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R&D Systems soluble cd14 protein
Soluble Cd14 Protein, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/recombinant+soluble+cd14/pmc06738877-110-9-21?v=R%26D+Systems
Average 93 stars, based on 34 article reviews

soluble cd14 protein - by Bioz Stars, 2026-08

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$Synergistic effect between huLTF and LTF-Abs. ( A ) Purified human monocytes (Mo, upper panels) and differentiated macrophages (M Ф , lower panels) were stained with FITC-conjugated mAbs against human CD14 or CD11b followed by FACS analysis. ( B ) Freshly fractionated human monocytes and ( C ) human monocytes-differentiated macrophages were incubated with 15 μg/ml huLTF, or RA-IgG, or RA-IgG-IC (mixture of huLTF plus RA-IgG), or huLTF plus IVIG (15 μg/ml for each protein), for 24 h. ( D ) Fractionated human monocytes and ( E ) human monocytes-differentiated macrophages were incubated with 15 μg/ml huLTF, or mouse anti-huLTF mAb M860, or rabbit anti-huLTF polyclonal IgG L3262, or a mixture of huLTF plus M860 ( LTF + M860 ) or L3262 ( LTF + L3262 ) for 24 h. Cells cultured in medium only ( Med ) or stimulated with 3 μg/ml LPS were included as controls. TNF-α concentration in the culture supernatant was then determined by ELISA. Data are mean concentration ± SEM from triplicate wells and these are representatives of at least 3 independent experiments. * p < 0.05, ** p < 0.01 compared with medium controls.$

Journal: Scientific Reports

Article Title: Extraordinarily potent proinflammatory properties of lactoferrin-containing immunocomplexes against human monocytes and macrophages

doi: 10.1038/s41598-017-04275-7

Figure Lengend Snippet: Synergistic effect between huLTF and LTF-Abs. ( A ) Purified human monocytes (Mo, upper panels) and differentiated macrophages (M Ф , lower panels) were stained with FITC-conjugated mAbs against human CD14 or CD11b followed by FACS analysis. ( B ) Freshly fractionated human monocytes and ( C ) human monocytes-differentiated macrophages were incubated with 15 μg/ml huLTF, or RA-IgG, or RA-IgG-IC (mixture of huLTF plus RA-IgG), or huLTF plus IVIG (15 μg/ml for each protein), for 24 h. ( D ) Fractionated human monocytes and ( E ) human monocytes-differentiated macrophages were incubated with 15 μg/ml huLTF, or mouse anti-huLTF mAb M860, or rabbit anti-huLTF polyclonal IgG L3262, or a mixture of huLTF plus M860 ( LTF + M860 ) or L3262 ( LTF + L3262 ) for 24 h. Cells cultured in medium only ( Med ) or stimulated with 3 μg/ml LPS were included as controls. TNF-α concentration in the culture supernatant was then determined by ELISA. Data are mean concentration ± SEM from triplicate wells and these are representatives of at least 3 independent experiments. * p < 0.05, ** p < 0.01 compared with medium controls.

Article Snippet: Recombinant soluble human CD14 was from Peprotech.

Techniques: Purification, Staining, Incubation, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

Role of CD32a and mCD14 in LTF-IC-mediated monocyte activation. Human monocytes were stimulated with 30 μg/ml huLTF-M860-IC ( A ), or RA-IgG-IC ( B ), in the presence or absence ( Med ) of blocking mAbs (15 μg/ml) against CD16 (B73.1, eBioscience), or CD32 (IV.3, eBioscience), or CD64 (10.1, Stem Cell Tech) for 18 h, followed by quantitation of TNF-α in the culture supernatant by ELISA. ( C ) Monocytes stimulated with 3 μg/ml LPS in the presence or absence ( Med ) of anti-CD32a mAb (15 μg/ml), or isotype control Abs as controls. ( D ) Human monocytes were stained with 30 μg/ml M860-IC containing FITC-conjugated huLTF in the presence or absence ( M860-IC ) of 10 μg/ml soluble recombinant human CD14 ( M860-IC + sCD14 ) or OVA ( M860-IC + OVA ) for 1 h at 4 °C, followed by FACS analysis. Unstained cells were included as negative control (filled histogram). ( E ) Human monocytes were treated with 30 μg/ml huLTF-M860 IC in the presence of mouse mAbs (15 μg/ml) against human LRP-1 (α LRP1 ), nucleolin-1 (α Nucleolin ), or CD14 (α CD14 ) for 18 h, followed by quantitation of TNF-α levels in the culture supernatants by ELISA. Cells treated with 1 μg/ml PMA in the presence of αCD14 mAb or isotype control mAb were included for specificity controls. ( F ) The anti-CD14 mAb was titrated against LTF-M860 IC (30 μg/ml) in monocyte activation assays with mouse IgG1 (1 μg/ml) as isotype control. Readings from unstimulated cells ( Med ) are also indicated in the figure. ( G ) TLR4-specific inhibitor CLI-095 was titrated against LTF-M860 IC (30 μg/ml) in monocyte activation assays monitoring TNF-α production. Monocytes stimulated with 5 μg/ml zymosan in the presence or absence of 1 μg/ml CLI-095 were included as specificity control. For ELISAs, values are mean TNF-α concentration (ng/ml) ± SEM from triplicate cultures. * p < 0.05, ** p < 0.01 compared with M860-IC- or LPS-stimulated cells in the presence of isotype control Abs or in the absence of inhibiting agents. NS : not statistically significant. Results are representative of three independent experiments.

