bovine il 17a do it yourself elisa kit  (Kingfisher Biotech)


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    Kingfisher Biotech bovine il 17a do it yourself elisa kit
    UA suppresses mitogen-induced <t>IL-17A</t> production by bovine PBMCs. Peripheral blood mononuclear cells from three healthy bovids were plated at 3 × 10 5 cells per well in a 96-well plate and incubated with Concanavalin A, vehicle control (DMSO), or escalating concentrations of UA dissolved in DMSO for 3 days. On day three, cell supernatants were collected, and the remaining cell pellet was re-suspended with an MTT solution using the manufacturer's protocol to subsequently analyze the viability of the cells after the incubation period. (A) Mean absorbance values at 570 nm ± SEM following the MTT incubation and solubilization. Significance was determined using a one-way ANOVA with Tukey's multiple comparisons; (ns) indicates no significant differences were observed. (B) The supernatants from the 3-day incubation were analyzed for IL-17A concentration by <t>ELISA.</t> Data represent mean IL-17A concentration (in ng/mL) ± SEM detected in the supernatants. The data were analyzed using a standard one-way ANOVA with Tukey's multiple comparisons; * p
    Bovine Il 17a Do It Yourself Elisa Kit, supplied by Kingfisher Biotech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "The Effects of Ursolic Acid Treatment on Immunopathogenesis Following Mannheimia haemolytica Infections"

    Article Title: The Effects of Ursolic Acid Treatment on Immunopathogenesis Following Mannheimia haemolytica Infections

    Journal: Frontiers in Veterinary Science

    doi: 10.3389/fvets.2021.782872

    UA suppresses mitogen-induced IL-17A production by bovine PBMCs. Peripheral blood mononuclear cells from three healthy bovids were plated at 3 × 10 5 cells per well in a 96-well plate and incubated with Concanavalin A, vehicle control (DMSO), or escalating concentrations of UA dissolved in DMSO for 3 days. On day three, cell supernatants were collected, and the remaining cell pellet was re-suspended with an MTT solution using the manufacturer's protocol to subsequently analyze the viability of the cells after the incubation period. (A) Mean absorbance values at 570 nm ± SEM following the MTT incubation and solubilization. Significance was determined using a one-way ANOVA with Tukey's multiple comparisons; (ns) indicates no significant differences were observed. (B) The supernatants from the 3-day incubation were analyzed for IL-17A concentration by ELISA. Data represent mean IL-17A concentration (in ng/mL) ± SEM detected in the supernatants. The data were analyzed using a standard one-way ANOVA with Tukey's multiple comparisons; * p
    Figure Legend Snippet: UA suppresses mitogen-induced IL-17A production by bovine PBMCs. Peripheral blood mononuclear cells from three healthy bovids were plated at 3 × 10 5 cells per well in a 96-well plate and incubated with Concanavalin A, vehicle control (DMSO), or escalating concentrations of UA dissolved in DMSO for 3 days. On day three, cell supernatants were collected, and the remaining cell pellet was re-suspended with an MTT solution using the manufacturer's protocol to subsequently analyze the viability of the cells after the incubation period. (A) Mean absorbance values at 570 nm ± SEM following the MTT incubation and solubilization. Significance was determined using a one-way ANOVA with Tukey's multiple comparisons; (ns) indicates no significant differences were observed. (B) The supernatants from the 3-day incubation were analyzed for IL-17A concentration by ELISA. Data represent mean IL-17A concentration (in ng/mL) ± SEM detected in the supernatants. The data were analyzed using a standard one-way ANOVA with Tukey's multiple comparisons; * p

    Techniques Used: Incubation, MTT Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

    2) Product Images from "Bovine Gamma Delta T Cells Contribute to Exacerbated IL-17 Production in Response to Co-Infection with Bovine RSV and Mannheimia haemolytica"

    Article Title: Bovine Gamma Delta T Cells Contribute to Exacerbated IL-17 Production in Response to Co-Infection with Bovine RSV and Mannheimia haemolytica

