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  • 95
    Thermo Fisher lps
    5-ASA and AZTP restore epithelial barrier. ( A ) Paracellular permeability assay using FITC-Dextran: Differentiated and polarized T-84 monolayer grown in Transwell was treated with TNF-α (10 ng/ml), <t>IFN-γ</t> (100U/ml) or <t>LPS</t> (10 ng/ml) with or without pretreatment (5 h) with 5-ASA (5 mM) or AZTP (10 μM) n = 5 each treatment. Experiments were performed in biological duplicates and repeated three times. p value (≤0.05), * con vs treatment; # vs TNF-α or IFN-γ. One-way ANOVA with multiple comparisons were done using Bonferroni’s post hoc analysis. ( B , C ) PCR array analysis of T-84 cells treated with 5-ASA (5 mM) and AZTP(10 μM), in the presence or absence of TNF-α (10 ng/ml) ( D ) Immunofluorescence analysis of T-84 monolayer for the representative proteins of adherens junctions; AJs (E-cadherin), tight junctions; TJs (occludin), Desmosomes (Desmoglein-2). TNF-α treatment disrupted all junctions and resulted in protein internalization as indicated by arrows. 5-ASA and AZTP restored membranous expression of these proteins. Image magnification 400x. Scale 50μm. E-cadherin and Desmoglein were visualized using Alexa Fluor 488 secondary antibody. Occludin was visualized using Alexa Fluor 568 ( E ) qRT-PCR analysis of intestinal alkaline phosphatase as a marker of cellular differentiation in T-84 cells. The experiment was performed twice.
    Lps, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 2391 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore lipopolysaccharides lps
    SER inhibits pro-inflammatory but enhances anti-inflammatory signature and up-regulates Notch1 signaling in BMDM cultures ( A/B ) BMDM stimulated with <t>LPS</t> (100ng/ml, 6h) were pretreated with or without SER (5μg/ml, 24h). ( A ) Western blot-assisted detection of NICD, Hes1 and pIκBα (Ser32). β-actin expression serves as an internal control for normalization. The values under the bands represent the relative ratio of normalized intensity compared to cells + LPS. Representative of three experiments is shown. ( B ) Quantitative RT-PCR-assisted detection of mRNA coding for MCP1, IL1β, CXCL1, CXCL2, CXCL10 and Arg1. Data were normalized to β2M gene expression (n=4/group). ( C/D ) BMDM stimulated with <t>IL4</t> (10ng/ml, 24h) were pretreated with or without SER (5μg/ml, 24h). ( C ) Western blot-assisted detection of NICD and Hes1. β-actin expression served as an internal control for normalization. The values under the bands represent the relative ratio of normalized intensity compared to cells + IL4. Representative of three experiments is shown. ( D ) Quantitative RT-PCR-assisted detection of mRNA coding for Arg1, CD163 and CCL24. Data were normalized to β2M gene expression (n=4/group). Data are shown as mean±SD (*p
    Lipopolysaccharides Lps, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1332 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Millipore lipopolysaccaride lps
    Chemokine and TLR-4 expression in palmitic acid-treated C3A hepatocytes . C3A hepatocytes were cultured in medium containing <t>lipopolysaccharide</t> <t>(LPS)</t> (500 pg/ml), palmitic acid (PA; 100 mM) or the combination of PA and LPS for 24h. Expression of (A) IL-8 and (B) TLR-4 was assessed using pre-developed assays for real-time PCR and values were calculated using a comparative C T method according to the manufacturer instructions (Applied Biosystems). Data are presented as mean SEM of 4 experiments. Statistical analysis was performed using two-way ANOVA and Tukey's multiple comparisons test. *p
    Lipopolysaccaride Lps, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millipore lipolysaccharides lps
    Chemokine and TLR-4 expression in palmitic acid-treated C3A hepatocytes . C3A hepatocytes were cultured in medium containing <t>lipopolysaccharide</t> <t>(LPS)</t> (500 pg/ml), palmitic acid (PA; 100 mM) or the combination of PA and LPS for 24h. Expression of (A) IL-8 and (B) TLR-4 was assessed using pre-developed assays for real-time PCR and values were calculated using a comparative C T method according to the manufacturer instructions (Applied Biosystems). Data are presented as mean SEM of 4 experiments. Statistical analysis was performed using two-way ANOVA and Tukey's multiple comparisons test. *p
    Lipolysaccharides Lps, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    InvivoGen ultrapure lipolysaccharide lps
    TLR2 is involved in IL-12-dependent IFN-γ secretion by CD4 + cells co-cultured with ESAT-6, HspX and BCG-treated DCs. DCs cultured in the absence (filled bars) or presence (open bars) of 5 µg/ml TLR2-blocking antibody were treated for 24 hrs with Mtb, BCG alone or combined with HspX and ESAT6, 10 µg/ml Pam3CSK4 (Pam3) or 100 ng/ml <t>LPS.</t> (A) Supernatants were collected and IL-12 release was analyzed by ELISA. Results are the mean value+SD of four experiments. Statistical analysis: antibody-treated vs antibody-untreated cells, ns P > 0.05, *P
    Ultrapure Lipolysaccharide Lps, supplied by InvivoGen, used in various techniques. Bioz Stars score: 85/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology anti lipoplysaccharide lps antibody
    Galectin-3 binds to meningococci expressing <t>LPS</t> with a terminal LacNAc
    Anti Lipoplysaccharide Lps Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    List Biological Laboratories lps
    V-58 stimulates intracellular Mtb to produce <t>cAMP</t> J774.16 murine macrophages were infected with live Mtb H37Rv or CDC1551, and heat-killed CDC1551 were treated with either 0.1 % DMSO (black bars) or 10 μM V-58 (gray bars). cAMP levels were quantified by RIA from the A) intracellular bacteria and B) macrophage lysates. The data shown are the means of two biologically independent experiments. Error bars represent the standard deviation between replicates. C) Macrophages were infected with Mtb CDC1551 at the indicated MOI and treated with 10 ng/mL <t>LPS,</t> 250 μM 8-Br-cAMP or V-58 at the indicated concentrations for 18 h and supernatant TNF-α levels were determined by ELISA. Data are means of four biologically independent experiments and the error bars represent the standard deviation between experiments. Statistical significance was determined with a two-tailed unpaired t-test, *p≤0.05, **p≤0.005 and ****p≤0.0001.
    Lps, supplied by List Biological Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 604 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    InvivoGen hek blue lipoplysaccharide lps detection system
    V-58 stimulates intracellular Mtb to produce <t>cAMP</t> J774.16 murine macrophages were infected with live Mtb H37Rv or CDC1551, and heat-killed CDC1551 were treated with either 0.1 % DMSO (black bars) or 10 μM V-58 (gray bars). cAMP levels were quantified by RIA from the A) intracellular bacteria and B) macrophage lysates. The data shown are the means of two biologically independent experiments. Error bars represent the standard deviation between replicates. C) Macrophages were infected with Mtb CDC1551 at the indicated MOI and treated with 10 ng/mL <t>LPS,</t> 250 μM 8-Br-cAMP or V-58 at the indicated concentrations for 18 h and supernatant TNF-α levels were determined by ELISA. Data are means of four biologically independent experiments and the error bars represent the standard deviation between experiments. Statistical significance was determined with a two-tailed unpaired t-test, *p≤0.05, **p≤0.005 and ****p≤0.0001.
    Hek Blue Lipoplysaccharide Lps Detection System, supplied by InvivoGen, used in various techniques. Bioz Stars score: 95/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Labconco lpss
    V-58 stimulates intracellular Mtb to produce <t>cAMP</t> J774.16 murine macrophages were infected with live Mtb H37Rv or CDC1551, and heat-killed CDC1551 were treated with either 0.1 % DMSO (black bars) or 10 μM V-58 (gray bars). cAMP levels were quantified by RIA from the A) intracellular bacteria and B) macrophage lysates. The data shown are the means of two biologically independent experiments. Error bars represent the standard deviation between replicates. C) Macrophages were infected with Mtb CDC1551 at the indicated MOI and treated with 10 ng/mL <t>LPS,</t> 250 μM 8-Br-cAMP or V-58 at the indicated concentrations for 18 h and supernatant TNF-α levels were determined by ELISA. Data are means of four biologically independent experiments and the error bars represent the standard deviation between experiments. Statistical significance was determined with a two-tailed unpaired t-test, *p≤0.05, **p≤0.005 and ****p≤0.0001.
    Lpss, supplied by Labconco, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore reagents lps
    DHA pretreatment attenuates forepaw placing deficits among hypoxic ischemic pups exposed to <t>LPS</t> pretreatment
    Reagents Lps, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 434 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Enzo Biochem ultrapure lpss
    Proteinase K-agarose digestion ablates <t>HSP70</t> activity . (A) 200 μg rhHSP70 was incubated with digestion buffer (lane 1), Proteinase K-agarose beads (lane 2), or control agarose beads (lane 3) for 3 hr at 37°C, as described in Methods . Protein integrity and endotoxin content were determined by silver staining and the LAL assay, respectively. (B) 100 pg of ultrapure <t>LPS,</t> which is an approximation of the endotoxin content in 200 μg of rhHSP70 (see Results and Discussion ), was incubated with Proteinase K-agarose beads or control agarose beads, under the conditions described in A . The ultrapure LPS treated with Proteinase K-agarose beads (PK) or control agarose beads (CTRL) were then used to stimulate primary human macrophages for 16 hr at 100 pg/mL LPS final concentration in each culture. Cell culture supernatants were collected and analyzed for TNF-α. (C) The HSP70 preparations in A were used to stimulate primary human macrophages for 4 hr at 10 μg/mL final concentration. Cell culture supernatants were collected and analyzed for TNF-α. Endotoxin levels shown at bottom are the calculated amounts in the macrophage cultures based on the LAL determinations in A . Untreated macrophage cultures produced ≤ 100 pg/mL of TNF-α under these conditions. Results shown are representative of two independent digestion and stimulation experiments. *** p
    Ultrapure Lpss, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Bristol Myers lpss counseling
    Proteinase K-agarose digestion ablates <t>HSP70</t> activity . (A) 200 μg rhHSP70 was incubated with digestion buffer (lane 1), Proteinase K-agarose beads (lane 2), or control agarose beads (lane 3) for 3 hr at 37°C, as described in Methods . Protein integrity and endotoxin content were determined by silver staining and the LAL assay, respectively. (B) 100 pg of ultrapure <t>LPS,</t> which is an approximation of the endotoxin content in 200 μg of rhHSP70 (see Results and Discussion ), was incubated with Proteinase K-agarose beads or control agarose beads, under the conditions described in A . The ultrapure LPS treated with Proteinase K-agarose beads (PK) or control agarose beads (CTRL) were then used to stimulate primary human macrophages for 16 hr at 100 pg/mL LPS final concentration in each culture. Cell culture supernatants were collected and analyzed for TNF-α. (C) The HSP70 preparations in A were used to stimulate primary human macrophages for 4 hr at 10 μg/mL final concentration. Cell culture supernatants were collected and analyzed for TNF-α. Endotoxin levels shown at bottom are the calculated amounts in the macrophage cultures based on the LAL determinations in A . Untreated macrophage cultures produced ≤ 100 pg/mL of TNF-α under these conditions. Results shown are representative of two independent digestion and stimulation experiments. *** p