Journal: Scientific Reports

Article Title: Extraordinarily potent proinflammatory properties of lactoferrin-containing immunocomplexes against human monocytes and macrophages

doi: 10.1038/s41598-017-04275-7

Figure Lengend Snippet: Role of CD32a and mCD14 in LTF-IC-mediated monocyte activation. Human monocytes were stimulated with 30 μg/ml huLTF-M860-IC ( A ), or RA-IgG-IC ( B ), in the presence or absence ( Med ) of blocking mAbs (15 μg/ml) against CD16 (B73.1, eBioscience), or CD32 (IV.3, eBioscience), or CD64 (10.1, Stem Cell Tech) for 18 h, followed by quantitation of TNF-α in the culture supernatant by ELISA. ( C ) Monocytes stimulated with 3 μg/ml LPS in the presence or absence ( Med ) of anti-CD32a mAb (15 μg/ml), or isotype control Abs as controls. ( D ) Human monocytes were stained with 30 μg/ml M860-IC containing FITC-conjugated huLTF in the presence or absence ( M860-IC ) of 10 μg/ml soluble recombinant human CD14 ( M860-IC + sCD14 ) or OVA ( M860-IC + OVA ) for 1 h at 4 °C, followed by FACS analysis. Unstained cells were included as negative control (filled histogram). ( E ) Human monocytes were treated with 30 μg/ml huLTF-M860 IC in the presence of mouse mAbs (15 μg/ml) against human LRP-1 (α LRP1 ), nucleolin-1 (α Nucleolin ), or CD14 (α CD14 ) for 18 h, followed by quantitation of TNF-α levels in the culture supernatants by ELISA. Cells treated with 1 μg/ml PMA in the presence of αCD14 mAb or isotype control mAb were included for specificity controls. ( F ) The anti-CD14 mAb was titrated against LTF-M860 IC (30 μg/ml) in monocyte activation assays with mouse IgG1 (1 μg/ml) as isotype control. Readings from unstimulated cells ( Med ) are also indicated in the figure. ( G ) TLR4-specific inhibitor CLI-095 was titrated against LTF-M860 IC (30 μg/ml) in monocyte activation assays monitoring TNF-α production. Monocytes stimulated with 5 μg/ml zymosan in the presence or absence of 1 μg/ml CLI-095 were included as specificity control. For ELISAs, values are mean TNF-α concentration (ng/ml) ± SEM from triplicate cultures. * p < 0.05, ** p < 0.01 compared with M860-IC- or LPS-stimulated cells in the presence of isotype control Abs or in the absence of inhibiting agents. NS : not statistically significant. Results are representative of three independent experiments.

Article Snippet: Recombinant soluble human CD14 was from Peprotech.

Techniques: Activation Assay, Blocking Assay, Quantitation Assay, Enzyme-linked Immunosorbent Assay, Control, Staining, Recombinant, Negative Control, Concentration Assay