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0151083

    In vitro infection with BRSV and M . haemolytica results in exacerbated IL-17 production. PBMC from BRSV vaccinated cows were stimulated with BRSV for 6 hours, then cultured with 0.1 MOI M . haemolytica in antibiotic free media for 4 hours. After four hours, antibiotics were added, and then incubations were continued for 18 hours (A-C) or 6 days (D and E). Control cultures were included that received neither pathogen, BRSV alone or M . haemolytica alone. (A-C) Cells were analyzed by qPCR for expression of IL-17 (A), IL-21 (B) and IL-22 (C). Cell culture supernatants from day 6 were analyzed by ELISA for IL-17 (D) and were diluted 1:1 and added to BT for 24 hours (E). BT were analyzed by qPCR for expression of IL-8 after overnight stimulation as in Fig 3 . Results in A-C are pooled from three independent experiments with n = 12. Results in D and E are from two experiments with n = 8. Data represent means ± SEM.
    Figure Legend Snippet: In vitro infection with BRSV and M . haemolytica results in exacerbated IL-17 production. PBMC from BRSV vaccinated cows were stimulated with BRSV for 6 hours, then cultured with 0.1 MOI M . haemolytica in antibiotic free media for 4 hours. After four hours, antibiotics were added, and then incubations were continued for 18 hours (A-C) or 6 days (D and E). Control cultures were included that received neither pathogen, BRSV alone or M . haemolytica alone. (A-C) Cells were analyzed by qPCR for expression of IL-17 (A), IL-21 (B) and IL-22 (C). Cell culture supernatants from day 6 were analyzed by ELISA for IL-17 (D) and were diluted 1:1 and added to BT for 24 hours (E). BT were analyzed by qPCR for expression of IL-8 after overnight stimulation as in Fig 3 . Results in A-C are pooled from three independent experiments with n = 12. Results in D and E are from two experiments with n = 8. Data represent means ± SEM.

    Techniques Used: In Vitro, Infection, Cell Culture, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay

    γδ T cells are the primary source of exacerbated IL-17 production in an in vitro model of BRDC. (A and B) PBMC from BRSV vaccinated cows were stimulated with BRSV for 6 hours, then with 0.1 MOI of heat-inactivated M . haemolytica (A) or 1 μg/mL purified LPS from M . haemolytica (B). Control cells remained unstimulated, were stimulated with only BRSV or were stimulated with only heat-inactivated M . haemolytica LPS. After six days, cell culture supernatants were analyzed by ELISA for IL-17. (C) PBMC from BRSV vaccinated cows were stimulated with BRSV for 6 hours, then cultured with 0.1 MOI P . multocida in antibiotic free media for 4 hours. After four hours, antibiotics were added, and then incubations were continued for 6 days. Control cultures were included that received neither pathogen, BRSV alone or P . multocida alone. Cell culture supernatants were analyzed by ELISA for IL-17. (D) PBMC were depleted of γδ T cells using MACS. Total PBMC and γδ T cell-depleted PBMC were then cultured in the presence of BRSV and M . haemolytica as above. After 6 days, cell culture supernatants were analyzed by ELISA. Results in A-C are from one experiment with n = 4. Results in D are pooled from 2 independent experiments with n = 8. Data represent means ± SEM.
    Figure Legend Snippet: γδ T cells are the primary source of exacerbated IL-17 production in an in vitro model of BRDC. (A and B) PBMC from BRSV vaccinated cows were stimulated with BRSV for 6 hours, then with 0.1 MOI of heat-inactivated M . haemolytica (A) or 1 μg/mL purified LPS from M . haemolytica (B). Control cells remained unstimulated, were stimulated with only BRSV or were stimulated with only heat-inactivated M . haemolytica LPS. After six days, cell culture supernatants were analyzed by ELISA for IL-17. (C) PBMC from BRSV vaccinated cows were stimulated with BRSV for 6 hours, then cultured with 0.1 MOI P . multocida in antibiotic free media for 4 hours. After four hours, antibiotics were added, and then incubations were continued for 6 days. Control cultures were included that received neither pathogen, BRSV alone or P . multocida alone. Cell culture supernatants were analyzed by ELISA for IL-17. (D) PBMC were depleted of γδ T cells using MACS. Total PBMC and γδ T cell-depleted PBMC were then cultured in the presence of BRSV and M . haemolytica as above. After 6 days, cell culture supernatants were analyzed by ELISA. Results in A-C are from one experiment with n = 4. Results in D are pooled from 2 independent experiments with n = 8. Data represent means ± SEM.

    Techniques Used: In Vitro, Purification, Cell Culture, Enzyme-linked Immunosorbent Assay, Magnetic Cell Separation

    Both CD4 T cells and γδ T cells produce IL-17 in response to BRSV. PBMC were isolated from control or BRSV vaccinated cows and labeled with Cell Trace Violet. Cells were then cultured for 6 days with BRSV. On day 6, CD4 T cells (A) and γδ T cells (B) were analyzed for virus-specific proliferation as measured by Cell Trace Violet dilution. Representative flow plots are shown in A and B. Aggregate results are shown in C. (D) CD4 T cells and γδ T cells from BRSV vaccinated or nonvaccinated animals were isolated by MACS and cultured in the presence of autologous APC ± BRSV. After 6 days, cell culture supernatants were analyzed by ELISA for IL-17. (E) CD4 T cells and γδ T cells were MACS purified from peripheral blood of calves infected or not with BRSV strain 375 for 7 days. Purified cells were cultured in the presence of autologous APC ± BRSV. After 6 days, cell culture supernatants were analyzed by ELISA for IL-17. For A-C, background levels of proliferation were subtracted and results are presented as change over mock. Results are pooled from two independent experiments. Data represent means ± SEM.
    Figure Legend Snippet: Both CD4 T cells and γδ T cells produce IL-17 in response to BRSV. PBMC were isolated from control or BRSV vaccinated cows and labeled with Cell Trace Violet. Cells were then cultured for 6 days with BRSV. On day 6, CD4 T cells (A) and γδ T cells (B) were analyzed for virus-specific proliferation as measured by Cell Trace Violet dilution. Representative flow plots are shown in A and B. Aggregate results are shown in C. (D) CD4 T cells and γδ T cells from BRSV vaccinated or nonvaccinated animals were isolated by MACS and cultured in the presence of autologous APC ± BRSV. After 6 days, cell culture supernatants were analyzed by ELISA for IL-17. (E) CD4 T cells and γδ T cells were MACS purified from peripheral blood of calves infected or not with BRSV strain 375 for 7 days. Purified cells were cultured in the presence of autologous APC ± BRSV. After 6 days, cell culture supernatants were analyzed by ELISA for IL-17. For A-C, background levels of proliferation were subtracted and results are presented as change over mock. Results are pooled from two independent experiments. Data represent means ± SEM.