    Lpss Counseling, supplied by Bristol Myers, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc lps
    Proteinase K-agarose digestion ablates <t>HSP70</t> activity . (A) 200 μg rhHSP70 was incubated with digestion buffer (lane 1), Proteinase K-agarose beads (lane 2), or control agarose beads (lane 3) for 3 hr at 37°C, as described in Methods . Protein integrity and endotoxin content were determined by silver staining and the LAL assay, respectively. (B) 100 pg of ultrapure <t>LPS,</t> which is an approximation of the endotoxin content in 200 μg of rhHSP70 (see Results and Discussion ), was incubated with Proteinase K-agarose beads or control agarose beads, under the conditions described in A . The ultrapure LPS treated with Proteinase K-agarose beads (PK) or control agarose beads (CTRL) were then used to stimulate primary human macrophages for 16 hr at 100 pg/mL LPS final concentration in each culture. Cell culture supernatants were collected and analyzed for TNF-α. (C) The HSP70 preparations in A were used to stimulate primary human macrophages for 4 hr at 10 μg/mL final concentration. Cell culture supernatants were collected and analyzed for TNF-α. Endotoxin levels shown at bottom are the calculated amounts in the macrophage cultures based on the LAL determinations in A . Untreated macrophage cultures produced ≤ 100 pg/mL of TNF-α under these conditions. Results shown are representative of two independent digestion and stimulation experiments. *** p
    Lps, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore bacterial lps
    Effects of carboxybutyrylated glucosamine (CGlcN) on the inhibition of lipopolysaccharide <t>(LPS)-induced</t> nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) production in RAW264.7 cells. (a) Cells cultured in phenol red and serum-free media were pretreated with different concentrations of CGlcN for 1 hr and stimulated with a 1-µg/ml final concentration of LPS for 48 hr. Conditioned medium was mixed with an equal amount of the <t>Griess</t> reagent and the absorbance, measured at 550 nm, represented the amount of (stable oxidation product of NO) in the medium. The values obtained were compared with those of standard concentrations of sodium nitrite dissolved in Dulbecco's modified Eagle's minimal essential medium (DMEM), and the concentrations of nitrite in conditioned media of sample-treated cells were calculated. (b) Cells growing in serum-free medium were pretreated with different concentrations of CGlcN for 1 hr and stimulated with LPS (1 µg/ml final concentration) for 24 hr. The amount of PGE 2 release was determined by the mouse PGE 2 enzyme-linked immunosorbent assay (ELISA) kit. BLK, LPS non-stimulated cells; LPS, LPS-stimulated cells. Statistical comparisons, * P
    Bacterial Lps, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 398 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    OriGene lipolysaccharide binding protein
    Effects of carboxybutyrylated glucosamine (CGlcN) on the inhibition of lipopolysaccharide <t>(LPS)-induced</t> nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) production in RAW264.7 cells. (a) Cells cultured in phenol red and serum-free media were pretreated with different concentrations of CGlcN for 1 hr and stimulated with a 1-µg/ml final concentration of LPS for 48 hr. Conditioned medium was mixed with an equal amount of the <t>Griess</t> reagent and the absorbance, measured at 550 nm, represented the amount of (stable oxidation product of NO) in the medium. The values obtained were compared with those of standard concentrations of sodium nitrite dissolved in Dulbecco's modified Eagle's minimal essential medium (DMEM), and the concentrations of nitrite in conditioned media of sample-treated cells were calculated. (b) Cells growing in serum-free medium were pretreated with different concentrations of CGlcN for 1 hr and stimulated with LPS (1 µg/ml final concentration) for 24 hr. The amount of PGE 2 release was determined by the mouse PGE 2 enzyme-linked immunosorbent assay (ELISA) kit. BLK, LPS non-stimulated cells; LPS, LPS-stimulated cells. Statistical comparisons, * P
    Lipolysaccharide Binding Protein, supplied by OriGene, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    InvivoGen ultra pure lps
    Sensitivity to C5a of PBMCs pre-exposed to TLR ligands. (A–C) Levels of IL-8 in culture supernatants of PBMCs (1.5×10 5 /well) (A) stimulated for 14 h with the indicated concentrations of C5a and (B) after washing and re-culture following pre-exposure (14 h) to <t>LPS</t> (100 pg/mL or as indicated), Pam 3 Cys (100 ng/mL), Zymosan (1 μg/mL), <t>Flagellin</t> (5 μg/mL), Imiquimod (3 μg/mL) or mock-pre-exposure (no TLR ligand). IL-8 concentrations were estimated by subtracting the background levels of IL-8 present in cultures not activated with C5a and pre-exposed or not to TLR ligands from the corresponding C5a-activated samples (IL-8 background levels (ng/mL): No ligand/No C5a, 1.6±0.7; +LPS, 2.3±1.2; +Pam 3 Cys, 1.5±0.6; +Zymosan, 1.3±0.9; +Flagellin, 6.9±2.5; +Imiquimod, 5.3±1.1; n ≥4). (C) IL-8 fold increases were determined by comparing IL-8 levels – after background subtraction – between C5a-stimulated (10 nM) cell samples pre-exposed and not pre-exposed to LPS. (D) Levels of IL-6 in culture supernatants of PBMCs stimulated for 14 h with the indicated concentrations of C5a, after washing and re-culture following pre-exposure to LPS. (E and F) Determination of (E) IL-6 and IL-8 mRNA levels in RNA samples and (F) NF-κB concentrations in the nuclear extracts of PBMCs pre-exposed or not to LPS and subsequently stimulated with C5a as described for A–D. (E) mRNA levels are relative to control (No LPS/No C5a). Results are from one experiment (+SD) representative of at least four for each ligand (A, B) or three (C–F). * p
    Ultra Pure Lps, supplied by InvivoGen, used in various techniques. Bioz Stars score: 92/100, based on 477 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore salmonella rough mutant lpss
    ). (B) OS structure of 15253 LOS. (C) Analysis of affinity purification by PAGE (silver staining). Lanes: IgG, purified human IgG (control); 1, bound to the LOS column; 2, unbound to the protein G column; 3, bound to the protein G column; 4, purified IgG after the inactivated matrix column. (D) Subclass analysis. The purified IgG was analyzed with a MAb IgG/IgA subclass typing kit. (E) ELISA analysis. Each well was coated with 15253 LOS, JW31R LOS, or S. minnesota Re 595 <t>LPS</t> (200 ng each). Anti-human IgG (AP conjugate) and p -nitrophosphate were used, and the absorbances (405 nm) at 70 min are shown.
    Salmonella Rough Mutant Lpss, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore lps lps
    ). (B) OS structure of 15253 LOS. (C) Analysis of affinity purification by PAGE (silver staining). Lanes: IgG, purified human IgG (control); 1, bound to the LOS column; 2, unbound to the protein G column; 3, bound to the protein G column; 4, purified IgG after the inactivated matrix column. (D) Subclass analysis. The purified IgG was analyzed with a MAb IgG/IgA subclass typing kit. (E) ELISA analysis. Each well was coated with 15253 LOS, JW31R LOS, or S. minnesota Re 595 <t>LPS</t> (200 ng each). Anti-human IgG (AP conjugate) and p -nitrophosphate were used, and the absorbances (405 nm) at 70 min are shown.
    Lps Lps, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    InvivoGen lps b5
    Antagonistic effects of the activators of TLRs, PI-3K/Akt, ERK1/2, p38/JNK, and NF-κB pathways on the anti-IAV activity of OMT. A549 cells were infected with IAV (MOI = 0.001), treated with or without ribavirin (25 μg/mL) and OMT (50 μg/mL), and simultaneously treated with or without TLR3 activator (polyI:C, 100 ng/mL), TLR4 activator <t>LPS-B5</t> (1 μg/mL), TLR7/8 activator R-848 (10 μg/mL), PI-3K/Akt activator (IGF-1, 100 ng/mL), ERK activator (EGF, 100 ng/mL), p38/JNK activator (Anisomycin, 10 μM), and NF-κB activator (PMA, 1 µg/mL). The test doses of all activators were determined according to previous reports and our preliminary tests. After 48 h, cell viability was determined by the SRB method ( A ) and IAV vRNA levels were determined by a qRT-PCR assay ( B ). Data shown were mean ± SD of three independent experiments performed in triplicate. * p
    Lps B5, supplied by InvivoGen, used in various techniques. Bioz Stars score: 94/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    InvivoGen lps eb
    Increased NF-κB p65 translocation into the cell nucleus in <t>ATI</t> and ATI TNM treated primary human macrophages. (A) Fluorescence microscopy images of macrophages treated for 2 h with <t>LPS</t> (positive control, 100 ng/mL), ATI or ATI TNM [12.5 μg/mL] and stained for NF-κB. In untreated conditions (medium) NF-κB resides mostly in the cytoplasm, whereas an increased translocation of NF-κB p65 into the nucleus can be observed for LPS, ATI, and ATI TNM treated cells (indicated by arrows). Brightness and contrast were adjusted to the same level for every image within the panel. Scale bar = 50 μm. (B) Quantitative evaluation of NF-κB translocation from the cell cytoplasm to the nucleus by Harmony high-content analysis software (Perkin Elmer). Shown are the means ± SEM from four independent experiments/donors, * P
    Lps Eb, supplied by InvivoGen, used in various techniques. Bioz Stars score: 99/100, based on 127 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    5-ASA and AZTP restore epithelial barrier. ( A ) Paracellular permeability assay using FITC-Dextran: Differentiated and polarized T-84 monolayer grown in Transwell was treated with TNF-α (10 ng/ml), IFN-γ (100U/ml) or LPS (10 ng/ml) with or without pretreatment (5 h) with 5-ASA (5 mM) or AZTP (10 μM) n = 5 each treatment. Experiments were performed in biological duplicates and repeated three times. p value (≤0.05), * con vs treatment; # vs TNF-α or IFN-γ. One-way ANOVA with multiple comparisons were done using Bonferroni’s post hoc analysis. ( B , C ) PCR array analysis of T-84 cells treated with 5-ASA (5 mM) and AZTP(10 μM), in the presence or absence of TNF-α (10 ng/ml) ( D ) Immunofluorescence analysis of T-84 monolayer for the representative proteins of adherens junctions; AJs (E-cadherin), tight junctions; TJs (occludin), Desmosomes (Desmoglein-2). TNF-α treatment disrupted all junctions and resulted in protein internalization as indicated by arrows. 5-ASA and AZTP restored membranous expression of these proteins. Image magnification 400x. Scale 50μm. E-cadherin and Desmoglein were visualized using Alexa Fluor 488 secondary antibody. Occludin was visualized using Alexa Fluor 568 ( E ) qRT-PCR analysis of intestinal alkaline phosphatase as a marker of cellular differentiation in T-84 cells. The experiment was performed twice.