LTF-IC endocytosis by human peripheral blood monocytes. Human monocytes were sequentially stained with 15 μg/ml FITC-LTF ( A ), mixture of FITC-OVA plus mAb M562 ( B ), or FITC-LTF plus mAb M860 ( C ) for 1 h at 37 °C and for the second half h with PE-labeled mAb against human CD14. The cells were imaged with a Nikon confocal microscope system A1. In functional assays ( D ), the monocytes were stimulated with 30 μg/ml LTF-M860 IC ( MDC/M860-IC ) or 3 μg/ml LPS ( MDC/LPS ) in the presence or absence of MDC for 18 h, followed by quantitation of TNF-α in the culture supernatant by ELISA. Values are mean concentration (ng/ml) ± SEM from triplicate cultures. ** p < 0.01 compared with the LPS/MDC group. For intracellular localization of the internalized LTF-ICs, human monocytes were stained with 30 μg/ml FITC-huLTF plus mAb M860 for 1 h at 37 °C, followed by treatment with red fluorescent ER tracker ( E , F , G ) or lysosome tracker ( H , J , K ). The specimens were observed under a confocal microscope through green and red channels, merged micrographs are shown on the right. Results are representative of 3 experiments.

Journal: Scientific Reports

Article Title: Extraordinarily potent proinflammatory properties of lactoferrin-containing immunocomplexes against human monocytes and macrophages

doi: 10.1038/s41598-017-04275-7

Figure Lengend Snippet: LTF-IC endocytosis by human peripheral blood monocytes. Human monocytes were sequentially stained with 15 μg/ml FITC-LTF ( A ), mixture of FITC-OVA plus mAb M562 ( B ), or FITC-LTF plus mAb M860 ( C ) for 1 h at 37 °C and for the second half h with PE-labeled mAb against human CD14. The cells were imaged with a Nikon confocal microscope system A1. In functional assays ( D ), the monocytes were stimulated with 30 μg/ml LTF-M860 IC ( MDC/M860-IC ) or 3 μg/ml LPS ( MDC/LPS ) in the presence or absence of MDC for 18 h, followed by quantitation of TNF-α in the culture supernatant by ELISA. Values are mean concentration (ng/ml) ± SEM from triplicate cultures. ** p < 0.01 compared with the LPS/MDC group. For intracellular localization of the internalized LTF-ICs, human monocytes were stained with 30 μg/ml FITC-huLTF plus mAb M860 for 1 h at 37 °C, followed by treatment with red fluorescent ER tracker ( E , F , G ) or lysosome tracker ( H , J , K ). The specimens were observed under a confocal microscope through green and red channels, merged micrographs are shown on the right. Results are representative of 3 experiments.

Article Snippet: Recombinant soluble human CD14 was from Peprotech.

Techniques: Staining, Labeling, Microscopy, Functional Assay, Quantitation Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay

Characterization of PS cells properties by flow cytometry and RT-PCR. PS cells, at passage 14, were labelled with antibodies against CD45 ( A ), cytokeratin 14 ( B ), cytokeratin 18 ( C ), TLR2 ( D ) and CD14 ( E ). Controls were performed either by omitting the primary antibody ( A , B , C ) or using unlabelled cells ( D , E ). As a control for CD45 labelling, bovine PBMC were labelled with the anti-CD45 antibody ( F ). Labelled cells are depicted as red curves while controls are represented as blue curves. ( G - H ) RNA was extracted from unstimulated PS cells and converted to cDNA. cDNAs for each of the indicated receptors were amplified by PCR and products were separated by agarose gel electrophoresis. MW: 50 bp molecular weight ladder.

Journal: Veterinary Research

Article Title: Investigating the contribution of IL-17A and IL-17F to the host response during Escherichia coli mastitis

doi: 10.1186/s13567-015-0201-4

Figure Lengend Snippet: Characterization of PS cells properties by flow cytometry and RT-PCR. PS cells, at passage 14, were labelled with antibodies against CD45 ( A ), cytokeratin 14 ( B ), cytokeratin 18 ( C ), TLR2 ( D ) and CD14 ( E ). Controls were performed either by omitting the primary antibody ( A , B , C ) or using unlabelled cells ( D , E ). As a control for CD45 labelling, bovine PBMC were labelled with the anti-CD45 antibody ( F ). Labelled cells are depicted as red curves while controls are represented as blue curves. ( G - H ) RNA was extracted from unstimulated PS cells and converted to cDNA. cDNAs for each of the indicated receptors were amplified by PCR and products were separated by agarose gel electrophoresis. MW: 50 bp molecular weight ladder.

Article Snippet: When required, 5 μg/mL of recombinant human soluble CD14 (CD14) (Biometec GmbH, Germany), 5 μg/mL of recombinant human LPS-binding protein (LBP) (Biometec GmbH) or fresh whole milk were used to supplement the culture medium of PS cells.