    Techniques Used: Isolation, Labeling, Cell Culture, Flow Cytometry, Magnetic Cell Separation, Enzyme-linked Immunosorbent Assay, Purification, Infection

    IL-17 and Th17 responses from BRSV vaccinated cattle. Peripheral blood was collected from cows receiving annual vaccinations with a multivalent vaccine containing live-attenuated BRSV (n = 8), or from control cows that were not included in the vaccination program due to inclusion in another study (n = 6). PMBC were isolated and stimulated with BRSV for 24 hours or 6 days, as in Fig 1 . RNA was isolated from the cells and analyzed by qPCR for expression of IL-17 (A, left panel), IL-21 (B) and IL-22 (C). Cell culture supernatants were also analyzed by ELISA for IL-17 (A, right panel). For qPCR analysis, results were normalized to the housekeeping gene RPS-9, and expressed relative to unstimulated control samples. Results were pooled from two independent experiments. Data represent means ± SEM.
    Figure Legend Snippet: IL-17 and Th17 responses from BRSV vaccinated cattle. Peripheral blood was collected from cows receiving annual vaccinations with a multivalent vaccine containing live-attenuated BRSV (n = 8), or from control cows that were not included in the vaccination program due to inclusion in another study (n = 6). PMBC were isolated and stimulated with BRSV for 24 hours or 6 days, as in Fig 1 . RNA was isolated from the cells and analyzed by qPCR for expression of IL-17 (A, left panel), IL-21 (B) and IL-22 (C). Cell culture supernatants were also analyzed by ELISA for IL-17 (A, right panel). For qPCR analysis, results were normalized to the housekeeping gene RPS-9, and expressed relative to unstimulated control samples. Results were pooled from two independent experiments. Data represent means ± SEM.

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

    WC1.1 + γδ T cells produce IL-17 in response to BRSV. γδ T cells were purified from the peripheral blood of BRSV vaccinated or control animals by FACS based upon their expression of the γδ T cell receptor and either expression of WC1.1, WC1.2 or lack of WC1. Cells were cultured in the presence of autologous APC ± BRSV for 6 days. Cell culture supernatants were then analyzed by ELISA for IL-17. (B) Cell culture supernatants from (A) were also diluted 1:1 and added to BT cells for 24 hours. After 24 hours, BT were analyzed by qPCR for expression of IL-8. For qPCR analysis, results were normalized to the housekeeping gene RPS-9, and expressed relative to unstimulated control samples. Results are pooled from two independent experiments. Data represent means ± SEM.
    Figure Legend Snippet: WC1.1 + γδ T cells produce IL-17 in response to BRSV. γδ T cells were purified from the peripheral blood of BRSV vaccinated or control animals by FACS based upon their expression of the γδ T cell receptor and either expression of WC1.1, WC1.2 or lack of WC1. Cells were cultured in the presence of autologous APC ± BRSV for 6 days. Cell culture supernatants were then analyzed by ELISA for IL-17. (B) Cell culture supernatants from (A) were also diluted 1:1 and added to BT cells for 24 hours. After 24 hours, BT were analyzed by qPCR for expression of IL-8. For qPCR analysis, results were normalized to the housekeeping gene RPS-9, and expressed relative to unstimulated control samples. Results are pooled from two independent experiments. Data represent means ± SEM.