    Journal: Scientific Reports

    Article Title: Mesalamine and azathioprine modulate junctional complexes and restore epithelial barrier function in intestinal inflammation

    doi: 10.1038/s41598-019-39401-0

    Figure Lengend Snippet: 5-ASA and AZTP restore epithelial barrier. ( A ) Paracellular permeability assay using FITC-Dextran: Differentiated and polarized T-84 monolayer grown in Transwell was treated with TNF-α (10 ng/ml), IFN-γ (100U/ml) or LPS (10 ng/ml) with or without pretreatment (5 h) with 5-ASA (5 mM) or AZTP (10 μM) n = 5 each treatment. Experiments were performed in biological duplicates and repeated three times. p value (≤0.05), * con vs treatment; # vs TNF-α or IFN-γ. One-way ANOVA with multiple comparisons were done using Bonferroni’s post hoc analysis. ( B , C ) PCR array analysis of T-84 cells treated with 5-ASA (5 mM) and AZTP(10 μM), in the presence or absence of TNF-α (10 ng/ml) ( D ) Immunofluorescence analysis of T-84 monolayer for the representative proteins of adherens junctions; AJs (E-cadherin), tight junctions; TJs (occludin), Desmosomes (Desmoglein-2). TNF-α treatment disrupted all junctions and resulted in protein internalization as indicated by arrows. 5-ASA and AZTP restored membranous expression of these proteins. Image magnification 400x. Scale 50μm. E-cadherin and Desmoglein were visualized using Alexa Fluor 488 secondary antibody. Occludin was visualized using Alexa Fluor 568 ( E ) qRT-PCR analysis of intestinal alkaline phosphatase as a marker of cellular differentiation in T-84 cells. The experiment was performed twice.

