polyclonal rabbit anti bovine il 17a antibodies  (Bio-Rad)

Journal: Scientific Reports

Article Title: Functionally distinct T-helper cell phenotypes predict resistance to different types of parasites in a wild mammal

doi: 10.1038/s41598-022-07149-9

Figure Lengend Snippet: Age- and/or sex-specific variation in ( A ) PMBC; ( B ) CD4 + cells; ( C ) CD4 + Tbet + cells; ( D ) CD4 + Gata3 + cells; ( E ) CD4 + RORγt + cells; ( F ) CD4 + FoxP3 + cells; ( G ) IFN-γ; ( H ) IL-4; ( I ) IL-17A; and ( J ) IL-10. Points show raw data, with green representing females and purple showing males. Large points connected by lines show estimates ± 95% CI from a model where age was fitted as a factor (with 2 or 4 levels) and lines with shaded areas show estimates ± 95% CI from models where age was fitted as a continuous variable; green lines represent females and purple lines males. For model details, see Supplementary Tables and .

Article Snippet: For the quantification of IL-17A, polyclonal rabbit anti-bovine IL-17A antibodies were used alongside bovine recombinant protein (Kingfisher Biotech, Inc., St. Paul, MN).

Techniques:

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: Commercial anti-IL-17A antibodies evaluated by intracellular staining for capacity to bind recombinant bovine and ovine IL-17A

Article Snippet: The plate was washed five times with PBS and 0.5 μg/mL biotinylated rabbit polyclonal anti-bovine IL-17A antibody (product code number PB0277B-50, Kingfisher Biotech) in PBS with 0.5% FBS was added to the wells and incubated for 2 h at RT.

Techniques: Staining, Recombinant, Conjugation Assay, Derivative Assay

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: Isotype control mabs and control pab used in the evaluation of the commercial anti-IL-17A antibodies

Article Snippet: The plate was washed five times with PBS and 0.5 μg/mL biotinylated rabbit polyclonal anti-bovine IL-17A antibody (product code number PB0277B-50, Kingfisher Biotech) in PBS with 0.5% FBS was added to the wells and incubated for 2 h at RT.

Techniques: Control, Staining, Conjugation Assay, Construct, Virus

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: Commercial antibodies used in the detection of native intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets

Article Snippet: The plate was washed five times with PBS and 0.5 μg/mL biotinylated rabbit polyclonal anti-bovine IL-17A antibody (product code number PB0277B-50, Kingfisher Biotech) in PBS with 0.5% FBS was added to the wells and incubated for 2 h at RT.

Techniques: Conjugation Assay, Marker

Journal: Veterinary Research

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

doi: 10.1186/s13567-017-0426-5

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

Article Snippet: The plate was washed five times with PBS and 0.5 μg/mL biotinylated rabbit polyclonal anti-bovine IL-17A antibody (product code number PB0277B-50, Kingfisher Biotech) in PBS with 0.5% FBS was added to the wells and incubated for 2 h at RT.

Techniques: Sequencing, Sampling, Comparison

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 plate was washed five times with PBS and 0.5 μg/mL biotinylated rabbit polyclonal anti-bovine IL-17A antibody (product code number PB0277B-50, Kingfisher Biotech) in PBS with 0.5% FBS was added to the wells and incubated for 2 h at RT.

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

Journal: Veterinary Research

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

doi: 10.1186/s13567-017-0426-5

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

Article Snippet: The plate was washed five times with PBS and 0.5 μg/mL biotinylated rabbit polyclonal anti-bovine IL-17A antibody (product code number PB0277B-50, Kingfisher Biotech) in PBS with 0.5% FBS was added to the wells and incubated for 2 h at RT.