    Techniques Used: Purification, FACS, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

    IL-17 and Th17 responses in the lungs and peripheral blood of calves infected with BRSV. Calves (n = 8) were infected via aerosol inoculation with BRSV Strain 375 (RSV) as described in Materials and Methods. Control calves remained uninfected (n = 8). On day 7 post-infection, the animals were sacrificed and the lungs analyzed by qPCR for expression of IL-17, IL-21 and IL-22 (A) and IL-8 (B). In separate experiments, PBMC were isolated from control (n = 8) and BRSV infected calves (n = 8) on day 7-post infection. PBMC were stimulated with BRSV for 24 hours and then analyzed for expression of IL-17 by qPCR (C); or for 6 days and then cell culture supernatants were analyzed by ELISA for expression of IL-17 (D). For qPCR analysis, results were normalized to the housekeeping gene RPS-9, and expressed relative to samples from uninfected control calves. Results were pooled from a total of three independent experiments. Data represent means ± SEM.
    Figure Legend Snippet: IL-17 and Th17 responses in the lungs and peripheral blood of calves infected with BRSV. Calves (n = 8) were infected via aerosol inoculation with BRSV Strain 375 (RSV) as described in Materials and Methods. Control calves remained uninfected (n = 8). On day 7 post-infection, the animals were sacrificed and the lungs analyzed by qPCR for expression of IL-17, IL-21 and IL-22 (A) and IL-8 (B). In separate experiments, PBMC were isolated from control (n = 8) and BRSV infected calves (n = 8) on day 7-post infection. PBMC were stimulated with BRSV for 24 hours and then analyzed for expression of IL-17 by qPCR (C); or for 6 days and then cell culture supernatants were analyzed by ELISA for expression of IL-17 (D). For qPCR analysis, results were normalized to the housekeeping gene RPS-9, and expressed relative to samples from uninfected control calves. Results were pooled from a total of three independent experiments. Data represent means ± SEM.

    Techniques Used: Infection, Real-time Polymerase Chain Reaction, Expressing, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay

    3) 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

    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

    4) 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

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

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

    Journal: Veterinary Research

    doi: 10.1186/s13567-017-0426-5

    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

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

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

    Journal: Veterinary Research

    doi: 10.1186/s13567-017-0426-5

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

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

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

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

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

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

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

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

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

    Techniques Used: Expressing, Staining

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

    Techniques Used: Sequencing, Sampling

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

    Techniques Used: Expressing, Staining

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    Kingfisher Biotech bovine il 17a do it yourself elisa kit
    UA suppresses mitogen-induced <t>IL-17A</t> production by bovine PBMCs. Peripheral blood mononuclear cells from three healthy bovids were plated at 3 × 10 5 cells per well in a 96-well plate and incubated with Concanavalin A, vehicle control (DMSO), or escalating concentrations of UA dissolved in DMSO for 3 days. On day three, cell supernatants were collected, and the remaining cell pellet was re-suspended with an MTT solution using the manufacturer's protocol to subsequently analyze the viability of the cells after the incubation period. (A) Mean absorbance values at 570 nm ± SEM following the MTT incubation and solubilization. Significance was determined using a one-way ANOVA with Tukey's multiple comparisons; (ns) indicates no significant differences were observed. (B) The supernatants from the 3-day incubation were analyzed for IL-17A concentration by <t>ELISA.</t> Data represent mean IL-17A concentration (in ng/mL) ± SEM detected in the supernatants. The data were analyzed using a standard one-way ANOVA with Tukey's multiple comparisons; * p
    Bovine Il 17a Do It Yourself Elisa Kit, supplied by Kingfisher Biotech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    UA suppresses mitogen-induced IL-17A production by bovine PBMCs. Peripheral blood mononuclear cells from three healthy bovids were plated at 3 × 10 5 cells per well in a 96-well plate and incubated with Concanavalin A, vehicle control (DMSO), or escalating concentrations of UA dissolved in DMSO for 3 days. On day three, cell supernatants were collected, and the remaining cell pellet was re-suspended with an MTT solution using the manufacturer's protocol to subsequently analyze the viability of the cells after the incubation period. (A) Mean absorbance values at 570 nm ± SEM following the MTT incubation and solubilization. Significance was determined using a one-way ANOVA with Tukey's multiple comparisons; (ns) indicates no significant differences were observed. (B) The supernatants from the 3-day incubation were analyzed for IL-17A concentration by ELISA. Data represent mean IL-17A concentration (in ng/mL) ± SEM detected in the supernatants. The data were analyzed using a standard one-way ANOVA with Tukey's multiple comparisons; * p

    Journal: Frontiers in Veterinary Science

    Article Title: The Effects of Ursolic Acid Treatment on Immunopathogenesis Following Mannheimia haemolytica Infections

    doi: 10.3389/fvets.2021.782872

    Figure Lengend Snippet: UA suppresses mitogen-induced IL-17A production by bovine PBMCs. Peripheral blood mononuclear cells from three healthy bovids were plated at 3 × 10 5 cells per well in a 96-well plate and incubated with Concanavalin A, vehicle control (DMSO), or escalating concentrations of UA dissolved in DMSO for 3 days. On day three, cell supernatants were collected, and the remaining cell pellet was re-suspended with an MTT solution using the manufacturer's protocol to subsequently analyze the viability of the cells after the incubation period. (A) Mean absorbance values at 570 nm ± SEM following the MTT incubation and solubilization. Significance was determined using a one-way ANOVA with Tukey's multiple comparisons; (ns) indicates no significant differences were observed. (B) The supernatants from the 3-day incubation were analyzed for IL-17A concentration by ELISA. Data represent mean IL-17A concentration (in ng/mL) ± SEM detected in the supernatants. The data were analyzed using a standard one-way ANOVA with Tukey's multiple comparisons; * p

    Article Snippet: IL-17A production by bovine PBMCs was determined using the Bovine IL-17A Do-It-Yourself ELISA kit (Kingfisher Biotech, Inc) following the manufacturer's published ELISA Technical Guide (Kingfisher Biotech, Inc).