Techniques: Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Control, Amplification, Agarose Gel Electrophoresis, Molecular Weight

Innate immune response of PS cells to different purified bacterial agonists. PS cells were incubated for 5 h with the indicated concentrations of different purified agonists. Response was analyzed in terms of expression of CCL20 ( A ), CXCL8 ( B ), TAP ( C ), LAP ( D ) genes by RT-qPCR or CXCL8 secretion by ELISA ( E , F ). Data presented are mean values and SD obtained from 3 independent experiments. Experiments were performed at passages 11, 12 and 24. PS cells were incubated for 5 hours in GM medium with different agonists at the indicated concentrations. (F) PS cells were incubated with ultrapure LPS (LPS) at 10 ng/mL in the presence of either 10% fetal calf serum, 5 μg/mL CD14 or 5 μg/mL LBP. Stimulation was also performed in whole milk instead of GM medium. * statistical significance ( P < 0.05) of stimulated versus unstimulated PS cells using exact permutation tests after global comparison using a Kruskal and Wallis test.

Journal: Veterinary Research

Article Title: Investigating the contribution of IL-17A and IL-17F to the host response during Escherichia coli mastitis

doi: 10.1186/s13567-015-0201-4

Figure Lengend Snippet: Innate immune response of PS cells to different purified bacterial agonists. PS cells were incubated for 5 h with the indicated concentrations of different purified agonists. Response was analyzed in terms of expression of CCL20 ( A ), CXCL8 ( B ), TAP ( C ), LAP ( D ) genes by RT-qPCR or CXCL8 secretion by ELISA ( E , F ). Data presented are mean values and SD obtained from 3 independent experiments. Experiments were performed at passages 11, 12 and 24. PS cells were incubated for 5 hours in GM medium with different agonists at the indicated concentrations. (F) PS cells were incubated with ultrapure LPS (LPS) at 10 ng/mL in the presence of either 10% fetal calf serum, 5 μg/mL CD14 or 5 μg/mL LBP. Stimulation was also performed in whole milk instead of GM medium. * statistical significance ( P < 0.05) of stimulated versus unstimulated PS cells using exact permutation tests after global comparison using a Kruskal and Wallis test.

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

IL-17A effect on the response of PS cells to LPS. PS cells were incubated for 5 h with 10 ng/mL of LPS in the presence or not of 100 ng/mL IL-17A and 5 μg/mL CD14. Response was analyzed in terms CXCL8 secretion ( A ) by ELISA or expression of CCL20 ( B ) and CXCL8 ( C ) by RT-qPCR. Data presented are mean values and SD obtained from 3 independent experiments. Experiments were performed at passages 15, 16 and 24. * statistical significance ( P < 0.05) calculated using exact permutation tests after global comparison using a Kruskal and Wallis test.

Journal: Veterinary Research

Article Title: Investigating the contribution of IL-17A and IL-17F to the host response during Escherichia coli mastitis

doi: 10.1186/s13567-015-0201-4

Figure Lengend Snippet: IL-17A effect on the response of PS cells to LPS. PS cells were incubated for 5 h with 10 ng/mL of LPS in the presence or not of 100 ng/mL IL-17A and 5 μg/mL CD14. Response was analyzed in terms CXCL8 secretion ( A ) by ELISA or expression of CCL20 ( B ) and CXCL8 ( C ) by RT-qPCR. Data presented are mean values and SD obtained from 3 independent experiments. Experiments were performed at passages 15, 16 and 24. * statistical significance ( P < 0.05) calculated using exact permutation tests after global comparison using a Kruskal and Wallis test.

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

Fig. 1. Quantity of sCD14 in the stomach contents (milk clot, left panel) and blood (right panel) of mouse pups ingesting milk with or without sCD14. On day six post-partum, pups

Journal: Immunobiology

Article Title: Ingested soluble CD14 contributes to the functional pool of circulating sCD14 in mice.

doi: 10.1016/j.imbio.2014.03.008

Figure Lengend Snippet: Fig. 1. Quantity of sCD14 in the stomach contents (milk clot, left panel) and blood (right panel) of mouse pups ingesting milk with or without sCD14. On day six post-partum, pups

Article Snippet: Human recombinant soluble CD14 (hrCD14, R&D Systems) was added to the apical well.

Techniques:

Fig. 3. Immune response to LPS following sCD14 ingestion in mouse pups, assessed via sCD14, TNF-alpha, and IL-6 levels. Pups were fostered to mothers expressing (WT) or

Journal: Immunobiology

Article Title: Ingested soluble CD14 contributes to the functional pool of circulating sCD14 in mice.

doi: 10.1016/j.imbio.2014.03.008

Figure Lengend Snippet: Fig. 3. Immune response to LPS following sCD14 ingestion in mouse pups, assessed via sCD14, TNF-alpha, and IL-6 levels. Pups were fostered to mothers expressing (WT) or

Article Snippet: Human recombinant soluble CD14 (hrCD14, R&D Systems) was added to the apical well.

