goat polyclonal anti il 6  (R&D Systems)

Journal: Cells

Article Title: Interleukin-1α Is a Critical Mediator of the Response of Human Bronchial Fibroblasts to Eosinophilic Inflammation

doi: 10.3390/cells10030528

Figure Lengend Snippet: Interleukin 6 (IL6) and IL8 expression and secretion by human bronchial fibroblasts stimulated with eosinophil-derived soluble mediators requires signaling via the IL1 receptor. Human bronchial fibroblasts (HBF) were incubated with IL1 receptor antagonist (IL1RA, 100 ng/mL) or vehicle (0.1% bovine serum albumin (BSA) in PBS) for 30 min, and subsequently stimulated with eosinophil supernatants (IL3 or IL3IgG) or basal medium (Ctrl). Twenty-four hours later, HBF supernatants were analyzed via ELISA for levels of IL8 ( A ) and IL6 ( B ) ( n = 8 for all conditions), and paired Student’s t -test was used to test for statistical significance (* p < 0.05). HBF lysates were analyzed for mRNA levels of CXCL8 ( C ) and IL6 ( D ) via RT-qPCR ( n = 3 for all conditions) and analyzed by setting IL3IgG as a reference (mean ± sd) and using unpaired Student’s t -test to test for statistical significance (* p < 0.05).

Article Snippet: For detection, biotinylated mouse monoclonal anti-human IL8 (clone G265-8, BD Biosciences) and biotinylated polyclonal goat anti-human IL6 (R&D Systems, Inc.) were utilized with sensitivities of less than 3 pg/mL for IL8 and IL6.

Techniques: Expressing, Derivative Assay, Incubation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Cells

Article Title: Interleukin-1α Is a Critical Mediator of the Response of Human Bronchial Fibroblasts to Eosinophilic Inflammation

doi: 10.3390/cells10030528

Figure Lengend Snippet: Dose response to recombinant IL1β and IL1α in human bronchial fibroblasts. Primary culture HBF were treated for 24 h with recombinant (r)IL1β ( 1 , 2 ) or rIL1α ( 3 , 4 ) using concentrations indicated in the figure. HBF supernatants were collected and release of IL6 and IL8 was analyzed via ELISA ( n = 2 technical replicates).

Article Snippet: For detection, biotinylated mouse monoclonal anti-human IL8 (clone G265-8, BD Biosciences) and biotinylated polyclonal goat anti-human IL6 (R&D Systems, Inc.) were utilized with sensitivities of less than 3 pg/mL for IL8 and IL6.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Interleukin-1α Is a Critical Mediator of the Response of Human Bronchial Fibroblasts to Eosinophilic Inflammation

doi: 10.3390/cells10030528

Figure Lengend Snippet: IL1β and IL1α neutralizing antibody validation and dose response. HBF were treated for 24 h with recombinant (r)IL1β (1 ng/mL; 1 , 2 ) or rIL1α (1 ng/mL; 3 , 4 ) that had been pre-incubated with IgG, IL1β (clone 4H5) or IL1α (clone 7D4) neutralizing antibodies (doses indicated in figure) for 30 min. After stimulation, HBF supernatants were assessed for IL6 and IL8 via ELISA ( n = 1).

Article Snippet: For detection, biotinylated mouse monoclonal anti-human IL8 (clone G265-8, BD Biosciences) and biotinylated polyclonal goat anti-human IL6 (R&D Systems, Inc.) were utilized with sensitivities of less than 3 pg/mL for IL8 and IL6.

Techniques: Recombinant, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Interleukin-1α Is a Critical Mediator of the Response of Human Bronchial Fibroblasts to Eosinophilic Inflammation

doi: 10.3390/cells10030528

Figure Lengend Snippet: Release of IL6 and IL8 by human bronchial fibroblasts stimulated with products from activated eosinophils is independent of IL1β. HBF were stimulated with eosinophil supernatants (IL3 or IL3IgG) or basal medium (Ctrl). IL3IgG eosinophil supernatants were incubated for 1 h with IgG control antibody (1 µg/mL) or IL1β neutralizing antibody (aIL1β, 1 µg/mL) prior to stimulation, as indicated. Twenty-four hours later, HBF supernatants were tested for IL8 ( A ) and IL6 ( B ) via ELISA ( n = 4 for all conditions). Paired Student’s t -test was used to test for statistically significant differences (NS = not significant).