    Techniques: Incubation, MTT Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

    In vitro infection with BRSV and M . haemolytica results in exacerbated IL-17 production. PBMC from BRSV vaccinated cows were stimulated with BRSV for 6 hours, then cultured with 0.1 MOI M . haemolytica in antibiotic free media for 4 hours. After four hours, antibiotics were added, and then incubations were continued for 18 hours (A-C) or 6 days (D and E). Control cultures were included that received neither pathogen, BRSV alone or M . haemolytica alone. (A-C) Cells were analyzed by qPCR for expression of IL-17 (A), IL-21 (B) and IL-22 (C). Cell culture supernatants from day 6 were analyzed by ELISA for IL-17 (D) and were diluted 1:1 and added to BT for 24 hours (E). BT were analyzed by qPCR for expression of IL-8 after overnight stimulation as in Fig 3 . Results in A-C are pooled from three independent experiments with n = 12. Results in D and E are from two experiments with n = 8. Data represent means ± SEM.

    Journal: PLoS ONE

    Article Title: Bovine Gamma Delta T Cells Contribute to Exacerbated IL-17 Production in Response to Co-Infection with Bovine RSV and Mannheimia haemolytica

    doi: 10.1371/journal.pone.0151083

    Figure Lengend Snippet: In vitro infection with BRSV and M . haemolytica results in exacerbated IL-17 production. PBMC from BRSV vaccinated cows were stimulated with BRSV for 6 hours, then cultured with 0.1 MOI M . haemolytica in antibiotic free media for 4 hours. After four hours, antibiotics were added, and then incubations were continued for 18 hours (A-C) or 6 days (D and E). Control cultures were included that received neither pathogen, BRSV alone or M . haemolytica alone. (A-C) Cells were analyzed by qPCR for expression of IL-17 (A), IL-21 (B) and IL-22 (C). Cell culture supernatants from day 6 were analyzed by ELISA for IL-17 (D) and were diluted 1:1 and added to BT for 24 hours (E). BT were analyzed by qPCR for expression of IL-8 after overnight stimulation as in Fig 3 . Results in A-C are pooled from three independent experiments with n = 12. Results in D and E are from two experiments with n = 8. Data represent means ± SEM.

    Article Snippet: IL-17 ELISA The Bovine IL-17A VetSet ELISA Development kit was purchased from Kingfisher Biotech, Inc. (St. Paul, MN).

    Techniques: In Vitro, Infection, Cell Culture, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay

    γδ T cells are the primary source of exacerbated IL-17 production in an in vitro model of BRDC. (A and B) PBMC from BRSV vaccinated cows were stimulated with BRSV for 6 hours, then with 0.1 MOI of heat-inactivated M . haemolytica (A) or 1 μg/mL purified LPS from M . haemolytica (B). Control cells remained unstimulated, were stimulated with only BRSV or were stimulated with only heat-inactivated M . haemolytica LPS. After six days, cell culture supernatants were analyzed by ELISA for IL-17. (C) PBMC from BRSV vaccinated cows were stimulated with BRSV for 6 hours, then cultured with 0.1 MOI P . multocida in antibiotic free media for 4 hours. After four hours, antibiotics were added, and then incubations were continued for 6 days. Control cultures were included that received neither pathogen, BRSV alone or P . multocida alone. Cell culture supernatants were analyzed by ELISA for IL-17. (D) PBMC were depleted of γδ T cells using MACS. Total PBMC and γδ T cell-depleted PBMC were then cultured in the presence of BRSV and M . haemolytica as above. After 6 days, cell culture supernatants were analyzed by ELISA. Results in A-C are from one experiment with n = 4. Results in D are pooled from 2 independent experiments with n = 8. Data represent means ± SEM.