Techniques: Expressing

Fig. 4. Quantity (A) and intactness (B) of sCD14 transferred across human intestinal monolayers

Journal: Immunobiology

Article Title: Ingested soluble CD14 contributes to the functional pool of circulating sCD14 in mice.

doi: 10.1016/j.imbio.2014.03.008

Figure Lengend Snippet: Fig. 4. Quantity (A) and intactness (B) of sCD14 transferred across human intestinal monolayers

Article Snippet: Human recombinant soluble CD14 (hrCD14, R&D Systems) was added to the apical well.

Techniques:

Fig. 5. Visualization of sCD14 within human intestinal cells treated with sCD14 in

Journal: Immunobiology

Article Title: Ingested soluble CD14 contributes to the functional pool of circulating sCD14 in mice.

doi: 10.1016/j.imbio.2014.03.008

Figure Lengend Snippet: Fig. 5. Visualization of sCD14 within human intestinal cells treated with sCD14 in

Article Snippet: Human recombinant soluble CD14 (hrCD14, R&D Systems) was added to the apical well.

Techniques:

Fig. 7. Effect of TLR4 knockdown on sCD14 transfer by human intestinal cells in vitro. Caco-2 cells were grown and differentiated as monolayers in Transwell assays, treated with TLR4 speciﬁc siRNA, scrambled siRNA or no siRNA, and TLR4 expression was

Journal: Immunobiology

Article Title: Ingested soluble CD14 contributes to the functional pool of circulating sCD14 in mice.

doi: 10.1016/j.imbio.2014.03.008

Figure Lengend Snippet: Fig. 7. Effect of TLR4 knockdown on sCD14 transfer by human intestinal cells in vitro. Caco-2 cells were grown and differentiated as monolayers in Transwell assays, treated with TLR4 speciﬁc siRNA, scrambled siRNA or no siRNA, and TLR4 expression was

Article Snippet: Human recombinant soluble CD14 (hrCD14, R&D Systems) was added to the apical well.

Techniques: Knockdown, In Vitro, Expressing

Fig. 8. Visualization of sCD14 and TLR4 within human intestinal cells treated with siRNA. Caco-2 cells were grown and differentiated as monolayers in Transwell assays and treated

Journal: Immunobiology

Article Title: Ingested soluble CD14 contributes to the functional pool of circulating sCD14 in mice.

doi: 10.1016/j.imbio.2014.03.008

Figure Lengend Snippet: Fig. 8. Visualization of sCD14 and TLR4 within human intestinal cells treated with siRNA. Caco-2 cells were grown and differentiated as monolayers in Transwell assays and treated

Article Snippet: Human recombinant soluble CD14 (hrCD14, R&D Systems) was added to the apical well.

Techniques:

Journal: Immunology

Article Title: Soluble CD14 is essential for lipopolysaccharide-dependent activation of human intestinal mast cells from macroscopically normal as well as Crohn's disease tissue

doi: 10.1111/imm.12299

Figure Lengend Snippet: Flow cytometric analysis of Toll-like receptor 4 (TLR4) and CD14 surface expression on human intestinal mast cells (hiMC). TLR4 expression (a) was determined by direct immunofluorescence using phycoerythrin-conjugated mouse anti-human CD284 antibody (dark grey profile). Expression of membrane bound CD14 (b) was assessed using FITC-conjugated mouse anti-human CD14 antibody (dark grey profile). Both stainings were compared with unstained hiMC (light grey profile). The data are expressed as cell number versus log fluorescence and are representative of three independent experiments.