Article Snippet: For detection, biotinylated mouse monoclonal anti-human IL8 (clone G265-8, BD Biosciences) and biotinylated polyclonal goat anti-human IL6 (R&D Systems, Inc.) were utilized with sensitivities of less than 3 pg/mL for IL8 and IL6.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Interleukin-1α Is a Critical Mediator of the Response of Human Bronchial Fibroblasts to Eosinophilic Inflammation

doi: 10.3390/cells10030528

Figure Lengend Snippet: Cross validation of IL1β with IL1α neutralizing antibody. HBF were treated with vehicle control (Ctrl) or recombinant (r)IL1β (1 ng/mL), pre-incubated with IgG (1 µg/mL), IL1β (clone 4H5, 1 µg/mL) or IL1α (clone 7D4, 1 µg/mL) neutralizing antibodies for 30 min. After stimulation, HBF supernatants were assessed for IL6 ( 1 ) and IL8 ( 2 ) via ELISA ( n = 2 technical replicates).

Article Snippet: For detection, biotinylated mouse monoclonal anti-human IL8 (clone G265-8, BD Biosciences) and biotinylated polyclonal goat anti-human IL6 (R&D Systems, Inc.) were utilized with sensitivities of less than 3 pg/mL for IL8 and IL6.

Techniques: Recombinant, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Interleukin-1α Is a Critical Mediator of the Response of Human Bronchial Fibroblasts to Eosinophilic Inflammation

doi: 10.3390/cells10030528

Figure Lengend Snippet: Release of IL6 and IL8 by human bronchial fibroblasts stimulated with eosinophil soluble mediators is dependent on activation by IL1α. HBF were stimulated with eosinophil supernatants (IL3 or IL3IgG) or basal medium (Ctrl). IL3IgG supernatants were incubated for 1 h with either IgG control antibody (1 µg/mL) or IL1α neutralizing antibody (aIL1α, 1 µg/mL) prior to stimulation, as indicated. Twenty-four hours later, HBF supernatants were collected, and IL8 ( A ) and IL6 ( B ) levels were analyzed via ELISA ( n = 4 for all conditions). Paired Student’s t -test was used to test for statistically significant differences (* p < 0.05).

Article Snippet: For detection, biotinylated mouse monoclonal anti-human IL8 (clone G265-8, BD Biosciences) and biotinylated polyclonal goat anti-human IL6 (R&D Systems, Inc.) were utilized with sensitivities of less than 3 pg/mL for IL8 and IL6.

Techniques: Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Interleukin-1α Is a Critical Mediator of the Response of Human Bronchial Fibroblasts to Eosinophilic Inflammation

doi: 10.3390/cells10030528

Figure Lengend Snippet: Eosinophil lysis products induce the production of IL6 by human bronchial fibroblasts through the IL1α-dependent nuclear factor kappa-light-chain-enhancer of activated B cell (NFκB) signaling mechanism. ( A ) IL3IgG supernatants were incubated for 1 h with either IgG control antibody (1 µg/mL), IL1α neutralizing antibody (aIL1α, 1 µg/mL) or IL1β neutralizing antibody (aIL1β, 1 µg/mL) prior to stimulation of HBF. HBF were alternatively incubated with basal medium (Ctrl), IL3 or IL3IgG eosinophil supernatants as a control. Thirty minutes after stimulation, HBF protein lysates were obtained and subjected to SDS-PAGE and Western blotting with indicated antibodies. ( B ) Densitometry of Western blots from A. ( n = 3 for all conditions) was done in ImageJ (mean ± sd) and analyzed by unpaired Student’s t -test with Bonferroni correction to determine statistically significant differences (* p < 0.025). ( C ) HBF were treated with IκB kinase inhibitor (10 µM BMS-345541 (BMS)) or vehicle (dimethyl sulfoxide (DMSO)) prior to stimulation with eosinophil supernatants for 24 h. HBF supernatants were collected ( n = 7 for all conditions) and levels of IL6 were determined via ELISA. Paired Student’s t -test was used to test for statistically significant differences (* p < 0.05).