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

Journal: Veterinary Research

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

doi: 10.1186/s13567-017-0426-5

Figure Lengend Snippet: Evaluation of commercial antibodies for the intracellular detection of recombinant bovine and ovine IL-17A. The eight commercial antibodies listed in Table 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 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 . Line colours representing different antibody treatments are given in parentheses: A Primary rabbit anti-bovine IL-17A pab PB0274B-100 at 1 μg/mL (A.1, red) or negative control primary anti-bovine CD34 pab (in-house) at an estimated 1 μg/mL equivalent (a, black) then detected with a secondary goat anti-rabbit alexafluor 488 at 1 μg/mL; B Directly conjugated mouse anti-human IL-17A eBio64DEC17-phycoerythrin (PE) mab (IgG1) at 2.5 μg/mL (B.1, red) and control IgG1 VPM21 mab (in-house) at an estimated 2.5 μg/mL equivalent (b, black) and detected with goat anti-mouse PE at 1 μg/mL; C Primary mouse anti-human IL-17A mabs MT44.6 (C.1, blue), MT241 (C.2, green), MT2770 (C.3, brown) and MT504 (C.4, red) [all IgG1] at 0.5 μg/mL and control IgG1 VPM21 mab (in-house) at an estimated 0.5 μg/mL equivalent (black), all detected with goat anti-mouse PE at 1 μg/mL; D Primary mouse anti-human IL-17A mabs #41809 (D.1, red) (IgG2b) and #41802 (D.2, blue) (IgG1) at 2.5 μg/mL and a mixture of control mabs VPM21 (IgG1) and VPM22 (IgG2b) at an estimated 2.5 μg/mL equivalent (d, black), all detected with goat anti-mouse PE at 1 μg/mL.

Article Snippet: The plate was washed five times with PBS and 0.5 μg/mL biotinylated rabbit polyclonal anti-bovine IL-17A antibody (product code number PB0277B-50, Kingfisher Biotech) in PBS with 0.5% FBS was added to the wells and incubated for 2 h at RT.

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

Journal: Veterinary Research

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

doi: 10.1186/s13567-017-0426-5

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

Article Snippet: The plate was washed five times with PBS and 0.5 μg/mL biotinylated rabbit polyclonal anti-bovine IL-17A antibody (product code number PB0277B-50, Kingfisher Biotech) in PBS with 0.5% FBS was added to the wells and incubated for 2 h at RT.

Techniques: Expressing, Staining

Journal: Veterinary Research

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

doi: 10.1186/s13567-017-0426-5

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

Article Snippet: The plate was washed five times with PBS and 0.5 μg/mL biotinylated rabbit polyclonal anti-bovine IL-17A antibody (product code number PB0277B-50, Kingfisher Biotech) in PBS with 0.5% FBS was added to the wells and incubated for 2 h at RT.

Techniques: Expressing, Staining

Journal: Veterinary Research

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

doi: 10.1186/s13567-017-0426-5

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

Article Snippet: The plate was washed five times with PBS and 0.5 μg/mL biotinylated rabbit polyclonal anti-bovine IL-17A antibody (product code number PB0277B-50, Kingfisher Biotech) in PBS with 0.5% FBS was added to the wells and incubated for 2 h at RT.

Techniques: Expressing, Two Tailed Test, MANN-WHITNEY

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: Commercial anti-IL-17A antibodies evaluated by intracellular staining for capacity to bind recombinant bovine and ovine IL-17A

Article Snippet: MultiScreen-IP Filter Plates (Merck Millipore, Hertfordshire, UK) were activated by addition of 70% ethanol (Fisher Scientific, Loughborough, UK) for a maximum of 2 min. Plates were washed five times with sterile ddH 2 O and then incubated with 50 μL/well of 5 μg/mL rabbit polyclonal anti-bovine IL-17A antibody (product code PB0274B-100, Kingfisher Biotech) for 18 h at 4 °C.

Techniques: Staining, Recombinant, Conjugation Assay, Derivative Assay

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: Isotype control mabs and control pab used in the evaluation of the commercial anti-IL-17A antibodies

Article Snippet: MultiScreen-IP Filter Plates (Merck Millipore, Hertfordshire, UK) were activated by addition of 70% ethanol (Fisher Scientific, Loughborough, UK) for a maximum of 2 min. Plates were washed five times with sterile ddH 2 O and then incubated with 50 μL/well of 5 μg/mL rabbit polyclonal anti-bovine IL-17A antibody (product code PB0274B-100, Kingfisher Biotech) for 18 h at 4 °C.