    Journal: PLoS ONE

    Article Title: Bovine Gamma Delta T Cells Contribute to Exacerbated IL-17 Production in Response to Co-Infection with Bovine RSV and Mannheimia haemolytica

    doi: 10.1371/journal.pone.0151083

    Figure Lengend Snippet: γδ T cells are the primary source of exacerbated IL-17 production in an in vitro model of BRDC. (A and B) PBMC from BRSV vaccinated cows were stimulated with BRSV for 6 hours, then with 0.1 MOI of heat-inactivated M . haemolytica (A) or 1 μg/mL purified LPS from M . haemolytica (B). Control cells remained unstimulated, were stimulated with only BRSV or were stimulated with only heat-inactivated M . haemolytica LPS. After six days, cell culture supernatants were analyzed by ELISA for IL-17. (C) PBMC from BRSV vaccinated cows were stimulated with BRSV for 6 hours, then cultured with 0.1 MOI P . multocida in antibiotic free media for 4 hours. After four hours, antibiotics were added, and then incubations were continued for 6 days. Control cultures were included that received neither pathogen, BRSV alone or P . multocida alone. Cell culture supernatants were analyzed by ELISA for IL-17. (D) PBMC were depleted of γδ T cells using MACS. Total PBMC and γδ T cell-depleted PBMC were then cultured in the presence of BRSV and M . haemolytica as above. After 6 days, cell culture supernatants were analyzed by ELISA. Results in A-C are from one experiment with n = 4. Results in D are pooled from 2 independent experiments with n = 8. Data represent means ± SEM.

    Article Snippet: IL-17 ELISA The Bovine IL-17A VetSet ELISA Development kit was purchased from Kingfisher Biotech, Inc. (St. Paul, MN).

    Techniques: In Vitro, Purification, Cell Culture, Enzyme-linked Immunosorbent Assay, Magnetic Cell Separation

    Both CD4 T cells and γδ T cells produce IL-17 in response to BRSV. PBMC were isolated from control or BRSV vaccinated cows and labeled with Cell Trace Violet. Cells were then cultured for 6 days with BRSV. On day 6, CD4 T cells (A) and γδ T cells (B) were analyzed for virus-specific proliferation as measured by Cell Trace Violet dilution. Representative flow plots are shown in A and B. Aggregate results are shown in C. (D) CD4 T cells and γδ T cells from BRSV vaccinated or nonvaccinated animals were isolated by MACS and cultured in the presence of autologous APC ± BRSV. After 6 days, cell culture supernatants were analyzed by ELISA for IL-17. (E) CD4 T cells and γδ T cells were MACS purified from peripheral blood of calves infected or not with BRSV strain 375 for 7 days. Purified cells were cultured in the presence of autologous APC ± BRSV. After 6 days, cell culture supernatants were analyzed by ELISA for IL-17. For A-C, background levels of proliferation were subtracted and results are presented as change over mock. Results are pooled from two independent experiments. Data represent means ± SEM.

    Journal: PLoS ONE

    Article Title: Bovine Gamma Delta T Cells Contribute to Exacerbated IL-17 Production in Response to Co-Infection with Bovine RSV and Mannheimia haemolytica

    doi: 10.1371/journal.pone.0151083

    Figure Lengend Snippet: Both CD4 T cells and γδ T cells produce IL-17 in response to BRSV. PBMC were isolated from control or BRSV vaccinated cows and labeled with Cell Trace Violet. Cells were then cultured for 6 days with BRSV. On day 6, CD4 T cells (A) and γδ T cells (B) were analyzed for virus-specific proliferation as measured by Cell Trace Violet dilution. Representative flow plots are shown in A and B. Aggregate results are shown in C. (D) CD4 T cells and γδ T cells from BRSV vaccinated or nonvaccinated animals were isolated by MACS and cultured in the presence of autologous APC ± BRSV. After 6 days, cell culture supernatants were analyzed by ELISA for IL-17. (E) CD4 T cells and γδ T cells were MACS purified from peripheral blood of calves infected or not with BRSV strain 375 for 7 days. Purified cells were cultured in the presence of autologous APC ± BRSV. After 6 days, cell culture supernatants were analyzed by ELISA for IL-17. For A-C, background levels of proliferation were subtracted and results are presented as change over mock. Results are pooled from two independent experiments. Data represent means ± SEM.

    Article Snippet: IL-17 ELISA The Bovine IL-17A VetSet ELISA Development kit was purchased from Kingfisher Biotech, Inc. (St. Paul, MN).

    Techniques: Isolation, Labeling, Cell Culture, Flow Cytometry, Magnetic Cell Separation, Enzyme-linked Immunosorbent Assay, Purification, Infection

    IL-17 and Th17 responses from BRSV vaccinated cattle. Peripheral blood was collected from cows receiving annual vaccinations with a multivalent vaccine containing live-attenuated BRSV (n = 8), or from control cows that were not included in the vaccination program due to inclusion in another study (n = 6). PMBC were isolated and stimulated with BRSV for 24 hours or 6 days, as in Fig 1 . RNA was isolated from the cells and analyzed by qPCR for expression of IL-17 (A, left panel), IL-21 (B) and IL-22 (C). Cell culture supernatants were also analyzed by ELISA for IL-17 (A, right panel). For qPCR analysis, results were normalized to the housekeeping gene RPS-9, and expressed relative to unstimulated control samples. Results were pooled from two independent experiments. Data represent means ± SEM.