Article Snippet: Reagents for performing the experiments were purchased from the following sources: phycoerythrin anti-human CD284 (Toll-like receptor 4; TLR4) from BioLegend (San Diego, CA), FITC anti-human CD14 from BD Biosciences (San Jose, CA); myeloma IgE from Calbiochem by Merck (Darmstadt, Germany); polyclonal anti-human IgE from U. Blank (INSERM, Paris, France); LPS from Escherichia coli serotype O111:B4 from Sigma-Aldrich; recombinant human soluble CD14 (sCD14), recombinant human interferon- γ (IFN- γ ) and recombinant human tumour necrosis factor- α (TNF- α ) from PeproTech; ionomycin and PMA from Sigma-Aldrich; TLR4 signalling inhibitor CLI-095 from InvivoGen (San Diego, CA); RNeasy Mini Kit from Qiagen (Hilden, Germany); SsoFast™ EVAGreen® Supermix from Bio-Rad Laboratories (Munich, Germany); cysteinyl leukotriene (CysLT) EIA kit and IL-1 β EIA kit from Enzo Life Sciences (Lorrach, Germany), CXCL8 Duo Set from R & D Systems (Abingdon, UK), Novex® magnetic Luminex® assay from Invitrogen (Frederick, MD).

Techniques: Expressing, Immunofluorescence, Fluorescence

In the presence of soluble CD14 (sCD14) in ascending concentrations, lipopolysaccharide (LPS) induced the cytokine expression in human intestinal mast cells (hiMC). The hiMC were treated with 1 μg/ml LPS (black bar) or sCD14 (light grey bar) solely, respectively, or with 1 μg/ml LPS plus sCD14 (0·001–1 μg/ml) (dark grey bars) for 3 hr to analyse the mRNA expression of CXCL8 (a), interleukin-1β (IL-1β) (b), IL-6 (c), CCL2 (d), CCL3 (e), and CCL4 (f). As positive control for determination of hiMC ability to induce cytokine mRNA expression, cells were treated with myeloma IgE for 90 min and afterwards stimulated with anti-human IgE for 90 min for cross-linking of high-affinity IgE receptor (FcεRI CL, black striped bar). Data represent mean ± SEM of five or six independent experiments. *P < 0·05 and **P < 0·001 indicate significant differences from hiMC treated with LPS (1 μg/ml) alone (black bar).

Journal: Immunology

Article Title: Soluble CD14 is essential for lipopolysaccharide-dependent activation of human intestinal mast cells from macroscopically normal as well as Crohn's disease tissue

doi: 10.1111/imm.12299

Figure Lengend Snippet: In the presence of soluble CD14 (sCD14) in ascending concentrations, lipopolysaccharide (LPS) induced the cytokine expression in human intestinal mast cells (hiMC). The hiMC were treated with 1 μg/ml LPS (black bar) or sCD14 (light grey bar) solely, respectively, or with 1 μg/ml LPS plus sCD14 (0·001–1 μg/ml) (dark grey bars) for 3 hr to analyse the mRNA expression of CXCL8 (a), interleukin-1β (IL-1β) (b), IL-6 (c), CCL2 (d), CCL3 (e), and CCL4 (f). As positive control for determination of hiMC ability to induce cytokine mRNA expression, cells were treated with myeloma IgE for 90 min and afterwards stimulated with anti-human IgE for 90 min for cross-linking of high-affinity IgE receptor (FcεRI CL, black striped bar). Data represent mean ± SEM of five or six independent experiments. *P < 0·05 and **P < 0·001 indicate significant differences from hiMC treated with LPS (1 μg/ml) alone (black bar).

Techniques: Expressing, Positive Control

Time-dependent expression and release of CXCL8 (a, b) and interleukin (IL)-1β (c) by human intestinal mast cells (hiMC) upon administration of lipopolysaccharide (LPS) in combination with soluble CD14 (sCD14). The hiMC were incubated with 1 μg/ml LPS (black bar) or sCD14 (light grey bar) solely for 6 hr, or with LPS plus sCD14 (each 1 μg/ml) (dark grey bars) for 3 hr, 6 hr and 12 hr to analyse protein production of CXCL8 (a) and IL-1β (c) in cell lysate and release of CXCL8 (b) in supernatants. In case of CXCL8 protein production, IgE receptors were cross-linked with myeloma IgE (90 min) and anti-human IgE for 6 hr (FcεRI CL, black striped bar) as stimulation control. For CXCL8 in cell lysates (a) and supernatants (b), data represent mean ± SEM of three independent experiments by treatments with LPS plus sCD14 (dark grey bars) and cross-linking of high-affinity IgE receptor (black striped bar). No CXCL8 protein was measured in cell lysates and supernatants of hiMC without any stimulation (a, b) and treated exclusively with LPS (b). In the case of cytosolic CXCL8 protein content (a), data represent mean ± SEM of two independent experiments for sole incubation with LPS (black bar) and sCD14 (light grey bar), respectively. After treatment of hiMC with sCD14 only (light grey bar), a single measurement was quantified and is represented for CXCL8 concentration in cell supernatants (b). According to the missing values concerning cytosolic content and release of CXCL8 by LPS treatment alone, no statistical analysis with the paired t-test was performed for these data. In case of cytosolic release of IL-1β and its precursor (c), data represent mean ± SEM of four independent experiments. *P < 0·05 indicate significant differences to hiMC treated with LPS [1 μg/ml] solely (black bar).