Techniques: Control, Staining, Conjugation Assay, Construct, Virus

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: Commercial antibodies used in the detection of native intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets

Article Snippet: MultiScreen-IP Filter Plates (Merck Millipore, Hertfordshire, UK) were activated by addition of 70% ethanol (Fisher Scientific, Loughborough, UK) for a maximum of 2 min. Plates were washed five times with sterile ddH 2 O and then incubated with 50 μL/well of 5 μg/mL rabbit polyclonal anti-bovine IL-17A antibody (product code PB0274B-100, Kingfisher Biotech) for 18 h at 4 °C.

Techniques: Conjugation Assay, Marker

Journal: Veterinary Research

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

doi: 10.1186/s13567-017-0426-5

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

Article Snippet: MultiScreen-IP Filter Plates (Merck Millipore, Hertfordshire, UK) were activated by addition of 70% ethanol (Fisher Scientific, Loughborough, UK) for a maximum of 2 min. Plates were washed five times with sterile ddH 2 O and then incubated with 50 μL/well of 5 μg/mL rabbit polyclonal anti-bovine IL-17A antibody (product code PB0274B-100, Kingfisher Biotech) for 18 h at 4 °C.

Techniques: Sequencing, Sampling, Comparison

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: MultiScreen-IP Filter Plates (Merck Millipore, Hertfordshire, UK) were activated by addition of 70% ethanol (Fisher Scientific, Loughborough, UK) for a maximum of 2 min. Plates were washed five times with sterile ddH 2 O and then incubated with 50 μL/well of 5 μg/mL rabbit polyclonal anti-bovine IL-17A antibody (product code PB0274B-100, Kingfisher Biotech) for 18 h at 4 °C.

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

Journal: Veterinary Research

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

doi: 10.1186/s13567-017-0426-5

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

Article Snippet: MultiScreen-IP Filter Plates (Merck Millipore, Hertfordshire, UK) were activated by addition of 70% ethanol (Fisher Scientific, Loughborough, UK) for a maximum of 2 min. Plates were washed five times with sterile ddH 2 O and then incubated with 50 μL/well of 5 μg/mL rabbit polyclonal anti-bovine IL-17A antibody (product code PB0274B-100, Kingfisher Biotech) for 18 h at 4 °C.

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

Journal: Veterinary Research

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

doi: 10.1186/s13567-017-0426-5

Figure Lengend Snippet: Evaluation of commercial antibodies for the intracellular detection of recombinant bovine and ovine IL-17A. The eight commercial antibodies listed in Table 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 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 . Line colours representing different antibody treatments are given in parentheses: A Primary rabbit anti-bovine IL-17A pab PB0274B-100 at 1 μg/mL (A.1, red) or negative control primary anti-bovine CD34 pab (in-house) at an estimated 1 μg/mL equivalent (a, black) then detected with a secondary goat anti-rabbit alexafluor 488 at 1 μg/mL; B Directly conjugated mouse anti-human IL-17A eBio64DEC17-phycoerythrin (PE) mab (IgG1) at 2.5 μg/mL (B.1, red) and control IgG1 VPM21 mab (in-house) at an estimated 2.5 μg/mL equivalent (b, black) and detected with goat anti-mouse PE at 1 μg/mL; C Primary mouse anti-human IL-17A mabs MT44.6 (C.1, blue), MT241 (C.2, green), MT2770 (C.3, brown) and MT504 (C.4, red) [all IgG1] at 0.5 μg/mL and control IgG1 VPM21 mab (in-house) at an estimated 0.5 μg/mL equivalent (black), all detected with goat anti-mouse PE at 1 μg/mL; D Primary mouse anti-human IL-17A mabs #41809 (D.1, red) (IgG2b) and #41802 (D.2, blue) (IgG1) at 2.5 μg/mL and a mixture of control mabs VPM21 (IgG1) and VPM22 (IgG2b) at an estimated 2.5 μg/mL equivalent (d, black), all detected with goat anti-mouse PE at 1 μg/mL.

Article Snippet: MultiScreen-IP Filter Plates (Merck Millipore, Hertfordshire, UK) were activated by addition of 70% ethanol (Fisher Scientific, Loughborough, UK) for a maximum of 2 min. Plates were washed five times with sterile ddH 2 O and then incubated with 50 μL/well of 5 μg/mL rabbit polyclonal anti-bovine IL-17A antibody (product code PB0274B-100, Kingfisher Biotech) for 18 h at 4 °C.

