fibroblast starvation medium  (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 experiments, HBF were plated at a density of 150,000 cells/mL in fibroblast growth medium for 24 h prior to serum starving in fibroblast starvation medium (FGM Bulletkit medium with gentamycin sulfate amphotericin B and 0.4% FBS) for 24 h. HBF inhibitors, including IL1 receptor antagonist (IL1RA, 100 ng/mL, R&D Systems, Inc.), inhibitor of Src-family kinases (PP2 and control (PP3), 10 μM, Cayman Chemical, Ann Arbor, MI, USA), IκB kinase inhibitor (BMS-345541, 10 μM, Cayman Chemical), and Janus-associated kinase (JAK) inhibitor (Ruxolitinib, 100 nM, Cayman Chemical), were added to HBF medium for 30 min prior to stimulation with eosinophil supernatant fluids or control medium (1:1 ratio with fibroblast starvation medium).

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 experiments, HBF were plated at a density of 150,000 cells/mL in fibroblast growth medium for 24 h prior to serum starving in fibroblast starvation medium (FGM Bulletkit medium with gentamycin sulfate amphotericin B and 0.4% FBS) for 24 h. HBF inhibitors, including IL1 receptor antagonist (IL1RA, 100 ng/mL, R&D Systems, Inc.), inhibitor of Src-family kinases (PP2 and control (PP3), 10 μM, Cayman Chemical, Ann Arbor, MI, USA), IκB kinase inhibitor (BMS-345541, 10 μM, Cayman Chemical), and Janus-associated kinase (JAK) inhibitor (Ruxolitinib, 100 nM, Cayman Chemical), were added to HBF medium for 30 min prior to stimulation with eosinophil supernatant fluids or control medium (1:1 ratio with fibroblast starvation medium).

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: 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 experiments, HBF were plated at a density of 150,000 cells/mL in fibroblast growth medium for 24 h prior to serum starving in fibroblast starvation medium (FGM Bulletkit medium with gentamycin sulfate amphotericin B and 0.4% FBS) for 24 h. HBF inhibitors, including IL1 receptor antagonist (IL1RA, 100 ng/mL, R&D Systems, Inc.), inhibitor of Src-family kinases (PP2 and control (PP3), 10 μM, Cayman Chemical, Ann Arbor, MI, USA), IκB kinase inhibitor (BMS-345541, 10 μM, Cayman Chemical), and Janus-associated kinase (JAK) inhibitor (Ruxolitinib, 100 nM, Cayman Chemical), were added to HBF medium for 30 min prior to stimulation with eosinophil supernatant fluids or control medium (1:1 ratio with fibroblast starvation medium).

Techniques: Incubation, Control, 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 experiments, HBF were plated at a density of 150,000 cells/mL in fibroblast growth medium for 24 h prior to serum starving in fibroblast starvation medium (FGM Bulletkit medium with gentamycin sulfate amphotericin B and 0.4% FBS) for 24 h. HBF inhibitors, including IL1 receptor antagonist (IL1RA, 100 ng/mL, R&D Systems, Inc.), inhibitor of Src-family kinases (PP2 and control (PP3), 10 μM, Cayman Chemical, Ann Arbor, MI, USA), IκB kinase inhibitor (BMS-345541, 10 μM, Cayman Chemical), and Janus-associated kinase (JAK) inhibitor (Ruxolitinib, 100 nM, Cayman Chemical), were added to HBF medium for 30 min prior to stimulation with eosinophil supernatant fluids or control medium (1:1 ratio with fibroblast starvation medium).

Techniques: Activation Assay, Incubation, Control, 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 experiments, HBF were plated at a density of 150,000 cells/mL in fibroblast growth medium for 24 h prior to serum starving in fibroblast starvation medium (FGM Bulletkit medium with gentamycin sulfate amphotericin B and 0.4% FBS) for 24 h. HBF inhibitors, including IL1 receptor antagonist (IL1RA, 100 ng/mL, R&D Systems, Inc.), inhibitor of Src-family kinases (PP2 and control (PP3), 10 μM, Cayman Chemical, Ann Arbor, MI, USA), IκB kinase inhibitor (BMS-345541, 10 μM, Cayman Chemical), and Janus-associated kinase (JAK) inhibitor (Ruxolitinib, 100 nM, Cayman Chemical), were added to HBF medium for 30 min prior to stimulation with eosinophil supernatant fluids or control medium (1:1 ratio with fibroblast starvation medium).