Journal: Immunology

Article Title: Soluble CD14 is essential for lipopolysaccharide-dependent activation of human intestinal mast cells from macroscopically normal as well as Crohn's disease tissue

doi: 10.1111/imm.12299

Figure Lengend Snippet: Time-dependent expression and release of CXCL8 (a, b) and interleukin (IL)-1β (c) by human intestinal mast cells (hiMC) upon administration of lipopolysaccharide (LPS) in combination with soluble CD14 (sCD14). The hiMC were incubated with 1 μg/ml LPS (black bar) or sCD14 (light grey bar) solely for 6 hr, or with LPS plus sCD14 (each 1 μg/ml) (dark grey bars) for 3 hr, 6 hr and 12 hr to analyse protein production of CXCL8 (a) and IL-1β (c) in cell lysate and release of CXCL8 (b) in supernatants. In case of CXCL8 protein production, IgE receptors were cross-linked with myeloma IgE (90 min) and anti-human IgE for 6 hr (FcεRI CL, black striped bar) as stimulation control. For CXCL8 in cell lysates (a) and supernatants (b), data represent mean ± SEM of three independent experiments by treatments with LPS plus sCD14 (dark grey bars) and cross-linking of high-affinity IgE receptor (black striped bar). No CXCL8 protein was measured in cell lysates and supernatants of hiMC without any stimulation (a, b) and treated exclusively with LPS (b). In the case of cytosolic CXCL8 protein content (a), data represent mean ± SEM of two independent experiments for sole incubation with LPS (black bar) and sCD14 (light grey bar), respectively. After treatment of hiMC with sCD14 only (light grey bar), a single measurement was quantified and is represented for CXCL8 concentration in cell supernatants (b). According to the missing values concerning cytosolic content and release of CXCL8 by LPS treatment alone, no statistical analysis with the paired t-test was performed for these data. In case of cytosolic release of IL-1β and its precursor (c), data represent mean ± SEM of four independent experiments. *P < 0·05 indicate significant differences to hiMC treated with LPS [1 μg/ml] solely (black bar).

Techniques: Expressing, Incubation, Concentration Assay

Down-regulation of induced mRNA expression of CXCL8 (a) and interleukin (IL)-1β (b) due to administration of Toll-like receptor 4 (TLR4) inhibitor before treatment with lipopolysaccharide (LPS) and soluble CD14 (sCD14). In advance of LPS/sCD14-stimulation, human intestinal mast cells (hiMC) were treated with the TLR4 inhibitor CLI-095 at concentrations of 0·1 and 1 μm for 60 min and afterwards incubated with LPS combined with sCD14 (each 1 μg/ml) (dark grey bars) for 3 hr to determine mRNA expression. Data represent mean ± SEM of six independent experiments. *P < 0·05 indicate significant differences between groups.

Journal: Immunology

Article Title: Soluble CD14 is essential for lipopolysaccharide-dependent activation of human intestinal mast cells from macroscopically normal as well as Crohn's disease tissue

doi: 10.1111/imm.12299

Figure Lengend Snippet: Down-regulation of induced mRNA expression of CXCL8 (a) and interleukin (IL)-1β (b) due to administration of Toll-like receptor 4 (TLR4) inhibitor before treatment with lipopolysaccharide (LPS) and soluble CD14 (sCD14). In advance of LPS/sCD14-stimulation, human intestinal mast cells (hiMC) were treated with the TLR4 inhibitor CLI-095 at concentrations of 0·1 and 1 μm for 60 min and afterwards incubated with LPS combined with sCD14 (each 1 μg/ml) (dark grey bars) for 3 hr to determine mRNA expression. Data represent mean ± SEM of six independent experiments. *P < 0·05 indicate significant differences between groups.