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

Journal: Veterinary Research

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

doi: 10.1186/s13567-017-0426-5

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

Article Snippet: MultiScreen-IP Filter Plates (Merck Millipore, Hertfordshire, UK) were activated by addition of 70% ethanol (Fisher Scientific, Loughborough, UK) for a maximum of 2 min. Plates were washed five times with sterile ddH 2 O and then incubated with 50 μL/well of 5 μg/mL rabbit polyclonal anti-bovine IL-17A antibody (product code PB0274B-100, Kingfisher Biotech) for 18 h at 4 °C.

Techniques: Expressing, Staining

Journal: Veterinary Research

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

doi: 10.1186/s13567-017-0426-5

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

Article Snippet: MultiScreen-IP Filter Plates (Merck Millipore, Hertfordshire, UK) were activated by addition of 70% ethanol (Fisher Scientific, Loughborough, UK) for a maximum of 2 min. Plates were washed five times with sterile ddH 2 O and then incubated with 50 μL/well of 5 μg/mL rabbit polyclonal anti-bovine IL-17A antibody (product code PB0274B-100, Kingfisher Biotech) for 18 h at 4 °C.

Techniques: Expressing, Staining

Journal: Veterinary Research

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

doi: 10.1186/s13567-017-0426-5

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

Article Snippet: MultiScreen-IP Filter Plates (Merck Millipore, Hertfordshire, UK) were activated by addition of 70% ethanol (Fisher Scientific, Loughborough, UK) for a maximum of 2 min. Plates were washed five times with sterile ddH 2 O and then incubated with 50 μL/well of 5 μg/mL rabbit polyclonal anti-bovine IL-17A antibody (product code PB0274B-100, Kingfisher Biotech) for 18 h at 4 °C.

Techniques: Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Tropical Animal Health and Production

Article Title: Morphological characterization and immunohistochemical detection of the proinflammatory cytokines IL-1β, IL-17A, and TNF-α in lung lesions associated with contagious bovine pleuropneumonia

doi: 10.1007/s11250-016-0994-9

Figure Lengend Snippet: Semi-quantitative assessment of inflammatory cytokines in the lung of infected and uninfected animals

Article Snippet: Bovine IL-1β, IL-17A, and TNF-α were detected by using biotinylated rabbit anti-bovine IL-1β polyclonal antibody (article no. ab23778, Abcam, UK), biotinylated rabbit anti-bovine IL-17A polyclonal antibody (article no. PBB0277B-050, Kingfisher Biotech, Inc., USA), and biotinylated rabbit anti-bovine TNF-α polyclonal antibodies (article no. AHP852B, AbD Serotec, Germany), respectively.

Techniques: Infection, Negative Control

Journal: Tropical Animal Health and Production

Article Title: Morphological characterization and immunohistochemical detection of the proinflammatory cytokines IL-1β, IL-17A, and TNF-α in lung lesions associated with contagious bovine pleuropneumonia

doi: 10.1007/s11250-016-0994-9

Figure Lengend Snippet: Representative qualitative results of the detection of the cytokines IL-1β, IL-17A, and TNF-α. The following polyclonal antibodies were used. a Anti- IL-1β: Lung: control animal; b Anti- IL-1β: Lung: acute CBPP lesion; c Anti- IL-17A: Lung: control animal; d Anti- IL-17A: Lung: acute CBPP lesion; e Anti- TNF-α: Lung: control animal; f Anti- TNF-α: Lung: acute CBPP lesion; g Anti- Mycoplasma : Lung: control animal; h Anti- Mycoplasma : Lung: acute CBPP lesion. Black bars in the lower right corner of images in a – f is the size standard representing 100 μm, black bars in images of g , h represent 20 μm. Arrows indicate positive immunoreactive signals ( reddish-brown )

Article Snippet: Bovine IL-1β, IL-17A, and TNF-α were detected by using biotinylated rabbit anti-bovine IL-1β polyclonal antibody (article no. ab23778, Abcam, UK), biotinylated rabbit anti-bovine IL-17A polyclonal antibody (article no. PBB0277B-050, Kingfisher Biotech, Inc., USA), and biotinylated rabbit anti-bovine TNF-α polyclonal antibodies (article no. AHP852B, AbD Serotec, Germany), respectively.

Techniques: Control