    Article Snippet: Proinflammatory mediators used were human recombinant IFN-γ (100U/ml), LPS (10 ng/ml; eBioscience, Austria) and TNF-α (10 ng/ml; Miltenyi Biotec, Germany).

    Techniques: Permeability, Polymerase Chain Reaction, Immunofluorescence, Expressing, Quantitative RT-PCR, Marker, Cell Differentiation

    SER inhibits pro-inflammatory but enhances anti-inflammatory signature and up-regulates Notch1 signaling in BMDM cultures ( A/B ) BMDM stimulated with LPS (100ng/ml, 6h) were pretreated with or without SER (5μg/ml, 24h). ( A ) Western blot-assisted detection of NICD, Hes1 and pIκBα (Ser32). β-actin expression serves as an internal control for normalization. The values under the bands represent the relative ratio of normalized intensity compared to cells + LPS. Representative of three experiments is shown. ( B ) Quantitative RT-PCR-assisted detection of mRNA coding for MCP1, IL1β, CXCL1, CXCL2, CXCL10 and Arg1. Data were normalized to β2M gene expression (n=4/group). ( C/D ) BMDM stimulated with IL4 (10ng/ml, 24h) were pretreated with or without SER (5μg/ml, 24h). ( C ) Western blot-assisted detection of NICD and Hes1. β-actin expression served as an internal control for normalization. The values under the bands represent the relative ratio of normalized intensity compared to cells + IL4. Representative of three experiments is shown. ( D ) Quantitative RT-PCR-assisted detection of mRNA coding for Arg1, CD163 and CCL24. Data were normalized to β2M gene expression (n=4/group). Data are shown as mean±SD (*p

    Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

    Article Title: Serelaxin Induces Notch1 Signaling and Alleviates Hepatocellular Damage in Orthotopic Liver Transplantation

    doi: 10.1111/ajt.14706

    Figure Lengend Snippet: SER inhibits pro-inflammatory but enhances anti-inflammatory signature and up-regulates Notch1 signaling in BMDM cultures ( A/B ) BMDM stimulated with LPS (100ng/ml, 6h) were pretreated with or without SER (5μg/ml, 24h). ( A ) Western blot-assisted detection of NICD, Hes1 and pIκBα (Ser32). β-actin expression serves as an internal control for normalization. The values under the bands represent the relative ratio of normalized intensity compared to cells + LPS. Representative of three experiments is shown. ( B ) Quantitative RT-PCR-assisted detection of mRNA coding for MCP1, IL1β, CXCL1, CXCL2, CXCL10 and Arg1. Data were normalized to β2M gene expression (n=4/group). ( C/D ) BMDM stimulated with IL4 (10ng/ml, 24h) were pretreated with or without SER (5μg/ml, 24h). ( C ) Western blot-assisted detection of NICD and Hes1. β-actin expression served as an internal control for normalization. The values under the bands represent the relative ratio of normalized intensity compared to cells + IL4. Representative of three experiments is shown. ( D ) Quantitative RT-PCR-assisted detection of mRNA coding for Arg1, CD163 and CCL24. Data were normalized to β2M gene expression (n=4/group). Data are shown as mean±SD (*p

    Article Snippet: Lipopolysaccharides (LPS) and Interleukin-4 (IL4) were obtained from Sigma Aldrich (St. Louis, MO).

    Techniques: Western Blot, Expressing, Quantitative RT-PCR

    Chemokine and TLR-4 expression in palmitic acid-treated C3A hepatocytes . C3A hepatocytes were cultured in medium containing lipopolysaccharide (LPS) (500 pg/ml), palmitic acid (PA; 100 mM) or the combination of PA and LPS for 24h. Expression of (A) IL-8 and (B) TLR-4 was assessed using pre-developed assays for real-time PCR and values were calculated using a comparative C T method according to the manufacturer instructions (Applied Biosystems). Data are presented as mean SEM of 4 experiments. Statistical analysis was performed using two-way ANOVA and Tukey's multiple comparisons test. *p

    Journal: BMC Physiology

    Article Title: Western diet enhances hepatic inflammation in mice exposed to cecal ligation and puncture

    doi: 10.1186/1472-6793-10-20

    Figure Lengend Snippet: Chemokine and TLR-4 expression in palmitic acid-treated C3A hepatocytes . C3A hepatocytes were cultured in medium containing lipopolysaccharide (LPS) (500 pg/ml), palmitic acid (PA; 100 mM) or the combination of PA and LPS for 24h. Expression of (A) IL-8 and (B) TLR-4 was assessed using pre-developed assays for real-time PCR and values were calculated using a comparative C T method according to the manufacturer instructions (Applied Biosystems). Data are presented as mean SEM of 4 experiments. Statistical analysis was performed using two-way ANOVA and Tukey's multiple comparisons test. *p

    Article Snippet: Lipopolysaccaride (LPS) from E. Coli (serotype 111:B4; Sigma-Aldrich, St. Louis, MO) was suspended in EMEM containing 1% BSA; the working concentration for cell treatment was 500 pg/ml.

    Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction

    A trapping mutant of PDI engages the known downstream electron acceptor, ERO1α, in mammalian cells (a) Autoradiograph of immunoglobulin-M from metabolically-labeled wildtype (α +/+ ;β +/+ ) and ERO1 mutant (α mut/mut ;β mut/mut ) lipopolysaccharide blasts. Cells were labeled with 35 S methionine-cysteine for 15 minutes and chased for the indicated time with unlabeled media before lysis and immunoprecipitation. The upper panel is a radiograph of a non-reducing gel and the lower panel is a reducing gel. The migration of IgM monomers (μ) dimers (2μ) and pentamers of dimers (5μ) is indicated. (b) Immunoblots of FLAG-tagged proteins (left) or endogenous ERO1α (right) immunopurified with the FLAG-M1 antibody from lysates of HEK 293T cells that were untransfected or transfected with expression plasmids of the indicated proteins. The upper panels are of reducing and the lower panels non-reducing gels. The low mobility complex containing ERO1α immunoreactive material in complex with the FLAG-tagged PDI C-terminal active site trapping mutant (FLAG-PDI C400S ) is noted by an asterisks on both non reducing gels.

    Journal: Molecular cell

    Article Title: Oxidative protein folding by an endoplasmic reticulum localized peroxiredoxin

    doi: 10.1016/j.molcel.2010.11.010

    Figure Lengend Snippet: A trapping mutant of PDI engages the known downstream electron acceptor, ERO1α, in mammalian cells (a) Autoradiograph of immunoglobulin-M from metabolically-labeled wildtype (α +/+ ;β +/+ ) and ERO1 mutant (α mut/mut ;β mut/mut ) lipopolysaccharide blasts. Cells were labeled with 35 S methionine-cysteine for 15 minutes and chased for the indicated time with unlabeled media before lysis and immunoprecipitation. The upper panel is a radiograph of a non-reducing gel and the lower panel is a reducing gel. The migration of IgM monomers (μ) dimers (2μ) and pentamers of dimers (5μ) is indicated. (b) Immunoblots of FLAG-tagged proteins (left) or endogenous ERO1α (right) immunopurified with the FLAG-M1 antibody from lysates of HEK 293T cells that were untransfected or transfected with expression plasmids of the indicated proteins. The upper panels are of reducing and the lower panels non-reducing gels. The low mobility complex containing ERO1α immunoreactive material in complex with the FLAG-tagged PDI C-terminal active site trapping mutant (FLAG-PDI C400S ) is noted by an asterisks on both non reducing gels.