Techniques: Expressing, Incubation

Journal: Immunology

Article Title: Soluble CD14 is essential for lipopolysaccharide-dependent activation of human intestinal mast cells from macroscopically normal as well as Crohn's disease tissue

doi: 10.1111/imm.12299

Figure Lengend Snippet: In the presence of soluble CD14 (sCD14) in ascending concentrations, lipopolysaccharide (LPS) induced the cytokine expression in human intestinal mast cells (hiMC) from patients with Crohn's disease. The hiMC were treated with 1 μg/ml LPS (black bar) or sCD14 (light grey bar) solely, respectively, or with 1 μg/ml LPS plus sCD14 (0·001–1 μg/ml) (dark grey bars) for 3 hr to analyse the mRNA expression of CXCL8 (a), interleukin-1β (IL-1β) (b), IL-6 (c), CCL2 (d), CCL3 (e), and CCL4 (f). As positive control for determination of hiMC ability to induce cytokine mRNA expression, cells were treated with myeloma IgE for 90 min and afterwards stimulated with anti-human IgE for 90 min for cross-linking of high-affinity IgE receptor (FcεRI CL, black striped bar). Data represent mean ± SEM of four to six independent experiments. *P < 0·05 indicates significant differences from hiMC treated with LPS (1 μg/ml) solely (black bar).

Techniques: Expressing, Positive Control

Comparison of CXCL8 mRNA (a) and protein (b) expression in human intestinal mast cells (hiMC) isolated from macroscopically normal tissue specimens (Co-MC) and from patients with Crohn's disease (CD-MC). For analysing mRNA expression of CXCL8 (a, b), Co-MC and CD-MC were treated with 1 μg/ml lipopolysaccharide (LPS; black bar) or soluble CD14 (sCD14) (light grey bar) solely for 6 hr, or with LPS plus sCD14 (each 1 μg/ml) (dark grey bars) for 90 min, 3 hr and 6 hr. As positive control for determination of the ability of hiMC to induce cytokine mRNA expression, cells were treated with myeloma IgE for 90 min and afterwards stimulated with anti-human IgE for 90 min for cross-linking of high-affinity IgE receptor (FcεRI CL, black striped bar). Data represent mean ± SEM of four or five independent experiments. *P < 0·05 indicates significant differences from hiMC treated with LPS (1 μg/ml) solely (black bar). Cytosolic content (c) and release of CXCL8 (d) were determined in cell lysate and supernatants, respectively. For this purpose, Co-MC (white bars) and CD-MC (black bars) were treated with LPS in combination with sCD14 (each 1 μg/ml) for 6 hr or with myeloma IgE for 90 min and afterwards stimulated with anti-human IgE for 6 hr for cross-linking of high-affinity IgE receptor (FcεRI CL), respectively. Data represent mean ± SEM of four to six independent experiments; a: P < 0·05 compared with Control (6 hr) of Co-MC, b: P < 0·05 compared with Control (6 hr) of CD-MC.

Journal: Immunology

Article Title: Soluble CD14 is essential for lipopolysaccharide-dependent activation of human intestinal mast cells from macroscopically normal as well as Crohn's disease tissue

doi: 10.1111/imm.12299

Figure Lengend Snippet: Comparison of CXCL8 mRNA (a) and protein (b) expression in human intestinal mast cells (hiMC) isolated from macroscopically normal tissue specimens (Co-MC) and from patients with Crohn's disease (CD-MC). For analysing mRNA expression of CXCL8 (a, b), Co-MC and CD-MC were treated with 1 μg/ml lipopolysaccharide (LPS; black bar) or soluble CD14 (sCD14) (light grey bar) solely for 6 hr, or with LPS plus sCD14 (each 1 μg/ml) (dark grey bars) for 90 min, 3 hr and 6 hr. As positive control for determination of the ability of hiMC to induce cytokine mRNA expression, cells were treated with myeloma IgE for 90 min and afterwards stimulated with anti-human IgE for 90 min for cross-linking of high-affinity IgE receptor (FcεRI CL, black striped bar). Data represent mean ± SEM of four or five independent experiments. *P < 0·05 indicates significant differences from hiMC treated with LPS (1 μg/ml) solely (black bar). Cytosolic content (c) and release of CXCL8 (d) were determined in cell lysate and supernatants, respectively. For this purpose, Co-MC (white bars) and CD-MC (black bars) were treated with LPS in combination with sCD14 (each 1 μg/ml) for 6 hr or with myeloma IgE for 90 min and afterwards stimulated with anti-human IgE for 6 hr for cross-linking of high-affinity IgE receptor (FcεRI CL), respectively. Data represent mean ± SEM of four to six independent experiments; a: P < 0·05 compared with Control (6 hr) of Co-MC, b: P < 0·05 compared with Control (6 hr) of CD-MC.

Techniques: Expressing, Isolation, Positive Control

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