    Journal: PLoS ONE

    Article Title: Bovine Gamma Delta T Cells Contribute to Exacerbated IL-17 Production in Response to Co-Infection with Bovine RSV and Mannheimia haemolytica

    doi: 10.1371/journal.pone.0151083

    Figure Lengend Snippet: IL-17 and Th17 responses from BRSV vaccinated cattle. Peripheral blood was collected from cows receiving annual vaccinations with a multivalent vaccine containing live-attenuated BRSV (n = 8), or from control cows that were not included in the vaccination program due to inclusion in another study (n = 6). PMBC were isolated and stimulated with BRSV for 24 hours or 6 days, as in Fig 1 . RNA was isolated from the cells and analyzed by qPCR for expression of IL-17 (A, left panel), IL-21 (B) and IL-22 (C). Cell culture supernatants were also analyzed by ELISA for IL-17 (A, right panel). For qPCR analysis, results were normalized to the housekeeping gene RPS-9, and expressed relative to unstimulated control samples. Results were pooled from two independent experiments. Data represent means ± SEM.

    Article Snippet: IL-17 ELISA The Bovine IL-17A VetSet ELISA Development kit was purchased from Kingfisher Biotech, Inc. (St. Paul, MN).

    Techniques: Isolation, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

    WC1.1 + γδ T cells produce IL-17 in response to BRSV. γδ T cells were purified from the peripheral blood of BRSV vaccinated or control animals by FACS based upon their expression of the γδ T cell receptor and either expression of WC1.1, WC1.2 or lack of WC1. Cells were cultured in the presence of autologous APC ± BRSV for 6 days. Cell culture supernatants were then analyzed by ELISA for IL-17. (B) Cell culture supernatants from (A) were also diluted 1:1 and added to BT cells for 24 hours. After 24 hours, BT were analyzed by qPCR for expression of IL-8. For qPCR analysis, results were normalized to the housekeeping gene RPS-9, and expressed relative to unstimulated control samples. Results are pooled from two independent experiments. Data represent means ± SEM.

    Journal: PLoS ONE

    Article Title: Bovine Gamma Delta T Cells Contribute to Exacerbated IL-17 Production in Response to Co-Infection with Bovine RSV and Mannheimia haemolytica

    doi: 10.1371/journal.pone.0151083

    Figure Lengend Snippet: WC1.1 + γδ T cells produce IL-17 in response to BRSV. γδ T cells were purified from the peripheral blood of BRSV vaccinated or control animals by FACS based upon their expression of the γδ T cell receptor and either expression of WC1.1, WC1.2 or lack of WC1. Cells were cultured in the presence of autologous APC ± BRSV for 6 days. Cell culture supernatants were then analyzed by ELISA for IL-17. (B) Cell culture supernatants from (A) were also diluted 1:1 and added to BT cells for 24 hours. After 24 hours, BT were analyzed by qPCR for expression of IL-8. For qPCR analysis, results were normalized to the housekeeping gene RPS-9, and expressed relative to unstimulated control samples. Results are pooled from two independent experiments. Data represent means ± SEM.

    Article Snippet: IL-17 ELISA The Bovine IL-17A VetSet ELISA Development kit was purchased from Kingfisher Biotech, Inc. (St. Paul, MN).

    Techniques: Purification, FACS, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

    IL-17 and Th17 responses in the lungs and peripheral blood of calves infected with BRSV. Calves (n = 8) were infected via aerosol inoculation with BRSV Strain 375 (RSV) as described in Materials and Methods. Control calves remained uninfected (n = 8). On day 7 post-infection, the animals were sacrificed and the lungs analyzed by qPCR for expression of IL-17, IL-21 and IL-22 (A) and IL-8 (B). In separate experiments, PBMC were isolated from control (n = 8) and BRSV infected calves (n = 8) on day 7-post infection. PBMC were stimulated with BRSV for 24 hours and then analyzed for expression of IL-17 by qPCR (C); or for 6 days and then cell culture supernatants were analyzed by ELISA for expression of IL-17 (D). For qPCR analysis, results were normalized to the housekeeping gene RPS-9, and expressed relative to samples from uninfected control calves. Results were pooled from a total of three independent experiments. Data represent means ± SEM.

    Journal: PLoS ONE

    Article Title: Bovine Gamma Delta T Cells Contribute to Exacerbated IL-17 Production in Response to Co-Infection with Bovine RSV and Mannheimia haemolytica

    doi: 10.1371/journal.pone.0151083

    Figure Lengend Snippet: IL-17 and Th17 responses in the lungs and peripheral blood of calves infected with BRSV. Calves (n = 8) were infected via aerosol inoculation with BRSV Strain 375 (RSV) as described in Materials and Methods. Control calves remained uninfected (n = 8). On day 7 post-infection, the animals were sacrificed and the lungs analyzed by qPCR for expression of IL-17, IL-21 and IL-22 (A) and IL-8 (B). In separate experiments, PBMC were isolated from control (n = 8) and BRSV infected calves (n = 8) on day 7-post infection. PBMC were stimulated with BRSV for 24 hours and then analyzed for expression of IL-17 by qPCR (C); or for 6 days and then cell culture supernatants were analyzed by ELISA for expression of IL-17 (D). For qPCR analysis, results were normalized to the housekeeping gene RPS-9, and expressed relative to samples from uninfected control calves. Results were pooled from a total of three independent experiments. Data represent means ± SEM.