    Article Snippet: Splenic cells from wildtype and compound homozygous Ero1α;Ero1β mutant mice ( ) were cultured in RPMI-1640 at a density of 106 cells/ml and exposed to 50 μg/ml Lipopolysaccaride from E. Coli (Sigma, L2755) for 2–3 days.

    Techniques: Mutagenesis, Autoradiography, Metabolic Labelling, Labeling, Lysis, Immunoprecipitation, Migration, Western Blot, Transfection, Expressing

    TLR2 is involved in IL-12-dependent IFN-γ secretion by CD4 + cells co-cultured with ESAT-6, HspX and BCG-treated DCs. DCs cultured in the absence (filled bars) or presence (open bars) of 5 µg/ml TLR2-blocking antibody were treated for 24 hrs with Mtb, BCG alone or combined with HspX and ESAT6, 10 µg/ml Pam3CSK4 (Pam3) or 100 ng/ml LPS. (A) Supernatants were collected and IL-12 release was analyzed by ELISA. Results are the mean value+SD of four experiments. Statistical analysis: antibody-treated vs antibody-untreated cells, ns P > 0.05, *P

    Journal: PLoS ONE

    Article Title: ESAT-6 and HspX Improve the Effectiveness of BCG to Induce Human Dendritic Cells-Dependent Th1 and NK Cells Activation

    doi: 10.1371/journal.pone.0075684

    Figure Lengend Snippet: TLR2 is involved in IL-12-dependent IFN-γ secretion by CD4 + cells co-cultured with ESAT-6, HspX and BCG-treated DCs. DCs cultured in the absence (filled bars) or presence (open bars) of 5 µg/ml TLR2-blocking antibody were treated for 24 hrs with Mtb, BCG alone or combined with HspX and ESAT6, 10 µg/ml Pam3CSK4 (Pam3) or 100 ng/ml LPS. (A) Supernatants were collected and IL-12 release was analyzed by ELISA. Results are the mean value+SD of four experiments. Statistical analysis: antibody-treated vs antibody-untreated cells, ns P > 0.05, *P

    Article Snippet: Ultrapure lipolysaccharide (LPS) from E. coli (0111: B4 strain) and palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK4) were purchased from InvivoGen (San Diego, CA).

    Techniques: Cell Culture, Blocking Assay, Enzyme-linked Immunosorbent Assay

    Galectin-3 binds to meningococci expressing LPS with a terminal LacNAc

    Journal: Cellular microbiology

    Article Title: Galectin-3 binds Neisseria meningitidis and increases interaction with phagocytic cells

    doi: 10.1111/j.1462-5822.2012.01838.x

    Figure Lengend Snippet: Galectin-3 binds to meningococci expressing LPS with a terminal LacNAc

    Article Snippet: For labelling of bacteria the cells were incubated for 1 hr with an anti-lipoplysaccharide (LPS) antibody (α-L3,7,9) and with an anti-galectin-3 antibody (Santa Cruz Biotechnology), after which the cells were washed twice in PBS then incubated for 30 min with an anti-mouse Alexa Fluor 488-conjugated antibody and anti-human Alexa Fluor 555-conjugated antibody (Invitrogen) to label bacteria and cells respectively.

    Techniques: Expressing

    Full length lipopolysaccharide is required for galectin-3 binding to N. meningitidis

    Journal: Cellular microbiology

    Article Title: Galectin-3 binds Neisseria meningitidis and increases interaction with phagocytic cells

    doi: 10.1111/j.1462-5822.2012.01838.x

    Figure Lengend Snippet: Full length lipopolysaccharide is required for galectin-3 binding to N. meningitidis

    Article Snippet: For labelling of bacteria the cells were incubated for 1 hr with an anti-lipoplysaccharide (LPS) antibody (α-L3,7,9) and with an anti-galectin-3 antibody (Santa Cruz Biotechnology), after which the cells were washed twice in PBS then incubated for 30 min with an anti-mouse Alexa Fluor 488-conjugated antibody and anti-human Alexa Fluor 555-conjugated antibody (Invitrogen) to label bacteria and cells respectively.

    Techniques: Binding Assay

    V-58 stimulates intracellular Mtb to produce cAMP J774.16 murine macrophages were infected with live Mtb H37Rv or CDC1551, and heat-killed CDC1551 were treated with either 0.1 % DMSO (black bars) or 10 μM V-58 (gray bars). cAMP levels were quantified by RIA from the A) intracellular bacteria and B) macrophage lysates. The data shown are the means of two biologically independent experiments. Error bars represent the standard deviation between replicates. C) Macrophages were infected with Mtb CDC1551 at the indicated MOI and treated with 10 ng/mL LPS, 250 μM 8-Br-cAMP or V-58 at the indicated concentrations for 18 h and supernatant TNF-α levels were determined by ELISA. Data are means of four biologically independent experiments and the error bars represent the standard deviation between experiments. Statistical significance was determined with a two-tailed unpaired t-test, *p≤0.05, **p≤0.005 and ****p≤0.0001.

    Journal: Molecular microbiology

    Article Title: Chemical activation of adenylyl cyclase Rv1625c inhibits growth of Mycobacterium tuberculosis on cholesterol and modulates intramacrophage signaling

    doi: 10.1111/mmi.13701

    Figure Lengend Snippet: V-58 stimulates intracellular Mtb to produce cAMP J774.16 murine macrophages were infected with live Mtb H37Rv or CDC1551, and heat-killed CDC1551 were treated with either 0.1 % DMSO (black bars) or 10 μM V-58 (gray bars). cAMP levels were quantified by RIA from the A) intracellular bacteria and B) macrophage lysates. The data shown are the means of two biologically independent experiments. Error bars represent the standard deviation between replicates. C) Macrophages were infected with Mtb CDC1551 at the indicated MOI and treated with 10 ng/mL LPS, 250 μM 8-Br-cAMP or V-58 at the indicated concentrations for 18 h and supernatant TNF-α levels were determined by ELISA. Data are means of four biologically independent experiments and the error bars represent the standard deviation between experiments. Statistical significance was determined with a two-tailed unpaired t-test, *p≤0.05, **p≤0.005 and ****p≤0.0001.

    Article Snippet: Following 2 h of infection, the cultures were washed twice with warm supplemented DMEM, replenished with the same medium containing 8Br-cAMP (Sigma) at the indicated concentrations, LPS 10 ng/ml (List Biological Laboratories), V-58 at the indicated concentrations, or DMSO vehicle control.

    Techniques: Infection, Standard Deviation, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    DHA pretreatment attenuates forepaw placing deficits among hypoxic ischemic pups exposed to LPS pretreatment

    Journal: American journal of obstetrics and gynecology

    Article Title: Docosahexaenoic acid Confers Neuroprotection in a Rat Model of Perinatal Hypoxia-ischemia potentiated by E. coli lipopolysaccharide-induced systemic inflammation

    doi: 10.1016/j.ajog.2009.01.020

    Figure Lengend Snippet: DHA pretreatment attenuates forepaw placing deficits among hypoxic ischemic pups exposed to LPS pretreatment

    Article Snippet: [ ] Rats received intraperitoneal Escherichia coli lipolysaccharide (LPS) 055:B5 (0.1 mg/kg) (Sigma, St. Louis) followed 2.5 hours later by right carotid artery ligation.

    Techniques:

    Proteinase K-agarose digestion ablates HSP70 activity . (A) 200 μg rhHSP70 was incubated with digestion buffer (lane 1), Proteinase K-agarose beads (lane 2), or control agarose beads (lane 3) for 3 hr at 37°C, as described in Methods . Protein integrity and endotoxin content were determined by silver staining and the LAL assay, respectively. (B) 100 pg of ultrapure LPS, which is an approximation of the endotoxin content in 200 μg of rhHSP70 (see Results and Discussion ), was incubated with Proteinase K-agarose beads or control agarose beads, under the conditions described in A . The ultrapure LPS treated with Proteinase K-agarose beads (PK) or control agarose beads (CTRL) were then used to stimulate primary human macrophages for 16 hr at 100 pg/mL LPS final concentration in each culture. Cell culture supernatants were collected and analyzed for TNF-α. (C) The HSP70 preparations in A were used to stimulate primary human macrophages for 4 hr at 10 μg/mL final concentration. Cell culture supernatants were collected and analyzed for TNF-α. Endotoxin levels shown at bottom are the calculated amounts in the macrophage cultures based on the LAL determinations in A . Untreated macrophage cultures produced ≤ 100 pg/mL of TNF-α under these conditions. Results shown are representative of two independent digestion and stimulation experiments. *** p

    Journal: Journal of Inflammation (London, England)

    Article Title: Stimulation of TLR4 by recombinant HSP70 requires structural integrity of the HSP70 protein itself

    doi: 10.1186/1476-9255-9-11

    Figure Lengend Snippet: Proteinase K-agarose digestion ablates HSP70 activity . (A) 200 μg rhHSP70 was incubated with digestion buffer (lane 1), Proteinase K-agarose beads (lane 2), or control agarose beads (lane 3) for 3 hr at 37°C, as described in Methods . Protein integrity and endotoxin content were determined by silver staining and the LAL assay, respectively. (B) 100 pg of ultrapure LPS, which is an approximation of the endotoxin content in 200 μg of rhHSP70 (see Results and Discussion ), was incubated with Proteinase K-agarose beads or control agarose beads, under the conditions described in A . The ultrapure LPS treated with Proteinase K-agarose beads (PK) or control agarose beads (CTRL) were then used to stimulate primary human macrophages for 16 hr at 100 pg/mL LPS final concentration in each culture. Cell culture supernatants were collected and analyzed for TNF-α. (C) The HSP70 preparations in A were used to stimulate primary human macrophages for 4 hr at 10 μg/mL final concentration. Cell culture supernatants were collected and analyzed for TNF-α. Endotoxin levels shown at bottom are the calculated amounts in the macrophage cultures based on the LAL determinations in A . Untreated macrophage cultures produced ≤ 100 pg/mL of TNF-α under these conditions. Results shown are representative of two independent digestion and stimulation experiments. *** p

    Article Snippet: Reagents Recombinant low-endotoxin human HSP70 (cat. # ADI-ESP-555-F; lot # 05040922) and ultrapure LPSs (cat. # ALX-581-007-L002 and ALX-581-013-L002) were from Enzo Life Sciences (Plymouth Meeting, PA).

    Techniques: Activity Assay, Incubation, Silver Staining, LAL Assay, Concentration Assay, Cell Culture, Produced

    Effects of carboxybutyrylated glucosamine (CGlcN) on the inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) production in RAW264.7 cells. (a) Cells cultured in phenol red and serum-free media were pretreated with different concentrations of CGlcN for 1 hr and stimulated with a 1-µg/ml final concentration of LPS for 48 hr. Conditioned medium was mixed with an equal amount of the Griess reagent and the absorbance, measured at 550 nm, represented the amount of (stable oxidation product of NO) in the medium. The values obtained were compared with those of standard concentrations of sodium nitrite dissolved in Dulbecco's modified Eagle's minimal essential medium (DMEM), and the concentrations of nitrite in conditioned media of sample-treated cells were calculated. (b) Cells growing in serum-free medium were pretreated with different concentrations of CGlcN for 1 hr and stimulated with LPS (1 µg/ml final concentration) for 24 hr. The amount of PGE 2 release was determined by the mouse PGE 2 enzyme-linked immunosorbent assay (ELISA) kit. BLK, LPS non-stimulated cells; LPS, LPS-stimulated cells. Statistical comparisons, * P

    Journal: Immunology

    Article Title: Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 in lipopolysaccharide-stimulated RAW264.7 cells by carboxybutyrylated glucosamine takes place via down-regulation of mitogen-activated protein kinase-mediated nuclear factor-?B signaling

    doi: 10.1111/j.1365-2567.2007.02683.x

    Figure Lengend Snippet: Effects of carboxybutyrylated glucosamine (CGlcN) on the inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) production in RAW264.7 cells. (a) Cells cultured in phenol red and serum-free media were pretreated with different concentrations of CGlcN for 1 hr and stimulated with a 1-µg/ml final concentration of LPS for 48 hr. Conditioned medium was mixed with an equal amount of the Griess reagent and the absorbance, measured at 550 nm, represented the amount of (stable oxidation product of NO) in the medium. The values obtained were compared with those of standard concentrations of sodium nitrite dissolved in Dulbecco's modified Eagle's minimal essential medium (DMEM), and the concentrations of nitrite in conditioned media of sample-treated cells were calculated. (b) Cells growing in serum-free medium were pretreated with different concentrations of CGlcN for 1 hr and stimulated with LPS (1 µg/ml final concentration) for 24 hr. The amount of PGE 2 release was determined by the mouse PGE 2 enzyme-linked immunosorbent assay (ELISA) kit. BLK, LPS non-stimulated cells; LPS, LPS-stimulated cells. Statistical comparisons, * P

    Article Snippet: The MTT reagent [(3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide)], the Griess reagent and bacterial LPS were purchased from Sigma Chemical Co. Primary and secondary antibodies used for Western blot analysis were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) and Amersham Pharmacia Biosciences (Piscataway, NJ), respectively.

    Techniques: Inhibition, Cell Culture, Concentration Assay, Modification, Enzyme-linked Immunosorbent Assay

    Sensitivity to C5a of PBMCs pre-exposed to TLR ligands. (A–C) Levels of IL-8 in culture supernatants of PBMCs (1.5×10 5 /well) (A) stimulated for 14 h with the indicated concentrations of C5a and (B) after washing and re-culture following pre-exposure (14 h) to LPS (100 pg/mL or as indicated), Pam 3 Cys (100 ng/mL), Zymosan (1 μg/mL), Flagellin (5 μg/mL), Imiquimod (3 μg/mL) or mock-pre-exposure (no TLR ligand). IL-8 concentrations were estimated by subtracting the background levels of IL-8 present in cultures not activated with C5a and pre-exposed or not to TLR ligands from the corresponding C5a-activated samples (IL-8 background levels (ng/mL): No ligand/No C5a, 1.6±0.7; +LPS, 2.3±1.2; +Pam 3 Cys, 1.5±0.6; +Zymosan, 1.3±0.9; +Flagellin, 6.9±2.5; +Imiquimod, 5.3±1.1; n ≥4). (C) IL-8 fold increases were determined by comparing IL-8 levels – after background subtraction – between C5a-stimulated (10 nM) cell samples pre-exposed and not pre-exposed to LPS. (D) Levels of IL-6 in culture supernatants of PBMCs stimulated for 14 h with the indicated concentrations of C5a, after washing and re-culture following pre-exposure to LPS. (E and F) Determination of (E) IL-6 and IL-8 mRNA levels in RNA samples and (F) NF-κB concentrations in the nuclear extracts of PBMCs pre-exposed or not to LPS and subsequently stimulated with C5a as described for A–D. (E) mRNA levels are relative to control (No LPS/No C5a). Results are from one experiment (+SD) representative of at least four for each ligand (A, B) or three (C–F). * p

    Journal: European Journal of Immunology

    Article Title: TLR activation enhances C5a-induced pro-inflammatory responses by negatively modulating the second C5a receptor, C5L2

    doi: 10.1002/eji.201041350

    Figure Lengend Snippet: Sensitivity to C5a of PBMCs pre-exposed to TLR ligands. (A–C) Levels of IL-8 in culture supernatants of PBMCs (1.5×10 5 /well) (A) stimulated for 14 h with the indicated concentrations of C5a and (B) after washing and re-culture following pre-exposure (14 h) to LPS (100 pg/mL or as indicated), Pam 3 Cys (100 ng/mL), Zymosan (1 μg/mL), Flagellin (5 μg/mL), Imiquimod (3 μg/mL) or mock-pre-exposure (no TLR ligand). IL-8 concentrations were estimated by subtracting the background levels of IL-8 present in cultures not activated with C5a and pre-exposed or not to TLR ligands from the corresponding C5a-activated samples (IL-8 background levels (ng/mL): No ligand/No C5a, 1.6±0.7; +LPS, 2.3±1.2; +Pam 3 Cys, 1.5±0.6; +Zymosan, 1.3±0.9; +Flagellin, 6.9±2.5; +Imiquimod, 5.3±1.1; n ≥4). (C) IL-8 fold increases were determined by comparing IL-8 levels – after background subtraction – between C5a-stimulated (10 nM) cell samples pre-exposed and not pre-exposed to LPS. (D) Levels of IL-6 in culture supernatants of PBMCs stimulated for 14 h with the indicated concentrations of C5a, after washing and re-culture following pre-exposure to LPS. (E and F) Determination of (E) IL-6 and IL-8 mRNA levels in RNA samples and (F) NF-κB concentrations in the nuclear extracts of PBMCs pre-exposed or not to LPS and subsequently stimulated with C5a as described for A–D. (E) mRNA levels are relative to control (No LPS/No C5a). Results are from one experiment (+SD) representative of at least four for each ligand (A, B) or three (C–F). * p

    Article Snippet: For cell activation experiments, triplicate cell aliquots (1.5×105 cells/well, unless stated otherwise) were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% heat-inactivated (56°C, 30 min) FCS (HyClone; < 0.06 U/mL endotoxin) and 2 mM glutamine (complete medium), and stimulated at 37°C for 14 h or the time indicated with optimal concentrations of ultra-pure LPS (E. coli O111:B4 strain), zymosan, flagellin, imiquimod – all from Invivogen – Pam3 -Cys-Ser-(Lys)4 HCl (Pam3 Cys; EMC microcollections GmbH) as indicated, or medium alone (mock stimulation).

    Techniques:

    DAB2 is downregulated in human and murine dendritic cells after Toll-like receptor activation through MyD88 and TRIF pathways. (A) Bone-marrow derived dendritic cells (BMDC) differentiated from C57BL/6J mice, DC2.4 cells, and human monocytes-derived dendritic cells were incubated with 100 ng/mL LPS, and DAB2 expression was quantified using western blotting . (B) DAB2 expression in BMDCs treated with agonist for extracellular TLR or (C) agonist for intracellular TLR. (D,E) DAB2 expression after treatment with HKLM, HMW, or LPS in BMDC differentiated from WT, MyD88 −/− , and or TRIF −/− C57BL/6 mice. Bar graphs represent mean values of three independent experiments combined ( n = 3 mice, ** p

    Journal: Frontiers in Immunology

    Article Title: Rapid Downregulation of DAB2 by Toll-Like Receptor Activation Contributes to a Pro-Inflammatory Switch in Activated Dendritic Cells

    doi: 10.3389/fimmu.2019.00304

    Figure Lengend Snippet: DAB2 is downregulated in human and murine dendritic cells after Toll-like receptor activation through MyD88 and TRIF pathways. (A) Bone-marrow derived dendritic cells (BMDC) differentiated from C57BL/6J mice, DC2.4 cells, and human monocytes-derived dendritic cells were incubated with 100 ng/mL LPS, and DAB2 expression was quantified using western blotting . (B) DAB2 expression in BMDCs treated with agonist for extracellular TLR or (C) agonist for intracellular TLR. (D,E) DAB2 expression after treatment with HKLM, HMW, or LPS in BMDC differentiated from WT, MyD88 −/− , and or TRIF −/− C57BL/6 mice. Bar graphs represent mean values of three independent experiments combined ( n = 3 mice, ** p

    Article Snippet: BMDCs were also treated for a short period with 100 ng/mL LPS (InvivoGen) or pre-treated with 100 nM bafilomycin A1 (Sigma Aldrich) or 2 μM MG132 (Sigma Aldrich) for 30 min before treatment with 100 ng/mL LPS for 1 h. To study Dab2 protein and mRNA stability, BMDCs were treated with 5 μg/mL cycloheximide (Sigma Aldrich) or 10 μg/mL actinomycin D (Santa Cruz Biotechnology) for different durations.

    Techniques: Activation Assay, Derivative Assay, Mouse Assay, Incubation, Expressing, Western Blot

    TLR4 activation represses both DAB2 protein and gene expression with different dynamics. (A) BMDCs were incubated with 100 ng/mL LPS for up to 90 min and DAB2 protein expression was evaluated by Western blotting. (B) Dab2 mRNA expression was evaluated after incubation with 100 ng/mL LPS for up to 16 h. Dab2 and Tbp (as an internal control) gene expression was quantified using qRT-PCR. * p

    Journal: Frontiers in Immunology

    Article Title: Rapid Downregulation of DAB2 by Toll-Like Receptor Activation Contributes to a Pro-Inflammatory Switch in Activated Dendritic Cells

    doi: 10.3389/fimmu.2019.00304

    Figure Lengend Snippet: TLR4 activation represses both DAB2 protein and gene expression with different dynamics. (A) BMDCs were incubated with 100 ng/mL LPS for up to 90 min and DAB2 protein expression was evaluated by Western blotting. (B) Dab2 mRNA expression was evaluated after incubation with 100 ng/mL LPS for up to 16 h. Dab2 and Tbp (as an internal control) gene expression was quantified using qRT-PCR. * p

    Article Snippet: BMDCs were also treated for a short period with 100 ng/mL LPS (InvivoGen) or pre-treated with 100 nM bafilomycin A1 (Sigma Aldrich) or 2 μM MG132 (Sigma Aldrich) for 30 min before treatment with 100 ng/mL LPS for 1 h. To study Dab2 protein and mRNA stability, BMDCs were treated with 5 μg/mL cycloheximide (Sigma Aldrich) or 10 μg/mL actinomycin D (Santa Cruz Biotechnology) for different durations.

    Techniques: Activation Assay, Expressing, Incubation, Western Blot, Quantitative RT-PCR

    ). (B) OS structure of 15253 LOS. (C) Analysis of affinity purification by PAGE (silver staining). Lanes: IgG, purified human IgG (control); 1, bound to the LOS column; 2, unbound to the protein G column; 3, bound to the protein G column; 4, purified IgG after the inactivated matrix column. (D) Subclass analysis. The purified IgG was analyzed with a MAb IgG/IgA subclass typing kit. (E) ELISA analysis. Each well was coated with 15253 LOS, JW31R LOS, or S. minnesota Re 595 LPS (200 ng each). Anti-human IgG (AP conjugate) and p -nitrophosphate were used, and the absorbances (405 nm) at 70 min are shown.

    Journal: Infection and Immunity

    Article Title: The Oligosaccharide of Gonococcal Lipooligosaccharide Contains Several Epitopes That Are Recognized by Human Antibodies ▿

    doi: 10.1128/IAI.01445-09

    Figure Lengend Snippet: ). (B) OS structure of 15253 LOS. (C) Analysis of affinity purification by PAGE (silver staining). Lanes: IgG, purified human IgG (control); 1, bound to the LOS column; 2, unbound to the protein G column; 3, bound to the protein G column; 4, purified IgG after the inactivated matrix column. (D) Subclass analysis. The purified IgG was analyzed with a MAb IgG/IgA subclass typing kit. (E) ELISA analysis. Each well was coated with 15253 LOS, JW31R LOS, or S. minnesota Re 595 LPS (200 ng each). Anti-human IgG (AP conjugate) and p -nitrophosphate were used, and the absorbances (405 nm) at 70 min are shown.

    Article Snippet: Escherichia coli LPS and the following Salmonella rough mutant LPSs were purchased from Sigma (St. Louis, MO): Ra ( S almonella enterica serovar Typhimurium TV119), Rc (“ S. minnesota ” R5), Rd ( S. minnesota R7), and Re ( S. minnesota Re 595).

    Techniques: Affinity Purification, Polyacrylamide Gel Electrophoresis, Silver Staining, Purification, Enzyme-linked Immunosorbent Assay

    Antagonistic effects of the activators of TLRs, PI-3K/Akt, ERK1/2, p38/JNK, and NF-κB pathways on the anti-IAV activity of OMT. A549 cells were infected with IAV (MOI = 0.001), treated with or without ribavirin (25 μg/mL) and OMT (50 μg/mL), and simultaneously treated with or without TLR3 activator (polyI:C, 100 ng/mL), TLR4 activator LPS-B5 (1 μg/mL), TLR7/8 activator R-848 (10 μg/mL), PI-3K/Akt activator (IGF-1, 100 ng/mL), ERK activator (EGF, 100 ng/mL), p38/JNK activator (Anisomycin, 10 μM), and NF-κB activator (PMA, 1 µg/mL). The test doses of all activators were determined according to previous reports and our preliminary tests. After 48 h, cell viability was determined by the SRB method ( A ) and IAV vRNA levels were determined by a qRT-PCR assay ( B ). Data shown were mean ± SD of three independent experiments performed in triplicate. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Oxymatrine Inhibits Influenza A Virus Replication and Inflammation via TLR4, p38 MAPK and NF-κB Pathways

    doi: 10.3390/ijms19040965

    Figure Lengend Snippet: Antagonistic effects of the activators of TLRs, PI-3K/Akt, ERK1/2, p38/JNK, and NF-κB pathways on the anti-IAV activity of OMT. A549 cells were infected with IAV (MOI = 0.001), treated with or without ribavirin (25 μg/mL) and OMT (50 μg/mL), and simultaneously treated with or without TLR3 activator (polyI:C, 100 ng/mL), TLR4 activator LPS-B5 (1 μg/mL), TLR7/8 activator R-848 (10 μg/mL), PI-3K/Akt activator (IGF-1, 100 ng/mL), ERK activator (EGF, 100 ng/mL), p38/JNK activator (Anisomycin, 10 μM), and NF-κB activator (PMA, 1 µg/mL). The test doses of all activators were determined according to previous reports and our preliminary tests. After 48 h, cell viability was determined by the SRB method ( A ) and IAV vRNA levels were determined by a qRT-PCR assay ( B ). Data shown were mean ± SD of three independent experiments performed in triplicate. * p

    Article Snippet: Poly(I:C) (tlrl-pic), LPS-B5 (tlrl-pb5lps), R-848 (tlrl-r848) and PMA (tlrl-pma) were purchased from InvivoGen (San Diego, CA, USA).

    Techniques: Activity Assay, Infection, Sulforhodamine B Assay, Quantitative RT-PCR

    Increased NF-κB p65 translocation into the cell nucleus in ATI and ATI TNM treated primary human macrophages. (A) Fluorescence microscopy images of macrophages treated for 2 h with LPS (positive control, 100 ng/mL), ATI or ATI TNM [12.5 μg/mL] and stained for NF-κB. In untreated conditions (medium) NF-κB resides mostly in the cytoplasm, whereas an increased translocation of NF-κB p65 into the nucleus can be observed for LPS, ATI, and ATI TNM treated cells (indicated by arrows). Brightness and contrast were adjusted to the same level for every image within the panel. Scale bar = 50 μm. (B) Quantitative evaluation of NF-κB translocation from the cell cytoplasm to the nucleus by Harmony high-content analysis software (Perkin Elmer). Shown are the means ± SEM from four independent experiments/donors, * P

    Journal: Frontiers in Immunology

    Article Title: Nitration of Wheat Amylase Trypsin Inhibitors Increases Their Innate and Adaptive Immunostimulatory Potential in vitro

    doi: 10.3389/fimmu.2018.03174

    Figure Lengend Snippet: Increased NF-κB p65 translocation into the cell nucleus in ATI and ATI TNM treated primary human macrophages. (A) Fluorescence microscopy images of macrophages treated for 2 h with LPS (positive control, 100 ng/mL), ATI or ATI TNM [12.5 μg/mL] and stained for NF-κB. In untreated conditions (medium) NF-κB resides mostly in the cytoplasm, whereas an increased translocation of NF-κB p65 into the nucleus can be observed for LPS, ATI, and ATI TNM treated cells (indicated by arrows). Brightness and contrast were adjusted to the same level for every image within the panel. Scale bar = 50 μm. (B) Quantitative evaluation of NF-κB translocation from the cell cytoplasm to the nucleus by Harmony high-content analysis software (Perkin Elmer). Shown are the means ± SEM from four independent experiments/donors, * P

    Article Snippet: After 6 days, cells were stimulated for 2 h either with ATI, ATI TNM [12.5 μg/mL], or with LPS EB (100 ng/mL, Invivogen) as a positive control.

    Techniques: Translocation Assay, Fluorescence, Microscopy, Positive Control, Staining, High Content Screening, Software

    Different nitrated ATI induce distinct TLR4 activation in HeLa TLR4 dual reporter cells. (A) HeLa TLR4 dual reporter cells were treated for 7 h with nitrated or unmodified ATI [7.5 μg/mL] or LPS [25 ng/mL] as a positive control. (B) Cells were pre-incubated for 2.5 h with TAK242 [0.36 μg/mL] or its solvent DMSO [4.4 μg/mL]. Then the cells were stimulated with nitrated or unmodified ATI [15 μg/mL], or LPS [25 ng/mL] for 7 h. The relative luciferase activity was calculated by dividing the Renilla luciferase (TLR4) signal by the Firefly luciferase (viability) signal. The resulting values were normalized to the value obtained for LPS- treated cells. Shown are the means ± SD of three independent experiments measured in triplicates using two independently nitrated probes. *** P

    Journal: Frontiers in Immunology

    Article Title: Nitration of Wheat Amylase Trypsin Inhibitors Increases Their Innate and Adaptive Immunostimulatory Potential in vitro

    doi: 10.3389/fimmu.2018.03174

    Figure Lengend Snippet: Different nitrated ATI induce distinct TLR4 activation in HeLa TLR4 dual reporter cells. (A) HeLa TLR4 dual reporter cells were treated for 7 h with nitrated or unmodified ATI [7.5 μg/mL] or LPS [25 ng/mL] as a positive control. (B) Cells were pre-incubated for 2.5 h with TAK242 [0.36 μg/mL] or its solvent DMSO [4.4 μg/mL]. Then the cells were stimulated with nitrated or unmodified ATI [15 μg/mL], or LPS [25 ng/mL] for 7 h. The relative luciferase activity was calculated by dividing the Renilla luciferase (TLR4) signal by the Firefly luciferase (viability) signal. The resulting values were normalized to the value obtained for LPS- treated cells. Shown are the means ± SD of three independent experiments measured in triplicates using two independently nitrated probes. *** P

    Article Snippet: After 6 days, cells were stimulated for 2 h either with ATI, ATI TNM [12.5 μg/mL], or with LPS EB (100 ng/mL, Invivogen) as a positive control.

    Techniques: Activation Assay, Positive Control, Incubation, Luciferase, Activity Assay