il 17a, supplied by Kingfisher Biotech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bovine recombinant il 17a/product/Kingfisher Biotech
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bovine recombinant il 17a - by Bioz Stars, 2022-07
    86/100 stars

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

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

    Journal: Veterinary Research

    doi: 10.1186/s13567-014-0105-8

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

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

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

    Techniques Used: Expressing, Quantitative RT-PCR

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

    Techniques Used: Cell Culture, Expressing

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    Kingfisher Biotech bovine il 17a
    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 <t>IL-17A,</t> 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.
    Bovine Il 17a, supplied by Kingfisher Biotech, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bovine il 17a/product/Kingfisher Biotech
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    Price from $9.99 to $1999.99
    bovine il 17a - by Bioz Stars, 2022-07
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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
    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
      Buy from Supplier

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    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: Cells were incubated with 5 µg/mL affinity-purified rabbit antibodies to bovine IL-17A (Kingfisher Biotech) for 20 min at 4 °C followed by labelling with 2.5 µg/mL donkey anti-rabbit IgG-PE (Jackson Immunoresearch) for 20 min at 4 °C, and then were stained with the live/dead cell stain kit.

    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: Cells were incubated with 5 µg/mL affinity-purified rabbit antibodies to bovine IL-17A (Kingfisher Biotech) for 20 min at 4 °C followed by labelling with 2.5 µg/mL donkey anti-rabbit IgG-PE (Jackson Immunoresearch) for 20 min at 4 °C, and then were stained with the live/dead cell stain kit.

    Techniques: Flow Cytometry, 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: Cells were incubated with 5 µg/mL affinity-purified rabbit antibodies to bovine IL-17A (Kingfisher Biotech) for 20 min at 4 °C followed by labelling with 2.5 µg/mL donkey anti-rabbit IgG-PE (Jackson Immunoresearch) for 20 min at 4 °C, and then were stained with the live/dead cell stain kit.

    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: Cells were incubated with 5 µg/mL affinity-purified rabbit antibodies to bovine IL-17A (Kingfisher Biotech) for 20 min at 4 °C followed by labelling with 2.5 µg/mL donkey anti-rabbit IgG-PE (Jackson Immunoresearch) for 20 min at 4 °C, and then were stained with the live/dead cell stain kit.

    Techniques: Staining, Flow Cytometry, 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: Cells were incubated with 5 µg/mL affinity-purified rabbit antibodies to bovine IL-17A (Kingfisher Biotech) for 20 min at 4 °C followed by labelling with 2.5 µg/mL donkey anti-rabbit IgG-PE (Jackson Immunoresearch) for 20 min at 4 °C, and then were stained with the live/dead cell stain kit.

    Techniques: Cell Culture, Flow Cytometry, 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: Cells were incubated with 5 µg/mL affinity-purified rabbit antibodies to bovine IL-17A (Kingfisher Biotech) for 20 min at 4 °C followed by labelling with 2.5 µg/mL donkey anti-rabbit IgG-PE (Jackson Immunoresearch) for 20 min at 4 °C, and then were stained with the live/dead cell stain kit.

    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: Cells were incubated with 5 µg/mL affinity-purified rabbit antibodies to bovine IL-17A (Kingfisher Biotech) for 20 min at 4 °C followed by labelling with 2.5 µg/mL donkey anti-rabbit IgG-PE (Jackson Immunoresearch) for 20 min at 4 °C, and then were stained with the live/dead cell stain kit.

    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: Cells were incubated with 5 µg/mL affinity-purified rabbit antibodies to bovine IL-17A (Kingfisher Biotech) for 20 min at 4 °C followed by labelling with 2.5 µg/mL donkey anti-rabbit IgG-PE (Jackson Immunoresearch) for 20 min at 4 °C, and then were stained with the live/dead cell stain kit.

    Techniques: Isolation, Flow Cytometry, 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: Cells were incubated with 5 µg/mL affinity-purified rabbit antibodies to bovine IL-17A (Kingfisher Biotech) for 20 min at 4 °C followed by labelling with 2.5 µg/mL donkey anti-rabbit IgG-PE (Jackson Immunoresearch) for 20 min at 4 °C, and then were stained with the live/dead cell stain kit.

    Techniques: Staining, Incubation, Binding Assay

    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