    Article Snippet: IL-17 ELISA The Bovine IL-17A VetSet ELISA Development kit was purchased from Kingfisher Biotech, Inc. (St. Paul, MN).

    Techniques: Infection, Real-time Polymerase Chain Reaction, Expressing, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay

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

    Journal: Veterinary Research

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

    doi: 10.1186/s13567-017-0426-5

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

    Article Snippet: The culture supernatants from the technical replicates were harvested and pooled together after 96 h and stored at −20 °C prior to analysis by the commercial anti-bovine IL-17A ELISA (Kingfisher Biotech).

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

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0063471

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

    Article Snippet: The sequence of incubation steps was as follows: (1) rabbit antibodies to bovine IL-17A (Kingfisher Biotech) (1 µg/ml in PBS 0.1 M pH 7.3) overnight at 4°C; (2) blocking with 0.5% gelatin in PBS for 30 min at 37°C; (3) samples under test (used neat or half diluted in PBSG) and a series of dilutions of insect cell culture supernatant containing a known concentration of recombinant bovine IL-17A to establish the standard curve, for 2 h at 37°C; the concentration of the IL-17A in the insect cell culture supernatant obtained as described was determined with reference to commercialized recombinant bovine IL-17A (Kingfisher) used as standard in the ELISA, and was preferred to this latter owing to its better stability. (4) incubation with 1 µg/ml biotinylated affinity-purifed Ab to the N-terminal peptide of bovine IL-17A for 1 h at 37°C; (5) incubation with peroxidase-conjugated avidin (Neutravidin, Molecular Probes) diluted 1/20 000 for 30 min; (6) incubation with 3,3′,5,5′-tetramethylbenzidine (TMB) for ELISA (Uptima, Interchim, Montluçon, France).

    Techniques: Immunohistochemistry, Inhibition, Labeling, Negative Control

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0063471

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

    Article Snippet: The sequence of incubation steps was as follows: (1) rabbit antibodies to bovine IL-17A (Kingfisher Biotech) (1 µg/ml in PBS 0.1 M pH 7.3) overnight at 4°C; (2) blocking with 0.5% gelatin in PBS for 30 min at 37°C; (3) samples under test (used neat or half diluted in PBSG) and a series of dilutions of insect cell culture supernatant containing a known concentration of recombinant bovine IL-17A to establish the standard curve, for 2 h at 37°C; the concentration of the IL-17A in the insect cell culture supernatant obtained as described was determined with reference to commercialized recombinant bovine IL-17A (Kingfisher) used as standard in the ELISA, and was preferred to this latter owing to its better stability. (4) incubation with 1 µg/ml biotinylated affinity-purifed Ab to the N-terminal peptide of bovine IL-17A for 1 h at 37°C; (5) incubation with peroxidase-conjugated avidin (Neutravidin, Molecular Probes) diluted 1/20 000 for 30 min; (6) incubation with 3,3′,5,5′-tetramethylbenzidine (TMB) for ELISA (Uptima, Interchim, Montluçon, France).

    Techniques: Immunohistochemistry, Inhibition, Labeling, Negative Control

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0063471

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

    Article Snippet: The sequence of incubation steps was as follows: (1) rabbit antibodies to bovine IL-17A (Kingfisher Biotech) (1 µg/ml in PBS 0.1 M pH 7.3) overnight at 4°C; (2) blocking with 0.5% gelatin in PBS for 30 min at 37°C; (3) samples under test (used neat or half diluted in PBSG) and a series of dilutions of insect cell culture supernatant containing a known concentration of recombinant bovine IL-17A to establish the standard curve, for 2 h at 37°C; the concentration of the IL-17A in the insect cell culture supernatant obtained as described was determined with reference to commercialized recombinant bovine IL-17A (Kingfisher) used as standard in the ELISA, and was preferred to this latter owing to its better stability. (4) incubation with 1 µg/ml biotinylated affinity-purifed Ab to the N-terminal peptide of bovine IL-17A for 1 h at 37°C; (5) incubation with peroxidase-conjugated avidin (Neutravidin, Molecular Probes) diluted 1/20 000 for 30 min; (6) incubation with 3,3′,5,5′-tetramethylbenzidine (TMB) for ELISA (Uptima, Interchim, Montluçon, France).

    Techniques: Enzyme-linked Immunosorbent Assay