Techniques: Lysis, Incubation, Control, 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 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 experiments, HBF were plated at a density of 150,000 cells/mL in fibroblast growth medium for 24 h prior to serum starving in fibroblast starvation medium (FGM Bulletkit medium with gentamycin sulfate amphotericin B and 0.4% FBS) for 24 h. HBF inhibitors, including IL1 receptor antagonist (IL1RA, 100 ng/mL, R&D Systems, Inc.), inhibitor of Src-family kinases (PP2 and control (PP3), 10 μM, Cayman Chemical, Ann Arbor, MI, USA), IκB kinase inhibitor (BMS-345541, 10 μM, Cayman Chemical), and Janus-associated kinase (JAK) inhibitor (Ruxolitinib, 100 nM, Cayman Chemical), were added to HBF medium for 30 min prior to stimulation with eosinophil supernatant fluids or control medium (1:1 ratio with fibroblast starvation medium).

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

Journal: Frontiers in Immunology

Article Title: Male Macrophages and Fibroblasts from C57/BL6J Mice Are More Susceptible to Inflammatory Stimuli

doi: 10.3389/fimmu.2021.758767

Figure Lengend Snippet: Characterization of bone marrow derived macrophages (BMMs) and cardiac fibroblasts. (A) Flow cytometric analysis of the F4/80 and CD11b expression in BMMs. Representative images of BMMs stained against (B, C) F4/80 and (D, E) CD11b. Representative images of cardiac fibroblasts stained against (F, G) vimentin, (H, I) CD31 (endothelial marker; negative control) and (J, K) desmin (smooth muscle marker; negative control). Nuclei were stained with DAPI (C, E, G, I, K) Magnification: 40x; scale bar: 50 µm. Data are representative of 3 independent experiments with similar results.

Article Snippet: Fibroblasts were activated using 20 ng/ml TNF-α ( ) (PeproTech, Germany), 10 ng/ml TGF-β (PeproTech, Germany) ( ) or 10 ng/ml LPS (Sigma, Germany) for 24 h in fibroblast starvation medium (with 2.5% charcoal-stripped FCS, Biochrom, Germany).

Techniques: Derivative Assay, Expressing, Staining, Marker, Negative Control

Journal: Frontiers in Immunology

Article Title: Male Macrophages and Fibroblasts from C57/BL6J Mice Are More Susceptible to Inflammatory Stimuli

doi: 10.3389/fimmu.2021.758767

Figure Lengend Snippet: TNF-α, but not TGF-β, induces pro-fibrotic phenotype in mouse cardiac fibroblasts. Real-time PCR analyses of (A) MCP-1, (B) TGF-β, (C) IL-1β, and (D) Col1A1 performed with lysates from male and female mice cardiac fibroblasts treated with 20 ng/ml TNF-α for 24 h. Real-time PCR analyses of (E) MCP-1 and (F) Col1A1 performed with lysates from male and female mice cardiac fibroblasts treated with 10 ng/ml TGF-β for 24 h. Data are shown as means ± SEM (n = 6; independent experiments with technical duplicates). Data are normalized to the male untreated group. *p < 0.05, **p < 0.01, untreated vs. treated.

Article Snippet: Fibroblasts were activated using 20 ng/ml TNF-α ( ) (PeproTech, Germany), 10 ng/ml TGF-β (PeproTech, Germany) ( ) or 10 ng/ml LPS (Sigma, Germany) for 24 h in fibroblast starvation medium (with 2.5% charcoal-stripped FCS, Biochrom, Germany).

Techniques: Real-time Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Male Macrophages and Fibroblasts from C57/BL6J Mice Are More Susceptible to Inflammatory Stimuli

doi: 10.3389/fimmu.2021.758767

Figure Lengend Snippet: Pro-inflammatory environment promotes a pro-inflammatory and pro-fibrotic fibroblast phenotype. Real-time PCR analyses of (A) MCP-1, (B) TNF-α, (C) NFκB, and (D) IL-1β performed with lysates from male and female mice cardiac fibroblasts cultivated with conditioned medium from M1 polarized BMMs for 24 h. Data are shown as means ± SEM (n = 6; independent experiments with technical duplicates). Data are normalized to the male untreated group. **p < 0.01, ***p < 0.001, untreated vs. treated; ## p < 0.01, male vs. female.

Article Snippet: Fibroblasts were activated using 20 ng/ml TNF-α ( ) (PeproTech, Germany), 10 ng/ml TGF-β (PeproTech, Germany) ( ) or 10 ng/ml LPS (Sigma, Germany) for 24 h in fibroblast starvation medium (with 2.5% charcoal-stripped FCS, Biochrom, Germany).

Techniques: Real-time Polymerase Chain Reaction