Article Snippet: For detection, biotinylated mouse monoclonal anti-human IL8 (clone G265-8, BD Biosciences) and biotinylated polyclonal goat anti-human IL6 (R&D Systems, Inc.) were utilized with sensitivities of less than 3 pg/mL for IL8 and IL6.

Techniques: Lysis, Incubation, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Interleukin-1α Is a Critical Mediator of the Response of Human Bronchial Fibroblasts to Eosinophilic Inflammation

doi: 10.3390/cells10030528

Figure Lengend Snippet: Eosinophil-derived supernatants lead to the production of IL6 by HBF through the IL1-independent Janus Kinase (JAK)/Signal Transducer and Activator of Transcription Protein (STAT) signaling mechanism. ( A ) IL3IgG supernatants were incubated for 1 h with either IgG control antibody (1 µg/mL), IL1α neutralizing antibody (aIL1α, 1 µg/mL) or IL1β neutralizing antibody (aIL1β, 1 µg/mL) prior to stimulation of HBF. HBF were alternatively incubated with basal medium (Ctrl), IL3 or IL3IgG eosinophil supernatants as a control. Thirty minutes after stimulation, HBF protein lysates were obtained and subjected to SDS-PAGE and Western blotting with indicated antibodies. ( B ) Densitometry of Western blots from A. ( n = 3 for all conditions) was done in ImageJ (mean ± sd). C-D. HBF were treated with Janus-associated kinase (JAK) inhibitor (100 nM ruxolitinib (Ruxo)) or vehicle (ethanol) prior to stimulation with eosinophil supernatants for 24 h. mRNA levels of IL6 in HBF ( n = 3 for all conditions) were assessed via RT-qPCR ( C ). Levels of IL6 in HBF supernatant ( n = 3 for all conditions) were assessed via ELISA ( D ). Unpaired ( B , C ) or paired ( D ) Student’s t -test was used to test for statistically significant differences (* p < 0.05).

Article Snippet: For detection, biotinylated mouse monoclonal anti-human IL8 (clone G265-8, BD Biosciences) and biotinylated polyclonal goat anti-human IL6 (R&D Systems, Inc.) were utilized with sensitivities of less than 3 pg/mL for IL8 and IL6.

Techniques: Derivative Assay, Incubation, SDS Page, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Interleukin-1α Is a Critical Mediator of the Response of Human Bronchial Fibroblasts to Eosinophilic Inflammation

doi: 10.3390/cells10030528

Figure Lengend Snippet: Eosinophil lysis products lead to the release of IL8 by human bronchial fibroblasts through the Src family kinase signaling mechanism. HBF were treated with IκB kinase inhibitor (10 µM BMS-345541 (BMS)), or vehicle (DMSO) ( A ), or inhibitor of Src-family kinases (10 µM PP2 (or PP3 control)) ( B – D ) prior to stimulation with eosinophil supernatants. Twenty-four hours later, HBF lysates were assessed for levels of IL8 or IL6 ( n = 7 for all conditions in A; n = 3 for Ctrl and IL3 conditions and n = 4 for IL3IgG + PP3 and IL3IgG + PP2 conditions in ( B , D )) via ELISA ( A – D ). At the same time point, mRNA levels of CXCL8 in HBF ( n = 3 for all conditions) were assessed via RT-qPCR (mean ± sd). Paired ( A – D ) or unpaired ( C ) Student’s t -test was used to test for statistically significant differences (* p < 0.05).

Article Snippet: For detection, biotinylated mouse monoclonal anti-human IL8 (clone G265-8, BD Biosciences) and biotinylated polyclonal goat anti-human IL6 (R&D Systems, Inc.) were utilized with sensitivities of less than 3 pg/mL for IL8 and IL6.

Techniques: Lysis, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR