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  • 99
    Thermo Fisher msu crystals
    Impact of anti-CD44 antibody treatment on NLRP3 inflammasome activation and nuclear factor kappa B (NFκB) p65 subunit nuclear translocation in differentiated human <t>THP-1</t> macrophages following incubation with monosodium urate monohydrate <t>(MSU)</t> crystals and role of intracellular protein-phosphatase 2A (PP2A) in mediating anti-CD44 antibody’s effect. We utilized the IM7 clone, which recognizes all CD44 isoforms and causes enzyme-mediated shedding of the extracellular domain. THP-1 macrophages were treated with MSU crystals (100 μg/mL) ± anti-CD44 or isotype control (IC) antibodies (2 μg/mL for both antibodies). NF-κB p65 nuclear levels were determined following a 1-hour incubation using a DNA binding assay. Pro-caspase-1 and the p10 subunit of active caspase-1 were determined using Western Blotting. Active caspase-1 activity was determined using a specific activity assay. Cellular pro-IL-1β and mature IL-1β levels were determined using an ELISA. PP2A inhibition was performed using okadiac acid (5 nM). Data from 3 independent experiments are presented with mean and S.D. highlighted. *p
    Msu Crystals, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 96 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore monosodium urate msu crystals crystals suspended in phosphate buffered saline
    Impact of anti-CD44 antibody treatment on NLRP3 inflammasome activation and nuclear factor kappa B (NFκB) p65 subunit nuclear translocation in differentiated human <t>THP-1</t> macrophages following incubation with monosodium urate monohydrate <t>(MSU)</t> crystals and role of intracellular protein-phosphatase 2A (PP2A) in mediating anti-CD44 antibody’s effect. We utilized the IM7 clone, which recognizes all CD44 isoforms and causes enzyme-mediated shedding of the extracellular domain. THP-1 macrophages were treated with MSU crystals (100 μg/mL) ± anti-CD44 or isotype control (IC) antibodies (2 μg/mL for both antibodies). NF-κB p65 nuclear levels were determined following a 1-hour incubation using a DNA binding assay. Pro-caspase-1 and the p10 subunit of active caspase-1 were determined using Western Blotting. Active caspase-1 activity was determined using a specific activity assay. Cellular pro-IL-1β and mature IL-1β levels were determined using an ELISA. PP2A inhibition was performed using okadiac acid (5 nM). Data from 3 independent experiments are presented with mean and S.D. highlighted. *p
    Monosodium Urate Msu Crystals Crystals Suspended In Phosphate Buffered Saline, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    InvivoGen msu crystals
    Time and treatment-dependent phagocytosis of monosodium urate monohydrate <t>(MSU)</t> crystals by differentiated human <t>THP-1</t> macrophages using flow cytometry and impact of recombinant human proteoglycan-4 (rhPRG4) or bovine submaxillary mucin (BSM) treatments following 2 and 4-h incubations. Quantitative determination of MSU phagocytosis was performed using the percentage of cells in the P2 region of interest. Data represent the mean ± S.D. of four independent experiments. * p
    Msu Crystals, supplied by InvivoGen, used in various techniques. Bioz Stars score: 99/100, based on 135 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Crystal IS msu crystal
    ( a ) Optimization of the orientation angle of the linear polarizer ( γ ). The normalized output amplitude is plotted as a function of the sample fast-axis orientation, <t>α</t> , for different linear polarizer orientations ( γ = 50°, 55°, 65°, 75°). ( b ) The simulated normalized output images of <t>MSU</t> crystals at varying orientations, using γ = 65°. The MSU crystals are simulated as cylinders with a birefringence of | Δ n| = 0.1, diameter of 0.5 μm, length of 10 μm, and the fast axis is along the long axis of the crystals.
    Msu Crystal, supplied by Crystal IS, used in various techniques. Bioz Stars score: 89/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Caltag-Medsystems msu crystals
    <t>IL-1β</t> secretion by PBMCs following exposure to <t>MSU</t> crystals or SNPs. Secretion of IL-1β by PBMCs with (main figure) or without (inset) initial exposure to 10 ng/mL LPS for 3 h followed by exposure to MSU crystals (100 μg/mL) or SNPs (100 μg/mL) for a further 3 h. The supernatant was either collected immediately for analysis (3 h; open bars) or following a further 21 h of cell incubation in fresh (i.e. without added particles or MAMPs) TCM (3 + 21 h; black bars). All data are expressed as mean ± SEM ( n = 4). Results were analysed by paired T test in comparison to baseline (B), i.e. non-particle-exposed cells; * p
    Msu Crystals, supplied by Caltag-Medsystems, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Enzo Biochem msu crystals
    Gasdermin D pore formation and pyroptosis are not involved in EEA1 release. ( A ) Immunoblot analysis of EEA1 and IL-1β on cell lysate and cell-free supernatant from LPS-primed BMDMs from wild type or Casp1/11 −/− after stimulation with nigericin, ATP, E . coli or <t>MSU</t> crystals as indicated. IL-1β released to the extracellular medium was detected by ELISA (bottom). ( B ) LDH release from macrophages treated as in A. ( C ) Immunoblot analysis of EEA1 on cell lysate and cell-free supernatant from BMDMs wild-type and Gsdmd −/− after nigericin stimulation. ( D ) Immunoblot analysis of <t>caspase-1</t> and IL-1β on cell lysate and cell-free supernatant from wild-type or Gsdmd −/− BMDMs after nigericin stimulation in the presence or absence of the caspase-1 inhibitor Ac-YVAD. ( E , F ) ELISA for released IL-1β ( E ) or LDH ( F ) from macrophages treated as in D. ( G ) Immunoblot analysis of ASC and NLRP3 on cell-free supernatant from macrophages stimulated as in D. ( H ) Yo-Pro uptake in macrophages treated as in D. Blots are representative of three to four independent experiments. Nitrocellulose membranes were cropped after protein blotting and then inmunodetection was carried out separately for each antibody. Complete blots for IL-1β and Caspase-1 are showed as supplementary. Data are presented as average ± sem from n = 3 independent experiments.
    Msu Crystals, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 91/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Adipogen msu crystals
    OM-85 is a priming signal for multiple inflammasomes. ( a and b ) BMDCs were primed with 10 ng/ml LPS or the indicated doses of OM-85 (referring to μg/ml) for 4 h, followed by stimulation with alum (300 μg/ml), <t>MSU</t> (300 μg/ml), nigericin, (0.1 μM) for 2.5 h and <t>ATP</t> (500 μM) for 45 min. Release of IL-1β and IL-1α was measured by ELISA. ( c and d ) BMDCs were primed as described above, followed by transfection of poly(dA:dT) (0.6 µg). 6 h after, release of IL-1β ( c ) and IL-1α ( d ) in the supernatants was measured by ELISA. Results represent mean ± SD (n = 3 technical replicates) and are representative of at least three ( a and b ) and two ( c and d ) independent experiments. us = unstimulated. Statistical differences were calculated between the condition exposed to the inflammasome activator only (Alum, MSU, ATP, or Nigericin in the absence of LPS) and the conditions treated with LPS or OM-85 and the inflammasome activator by Student’s t-test adjusted by Bonferroni correction over 4 ( a and b ). Statistical differences were calculated between the condition exposed to poly(dA:dT) in the absence of LPS and the conditions treated with LPS or OM-85 and poly(dA:dT) as well as between the condition treated by LPS and poly(dA:dT) and by OM-85 (1000 μg/ml) and poly(dA:dT) using Student’s t-test adjusted by Bonferroni correction over 5 ( c and d ). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.
    Msu Crystals, supplied by Adipogen, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Enzo Biochem reagents msu crystals
    OM-85 is a priming signal for multiple inflammasomes. ( a and b ) BMDCs were primed with 10 ng/ml LPS or the indicated doses of OM-85 (referring to μg/ml) for 4 h, followed by stimulation with alum (300 μg/ml), <t>MSU</t> (300 μg/ml), nigericin, (0.1 μM) for 2.5 h and <t>ATP</t> (500 μM) for 45 min. Release of IL-1β and IL-1α was measured by ELISA. ( c and d ) BMDCs were primed as described above, followed by transfection of poly(dA:dT) (0.6 µg). 6 h after, release of IL-1β ( c ) and IL-1α ( d ) in the supernatants was measured by ELISA. Results represent mean ± SD (n = 3 technical replicates) and are representative of at least three ( a and b ) and two ( c and d ) independent experiments. us = unstimulated. Statistical differences were calculated between the condition exposed to the inflammasome activator only (Alum, MSU, ATP, or Nigericin in the absence of LPS) and the conditions treated with LPS or OM-85 and the inflammasome activator by Student’s t-test adjusted by Bonferroni correction over 4 ( a and b ). Statistical differences were calculated between the condition exposed to poly(dA:dT) in the absence of LPS and the conditions treated with LPS or OM-85 and poly(dA:dT) as well as between the condition treated by LPS and poly(dA:dT) and by OM-85 (1000 μg/ml) and poly(dA:dT) using Student’s t-test adjusted by Bonferroni correction over 5 ( c and d ). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.
    Reagents Msu Crystals, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 90/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Enzo Biochem monosodium urate crystal msu
    OM-85 is a priming signal for multiple inflammasomes. ( a and b ) BMDCs were primed with 10 ng/ml LPS or the indicated doses of OM-85 (referring to μg/ml) for 4 h, followed by stimulation with alum (300 μg/ml), <t>MSU</t> (300 μg/ml), nigericin, (0.1 μM) for 2.5 h and <t>ATP</t> (500 μM) for 45 min. Release of IL-1β and IL-1α was measured by ELISA. ( c and d ) BMDCs were primed as described above, followed by transfection of poly(dA:dT) (0.6 µg). 6 h after, release of IL-1β ( c ) and IL-1α ( d ) in the supernatants was measured by ELISA. Results represent mean ± SD (n = 3 technical replicates) and are representative of at least three ( a and b ) and two ( c and d ) independent experiments. us = unstimulated. Statistical differences were calculated between the condition exposed to the inflammasome activator only (Alum, MSU, ATP, or Nigericin in the absence of LPS) and the conditions treated with LPS or OM-85 and the inflammasome activator by Student’s t-test adjusted by Bonferroni correction over 4 ( a and b ). Statistical differences were calculated between the condition exposed to poly(dA:dT) in the absence of LPS and the conditions treated with LPS or OM-85 and poly(dA:dT) as well as between the condition treated by LPS and poly(dA:dT) and by OM-85 (1000 μg/ml) and poly(dA:dT) using Student’s t-test adjusted by Bonferroni correction over 5 ( c and d ). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.
    Monosodium Urate Crystal Msu, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Crystal IS monosodium urate msu crystal
    OM-85 is a priming signal for multiple inflammasomes. ( a and b ) BMDCs were primed with 10 ng/ml LPS or the indicated doses of OM-85 (referring to μg/ml) for 4 h, followed by stimulation with alum (300 μg/ml), <t>MSU</t> (300 μg/ml), nigericin, (0.1 μM) for 2.5 h and <t>ATP</t> (500 μM) for 45 min. Release of IL-1β and IL-1α was measured by ELISA. ( c and d ) BMDCs were primed as described above, followed by transfection of poly(dA:dT) (0.6 µg). 6 h after, release of IL-1β ( c ) and IL-1α ( d ) in the supernatants was measured by ELISA. Results represent mean ± SD (n = 3 technical replicates) and are representative of at least three ( a and b ) and two ( c and d ) independent experiments. us = unstimulated. Statistical differences were calculated between the condition exposed to the inflammasome activator only (Alum, MSU, ATP, or Nigericin in the absence of LPS) and the conditions treated with LPS or OM-85 and the inflammasome activator by Student’s t-test adjusted by Bonferroni correction over 4 ( a and b ). Statistical differences were calculated between the condition exposed to poly(dA:dT) in the absence of LPS and the conditions treated with LPS or OM-85 and poly(dA:dT) as well as between the condition treated by LPS and poly(dA:dT) and by OM-85 (1000 μg/ml) and poly(dA:dT) using Student’s t-test adjusted by Bonferroni correction over 5 ( c and d ). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.
    Monosodium Urate Msu Crystal, supplied by Crystal IS, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore monosodium urate msu crystals
    OM-85 is a priming signal for multiple inflammasomes. ( a and b ) BMDCs were primed with 10 ng/ml LPS or the indicated doses of OM-85 (referring to μg/ml) for 4 h, followed by stimulation with alum (300 μg/ml), <t>MSU</t> (300 μg/ml), nigericin, (0.1 μM) for 2.5 h and <t>ATP</t> (500 μM) for 45 min. Release of IL-1β and IL-1α was measured by ELISA. ( c and d ) BMDCs were primed as described above, followed by transfection of poly(dA:dT) (0.6 µg). 6 h after, release of IL-1β ( c ) and IL-1α ( d ) in the supernatants was measured by ELISA. Results represent mean ± SD (n = 3 technical replicates) and are representative of at least three ( a and b ) and two ( c and d ) independent experiments. us = unstimulated. Statistical differences were calculated between the condition exposed to the inflammasome activator only (Alum, MSU, ATP, or Nigericin in the absence of LPS) and the conditions treated with LPS or OM-85 and the inflammasome activator by Student’s t-test adjusted by Bonferroni correction over 4 ( a and b ). Statistical differences were calculated between the condition exposed to poly(dA:dT) in the absence of LPS and the conditions treated with LPS or OM-85 and poly(dA:dT) as well as between the condition treated by LPS and poly(dA:dT) and by OM-85 (1000 μg/ml) and poly(dA:dT) using Student’s t-test adjusted by Bonferroni correction over 5 ( c and d ). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.
    Monosodium Urate Msu Crystals, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore endotoxin free msu crystals
    OM-85 is a priming signal for multiple inflammasomes. ( a and b ) BMDCs were primed with 10 ng/ml LPS or the indicated doses of OM-85 (referring to μg/ml) for 4 h, followed by stimulation with alum (300 μg/ml), <t>MSU</t> (300 μg/ml), nigericin, (0.1 μM) for 2.5 h and <t>ATP</t> (500 μM) for 45 min. Release of IL-1β and IL-1α was measured by ELISA. ( c and d ) BMDCs were primed as described above, followed by transfection of poly(dA:dT) (0.6 µg). 6 h after, release of IL-1β ( c ) and IL-1α ( d ) in the supernatants was measured by ELISA. Results represent mean ± SD (n = 3 technical replicates) and are representative of at least three ( a and b ) and two ( c and d ) independent experiments. us = unstimulated. Statistical differences were calculated between the condition exposed to the inflammasome activator only (Alum, MSU, ATP, or Nigericin in the absence of LPS) and the conditions treated with LPS or OM-85 and the inflammasome activator by Student’s t-test adjusted by Bonferroni correction over 4 ( a and b ). Statistical differences were calculated between the condition exposed to poly(dA:dT) in the absence of LPS and the conditions treated with LPS or OM-85 and poly(dA:dT) as well as between the condition treated by LPS and poly(dA:dT) and by OM-85 (1000 μg/ml) and poly(dA:dT) using Student’s t-test adjusted by Bonferroni correction over 5 ( c and d ). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.
    Endotoxin Free Msu Crystals, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Impact of anti-CD44 antibody treatment on NLRP3 inflammasome activation and nuclear factor kappa B (NFκB) p65 subunit nuclear translocation in differentiated human THP-1 macrophages following incubation with monosodium urate monohydrate (MSU) crystals and role of intracellular protein-phosphatase 2A (PP2A) in mediating anti-CD44 antibody’s effect. We utilized the IM7 clone, which recognizes all CD44 isoforms and causes enzyme-mediated shedding of the extracellular domain. THP-1 macrophages were treated with MSU crystals (100 μg/mL) ± anti-CD44 or isotype control (IC) antibodies (2 μg/mL for both antibodies). NF-κB p65 nuclear levels were determined following a 1-hour incubation using a DNA binding assay. Pro-caspase-1 and the p10 subunit of active caspase-1 were determined using Western Blotting. Active caspase-1 activity was determined using a specific activity assay. Cellular pro-IL-1β and mature IL-1β levels were determined using an ELISA. PP2A inhibition was performed using okadiac acid (5 nM). Data from 3 independent experiments are presented with mean and S.D. highlighted. *p

    Journal: Scientific Reports

    Article Title: CD44 Receptor Mediates Urate Crystal Phagocytosis by Macrophages and Regulates Inflammation in A Murine Peritoneal Model of Acute Gout

    doi: 10.1038/s41598-020-62727-z

    Figure Lengend Snippet: Impact of anti-CD44 antibody treatment on NLRP3 inflammasome activation and nuclear factor kappa B (NFκB) p65 subunit nuclear translocation in differentiated human THP-1 macrophages following incubation with monosodium urate monohydrate (MSU) crystals and role of intracellular protein-phosphatase 2A (PP2A) in mediating anti-CD44 antibody’s effect. We utilized the IM7 clone, which recognizes all CD44 isoforms and causes enzyme-mediated shedding of the extracellular domain. THP-1 macrophages were treated with MSU crystals (100 μg/mL) ± anti-CD44 or isotype control (IC) antibodies (2 μg/mL for both antibodies). NF-κB p65 nuclear levels were determined following a 1-hour incubation using a DNA binding assay. Pro-caspase-1 and the p10 subunit of active caspase-1 were determined using Western Blotting. Active caspase-1 activity was determined using a specific activity assay. Cellular pro-IL-1β and mature IL-1β levels were determined using an ELISA. PP2A inhibition was performed using okadiac acid (5 nM). Data from 3 independent experiments are presented with mean and S.D. highlighted. *p

    Article Snippet: THP-1 macrophages were treated with MSU crystals (100 μg/mL) ± anti-CD44 or IC antibodies for 1 hour followed by cell harvest and extraction of nuclear protein (ThermoFisher Scientific).

    Techniques: Activation Assay, Translocation Assay, Incubation, DNA Binding Assay, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay, Inhibition

    Impact of monosodium urate monohydrate (MSU) crystal treatment on CD44 receptor expression and internalization in differentiated human THP-1 macrophages. CD44 expression was determined using qPCR, flow cytometry and ELISA at 3, 6, 12 and 24 hours following MSU treatment. CD44 cytosolic internalization was determined using confocal microscopy, and intracellular CD44 staining intensity was determined following incubation with MSU crystals for 3 and 6 hours. Data are from 3 independent experiments and are presented with mean and S.D. highlighted. *p

    Journal: Scientific Reports

    Article Title: CD44 Receptor Mediates Urate Crystal Phagocytosis by Macrophages and Regulates Inflammation in A Murine Peritoneal Model of Acute Gout

    doi: 10.1038/s41598-020-62727-z

    Figure Lengend Snippet: Impact of monosodium urate monohydrate (MSU) crystal treatment on CD44 receptor expression and internalization in differentiated human THP-1 macrophages. CD44 expression was determined using qPCR, flow cytometry and ELISA at 3, 6, 12 and 24 hours following MSU treatment. CD44 cytosolic internalization was determined using confocal microscopy, and intracellular CD44 staining intensity was determined following incubation with MSU crystals for 3 and 6 hours. Data are from 3 independent experiments and are presented with mean and S.D. highlighted. *p

    Article Snippet: THP-1 macrophages were treated with MSU crystals (100 μg/mL) ± anti-CD44 or IC antibodies for 1 hour followed by cell harvest and extraction of nuclear protein (ThermoFisher Scientific).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Staining, Incubation

    Impact of antibody-mediated CD44 receptor shedding on monosodium urate monohydrate (MSU) crystal phagocytosis by THP-1 macrophages, expression and production of interleukin-1 beta (IL-1β) and interleukin-8 (IL8). We utilized the IM7 clone, which recognizes all CD44 isoforms and causes enzyme-mediated shedding of the extracellular domain. THP-1 macrophages were incubated with MSU crystals (100 μg/mL) ± anti-CD44 or isotype control (IC) antibodies (2 μg/mL for both antibodies). MSU phagocytosis was determined by the change in side scatter in macrophages following a 4-hour incubation and expressed as percent positive cells that internalized MSU crystals. Representative flow cytometry plots are shown in supplementary figure 2. Gene expression and production of cytokines were determined following a 6-hour incubation. Data from 3–5 independent experiments are presented with mean and S.D. highlighted. Dashed line represents gene expression in untreated controls. *p

    Journal: Scientific Reports

    Article Title: CD44 Receptor Mediates Urate Crystal Phagocytosis by Macrophages and Regulates Inflammation in A Murine Peritoneal Model of Acute Gout

    doi: 10.1038/s41598-020-62727-z

    Figure Lengend Snippet: Impact of antibody-mediated CD44 receptor shedding on monosodium urate monohydrate (MSU) crystal phagocytosis by THP-1 macrophages, expression and production of interleukin-1 beta (IL-1β) and interleukin-8 (IL8). We utilized the IM7 clone, which recognizes all CD44 isoforms and causes enzyme-mediated shedding of the extracellular domain. THP-1 macrophages were incubated with MSU crystals (100 μg/mL) ± anti-CD44 or isotype control (IC) antibodies (2 μg/mL for both antibodies). MSU phagocytosis was determined by the change in side scatter in macrophages following a 4-hour incubation and expressed as percent positive cells that internalized MSU crystals. Representative flow cytometry plots are shown in supplementary figure 2. Gene expression and production of cytokines were determined following a 6-hour incubation. Data from 3–5 independent experiments are presented with mean and S.D. highlighted. Dashed line represents gene expression in untreated controls. *p

    Article Snippet: THP-1 macrophages were treated with MSU crystals (100 μg/mL) ± anti-CD44 or IC antibodies for 1 hour followed by cell harvest and extraction of nuclear protein (ThermoFisher Scientific).

    Techniques: Expressing, Incubation, Flow Cytometry

    Impact of intraperitoneal administration of monosodium urate monohydrate (MSU) crystals on inflammatory cell infiltration and production of interleukin-1 beta (IL-1β) and interleukin-1 receptor antagonist (IL-1Ra) in wildtype, Cd44 −/− and Nlrp3 −/− mice. MSU (2 mg in 200 μl PBS) or PBS (200 μl) were administered via the intraperitoneal route in wildtype (n = 8), Cd44 −/− (n = 8) or Nlrp3 −/− (n = 6) and peritoneal lavaging was performed at 4 hours. Total peritoneal lavage cell counts were determined. The number of infiltrated neutrophils and monocytes were determined using flow cytometry and probing for neutrophil markers; Ly6B.2 and Ly6G and monocyte markers; Cd11b and Ly6C. Representative flow cytometry plots of neutrophil markers are shown in supplementary figure 3. Peritoneal lavage IL-1β and IL-1Ra levels were determined by ELISA and the IL-1Ra/IL-1β ratios were computed. *p

    Journal: Scientific Reports

    Article Title: CD44 Receptor Mediates Urate Crystal Phagocytosis by Macrophages and Regulates Inflammation in A Murine Peritoneal Model of Acute Gout

    doi: 10.1038/s41598-020-62727-z

    Figure Lengend Snippet: Impact of intraperitoneal administration of monosodium urate monohydrate (MSU) crystals on inflammatory cell infiltration and production of interleukin-1 beta (IL-1β) and interleukin-1 receptor antagonist (IL-1Ra) in wildtype, Cd44 −/− and Nlrp3 −/− mice. MSU (2 mg in 200 μl PBS) or PBS (200 μl) were administered via the intraperitoneal route in wildtype (n = 8), Cd44 −/− (n = 8) or Nlrp3 −/− (n = 6) and peritoneal lavaging was performed at 4 hours. Total peritoneal lavage cell counts were determined. The number of infiltrated neutrophils and monocytes were determined using flow cytometry and probing for neutrophil markers; Ly6B.2 and Ly6G and monocyte markers; Cd11b and Ly6C. Representative flow cytometry plots of neutrophil markers are shown in supplementary figure 3. Peritoneal lavage IL-1β and IL-1Ra levels were determined by ELISA and the IL-1Ra/IL-1β ratios were computed. *p

    Article Snippet: THP-1 macrophages were treated with MSU crystals (100 μg/mL) ± anti-CD44 or IC antibodies for 1 hour followed by cell harvest and extraction of nuclear protein (ThermoFisher Scientific).

    Techniques: Mouse Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay

    Role of CD44 receptor in mediating monosodium urate monohydrate (MSU) crystal phagocytosis and downstream inflammation in bone marrow derived macrophages (BMDMs) from Cd44 +/+ and Cd44 −/− mice and impact of antibody-mediated receptor shedding on MSU phagocytosis by murine macrophages. BMDMs were treated with MSU (100 μg/mL) for 4 hours to evaluate phagocytosis and for 6 hours to evaluate interleukin-1 beta (IL-1β) gene expression and production. BMDMs were stimulated with lipopolysaccharide (LPS; 100 ng/ml) for 72 hours as a positive control. Lactate dehydrogenase (LDH) release from BMDMs was determined at 1 and 4 hours following incubation with MSU crystals and LDH activity level was used to calculate percent cytotoxicity. We utilized the IM7 clone, which recognizes all CD44 isoforms and causes shedding of the extracellular domain. Anti-CD44 and isotype control (IC) antibodies (2 μg/mL for both antibodies) were incubated with J774A.1 murine macrophages for 4 hours. Data from 3–5 independent experiments are presented with mean and S.D. highlighted. *p

    Journal: Scientific Reports

    Article Title: CD44 Receptor Mediates Urate Crystal Phagocytosis by Macrophages and Regulates Inflammation in A Murine Peritoneal Model of Acute Gout

    doi: 10.1038/s41598-020-62727-z

    Figure Lengend Snippet: Role of CD44 receptor in mediating monosodium urate monohydrate (MSU) crystal phagocytosis and downstream inflammation in bone marrow derived macrophages (BMDMs) from Cd44 +/+ and Cd44 −/− mice and impact of antibody-mediated receptor shedding on MSU phagocytosis by murine macrophages. BMDMs were treated with MSU (100 μg/mL) for 4 hours to evaluate phagocytosis and for 6 hours to evaluate interleukin-1 beta (IL-1β) gene expression and production. BMDMs were stimulated with lipopolysaccharide (LPS; 100 ng/ml) for 72 hours as a positive control. Lactate dehydrogenase (LDH) release from BMDMs was determined at 1 and 4 hours following incubation with MSU crystals and LDH activity level was used to calculate percent cytotoxicity. We utilized the IM7 clone, which recognizes all CD44 isoforms and causes shedding of the extracellular domain. Anti-CD44 and isotype control (IC) antibodies (2 μg/mL for both antibodies) were incubated with J774A.1 murine macrophages for 4 hours. Data from 3–5 independent experiments are presented with mean and S.D. highlighted. *p

    Article Snippet: THP-1 macrophages were treated with MSU crystals (100 μg/mL) ± anti-CD44 or IC antibodies for 1 hour followed by cell harvest and extraction of nuclear protein (ThermoFisher Scientific).

    Techniques: Derivative Assay, Mouse Assay, Expressing, Positive Control, Incubation, Activity Assay

    Impact of anti-CD44 antibody treatment on inflammatory cell infiltration and production of interleukin-1 beta (IL-1β) in murine peritoneal monosodium urate monohydrate (MSU) crystal inflammation model. MSU crystals (2 mg in 200 μL PBS), vehicle (Veh.; 200 μL PBS), anti-CD44 and isotype control (IC) antibodies (50 μg in 200 μL PBS) were administered via the intraperitoneal route. Experimental groups included untreated controls (n = 4), MSU + Veh. (n = 4), MSU + Anti-CD44 antibody (n = 5) and MSU + IC antibody (n = 3). Peritoneal lavaging was performed at 4 hours. Total peritoneal lavage cell counts were determined. The number of infiltrated neutrophils and monocytes were determined using flow cytometry and probing for neutrophil markers; Ly6B.2 and Ly6G and monocyte markers; Cd11b and Ly6C. Representative flow cytometry plots of neutrophil and monocytes markers are shown in Supplementary Fig. 4 . Peritoneal lavage IL-1β levels were determined by ELISA. *p

    Journal: Scientific Reports

    Article Title: CD44 Receptor Mediates Urate Crystal Phagocytosis by Macrophages and Regulates Inflammation in A Murine Peritoneal Model of Acute Gout

    doi: 10.1038/s41598-020-62727-z

    Figure Lengend Snippet: Impact of anti-CD44 antibody treatment on inflammatory cell infiltration and production of interleukin-1 beta (IL-1β) in murine peritoneal monosodium urate monohydrate (MSU) crystal inflammation model. MSU crystals (2 mg in 200 μL PBS), vehicle (Veh.; 200 μL PBS), anti-CD44 and isotype control (IC) antibodies (50 μg in 200 μL PBS) were administered via the intraperitoneal route. Experimental groups included untreated controls (n = 4), MSU + Veh. (n = 4), MSU + Anti-CD44 antibody (n = 5) and MSU + IC antibody (n = 3). Peritoneal lavaging was performed at 4 hours. Total peritoneal lavage cell counts were determined. The number of infiltrated neutrophils and monocytes were determined using flow cytometry and probing for neutrophil markers; Ly6B.2 and Ly6G and monocyte markers; Cd11b and Ly6C. Representative flow cytometry plots of neutrophil and monocytes markers are shown in Supplementary Fig. 4 . Peritoneal lavage IL-1β levels were determined by ELISA. *p

    Article Snippet: THP-1 macrophages were treated with MSU crystals (100 μg/mL) ± anti-CD44 or IC antibodies for 1 hour followed by cell harvest and extraction of nuclear protein (ThermoFisher Scientific).

    Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay

    Time and treatment-dependent phagocytosis of monosodium urate monohydrate (MSU) crystals by differentiated human THP-1 macrophages using flow cytometry and impact of recombinant human proteoglycan-4 (rhPRG4) or bovine submaxillary mucin (BSM) treatments following 2 and 4-h incubations. Quantitative determination of MSU phagocytosis was performed using the percentage of cells in the P2 region of interest. Data represent the mean ± S.D. of four independent experiments. * p

    Journal: Arthritis Research & Therapy

    Article Title: Recombinant human proteoglycan-4 reduces phagocytosis of urate crystals and downstream nuclear factor kappa B and inflammasome activation and production of cytokines and chemokines in human and murine macrophages

    doi: 10.1186/s13075-018-1693-x

    Figure Lengend Snippet: Time and treatment-dependent phagocytosis of monosodium urate monohydrate (MSU) crystals by differentiated human THP-1 macrophages using flow cytometry and impact of recombinant human proteoglycan-4 (rhPRG4) or bovine submaxillary mucin (BSM) treatments following 2 and 4-h incubations. Quantitative determination of MSU phagocytosis was performed using the percentage of cells in the P2 region of interest. Data represent the mean ± S.D. of four independent experiments. * p

    Article Snippet: THP-1 macrophages were treated with endotoxin-free MSU crystals (100μg/ml; Invivogen, USA) ± bovine submaxillary mucin (BSM; molecular mass > 1000 KDa) (Sigma Aldrich) (25 μg/ml) or rhPRG4 (molecular mass is approximately 240 KDa) (100 μg/ml) for 2 and 4 h at 37 °C. rhPRG4 is an endotoxin-free full-length product produced by CHO-M cells (Lubris, Framingham, MA, USA) [ ].

    Techniques: Flow Cytometry, Cytometry, Recombinant

    Impact of recombinant human proteoglycan-4 (rhPRG4) treatment on monosodium urate monohydrate (MSU) crystal-induced expression and production of proinflammatory cytokines and chemokines and nuclear factor kappa b (NFκB) p65 subunit nuclear translocation in THP-1 macrophages. Cytokines included interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α). Chemokines included interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1). Gene expression data are presented as fold induction of proinflammatory cytokines and chemokines gene expression compared to control untreated THP-1 macrophages. THP-1 macrophages were treated with MSU crystals (100μg/ml) ± rhPRG4 (100μg/ml) or bovine submaxillary mucin (BSM; 25μg/ml) for 6 h ( a through d ). NFκB p65 subunit nuclear translocation in THP-1 macrophages was performed at 1 h following MSU challenge (100μg/ml). Gene expression studies were performed at 6 h ( f through i ) and cytokine and chemokine media concentrations were determined at 24 h ( j through m ). Data represent the mean ± S.D. of three to four independent experiments with duplicate wells per group. *p

    Journal: Arthritis Research & Therapy

    Article Title: Recombinant human proteoglycan-4 reduces phagocytosis of urate crystals and downstream nuclear factor kappa B and inflammasome activation and production of cytokines and chemokines in human and murine macrophages

    doi: 10.1186/s13075-018-1693-x

    Figure Lengend Snippet: Impact of recombinant human proteoglycan-4 (rhPRG4) treatment on monosodium urate monohydrate (MSU) crystal-induced expression and production of proinflammatory cytokines and chemokines and nuclear factor kappa b (NFκB) p65 subunit nuclear translocation in THP-1 macrophages. Cytokines included interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α). Chemokines included interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1). Gene expression data are presented as fold induction of proinflammatory cytokines and chemokines gene expression compared to control untreated THP-1 macrophages. THP-1 macrophages were treated with MSU crystals (100μg/ml) ± rhPRG4 (100μg/ml) or bovine submaxillary mucin (BSM; 25μg/ml) for 6 h ( a through d ). NFκB p65 subunit nuclear translocation in THP-1 macrophages was performed at 1 h following MSU challenge (100μg/ml). Gene expression studies were performed at 6 h ( f through i ) and cytokine and chemokine media concentrations were determined at 24 h ( j through m ). Data represent the mean ± S.D. of three to four independent experiments with duplicate wells per group. *p

    Article Snippet: THP-1 macrophages were treated with endotoxin-free MSU crystals (100μg/ml; Invivogen, USA) ± bovine submaxillary mucin (BSM; molecular mass > 1000 KDa) (Sigma Aldrich) (25 μg/ml) or rhPRG4 (molecular mass is approximately 240 KDa) (100 μg/ml) for 2 and 4 h at 37 °C. rhPRG4 is an endotoxin-free full-length product produced by CHO-M cells (Lubris, Framingham, MA, USA) [ ].

    Techniques: Recombinant, Expressing, Translocation Assay

    Impact of recombinant human proteoglycan-4 (rhPRG4) treatment on monosodium urate monohydrate (MSU) crystal-induced NLRP3 inflammasome activation in THP-1 macrophages. THP-1 macrophages were treated with 100μg/ml MSU in the absence or presence of rhPRG4 (100 and 200μg/ml) for 12 h. H 2 O 2 (5 mM) was used as a positive control. Data represent the mean ± S.D. of three independent experiments. * p

    Journal: Arthritis Research & Therapy

    Article Title: Recombinant human proteoglycan-4 reduces phagocytosis of urate crystals and downstream nuclear factor kappa B and inflammasome activation and production of cytokines and chemokines in human and murine macrophages

    doi: 10.1186/s13075-018-1693-x

    Figure Lengend Snippet: Impact of recombinant human proteoglycan-4 (rhPRG4) treatment on monosodium urate monohydrate (MSU) crystal-induced NLRP3 inflammasome activation in THP-1 macrophages. THP-1 macrophages were treated with 100μg/ml MSU in the absence or presence of rhPRG4 (100 and 200μg/ml) for 12 h. H 2 O 2 (5 mM) was used as a positive control. Data represent the mean ± S.D. of three independent experiments. * p

    Article Snippet: THP-1 macrophages were treated with endotoxin-free MSU crystals (100μg/ml; Invivogen, USA) ± bovine submaxillary mucin (BSM; molecular mass > 1000 KDa) (Sigma Aldrich) (25 μg/ml) or rhPRG4 (molecular mass is approximately 240 KDa) (100 μg/ml) for 2 and 4 h at 37 °C. rhPRG4 is an endotoxin-free full-length product produced by CHO-M cells (Lubris, Framingham, MA, USA) [ ].

    Techniques: Recombinant, Activation Assay, Positive Control

    ( a ) Optimization of the orientation angle of the linear polarizer ( γ ). The normalized output amplitude is plotted as a function of the sample fast-axis orientation, α , for different linear polarizer orientations ( γ = 50°, 55°, 65°, 75°). ( b ) The simulated normalized output images of MSU crystals at varying orientations, using γ = 65°. The MSU crystals are simulated as cylinders with a birefringence of | Δ n| = 0.1, diameter of 0.5 μm, length of 10 μm, and the fast axis is along the long axis of the crystals.

    Journal: Scientific Reports

    Article Title: Wide-field imaging of birefringent synovial fluid crystals using lens-free polarized microscopy for gout diagnosis

    doi: 10.1038/srep28793

    Figure Lengend Snippet: ( a ) Optimization of the orientation angle of the linear polarizer ( γ ). The normalized output amplitude is plotted as a function of the sample fast-axis orientation, α , for different linear polarizer orientations ( γ = 50°, 55°, 65°, 75°). ( b ) The simulated normalized output images of MSU crystals at varying orientations, using γ = 65°. The MSU crystals are simulated as cylinders with a birefringence of | Δ n| = 0.1, diameter of 0.5 μm, length of 10 μm, and the fast axis is along the long axis of the crystals.

    Article Snippet: Moreover, the red curve is almost symmetrically distributed around unity, and thus, the brighter-than-background orientations of the MSU crystal roughly correspond to 0° <α < 90°, whereas the darker-than-background orientations of the MSU crystal roughly correspond to 90° <α < 180°.

    Techniques:

    ( a ) Simulation of the differential output as a function of the relative phase retardation φ , with the crystals aligned at 45° ( α = 45°). almost linearly reaches to the maximum/minimum when |φ| increases from 0 to ~ 0.22π, then turns backwards towards 0 as |φ| further increases to π. ( b ) Simulated image of a MSU crystal with larger diameter (2 μm) compared to Figs 4 and 5 . The effect of the nonlinearity is manifested by the hollow appearance of the simulated images. Nevertheless, the intense (bright/dark) edges provide enough contrast for crystal detection and identification.

    Journal: Scientific Reports

    Article Title: Wide-field imaging of birefringent synovial fluid crystals using lens-free polarized microscopy for gout diagnosis

    doi: 10.1038/srep28793

    Figure Lengend Snippet: ( a ) Simulation of the differential output as a function of the relative phase retardation φ , with the crystals aligned at 45° ( α = 45°). almost linearly reaches to the maximum/minimum when |φ| increases from 0 to ~ 0.22π, then turns backwards towards 0 as |φ| further increases to π. ( b ) Simulated image of a MSU crystal with larger diameter (2 μm) compared to Figs 4 and 5 . The effect of the nonlinearity is manifested by the hollow appearance of the simulated images. Nevertheless, the intense (bright/dark) edges provide enough contrast for crystal detection and identification.

    Article Snippet: Moreover, the red curve is almost symmetrically distributed around unity, and thus, the brighter-than-background orientations of the MSU crystal roughly correspond to 0° <α < 90°, whereas the darker-than-background orientations of the MSU crystal roughly correspond to 90° <α < 180°.

    Techniques:

    IL-1β secretion by PBMCs following exposure to MSU crystals or SNPs. Secretion of IL-1β by PBMCs with (main figure) or without (inset) initial exposure to 10 ng/mL LPS for 3 h followed by exposure to MSU crystals (100 μg/mL) or SNPs (100 μg/mL) for a further 3 h. The supernatant was either collected immediately for analysis (3 h; open bars) or following a further 21 h of cell incubation in fresh (i.e. without added particles or MAMPs) TCM (3 + 21 h; black bars). All data are expressed as mean ± SEM ( n = 4). Results were analysed by paired T test in comparison to baseline (B), i.e. non-particle-exposed cells; * p

    Journal: Particle and Fibre Toxicology

    Article Title: Pro-inflammatory adjuvant properties of pigment-grade titanium dioxide particles are augmented by a genotype that potentiates interleukin 1β processing

    doi: 10.1186/s12989-017-0232-2

    Figure Lengend Snippet: IL-1β secretion by PBMCs following exposure to MSU crystals or SNPs. Secretion of IL-1β by PBMCs with (main figure) or without (inset) initial exposure to 10 ng/mL LPS for 3 h followed by exposure to MSU crystals (100 μg/mL) or SNPs (100 μg/mL) for a further 3 h. The supernatant was either collected immediately for analysis (3 h; open bars) or following a further 21 h of cell incubation in fresh (i.e. without added particles or MAMPs) TCM (3 + 21 h; black bars). All data are expressed as mean ± SEM ( n = 4). Results were analysed by paired T test in comparison to baseline (B), i.e. non-particle-exposed cells; * p

    Article Snippet: Finally, paired T tests were used to compare supernatant levels of IL-1β for cells exposed to MSU crystals or SNPs versus non-particle-exposed control cells.

    Techniques: Incubation

    Gasdermin D pore formation and pyroptosis are not involved in EEA1 release. ( A ) Immunoblot analysis of EEA1 and IL-1β on cell lysate and cell-free supernatant from LPS-primed BMDMs from wild type or Casp1/11 −/− after stimulation with nigericin, ATP, E . coli or MSU crystals as indicated. IL-1β released to the extracellular medium was detected by ELISA (bottom). ( B ) LDH release from macrophages treated as in A. ( C ) Immunoblot analysis of EEA1 on cell lysate and cell-free supernatant from BMDMs wild-type and Gsdmd −/− after nigericin stimulation. ( D ) Immunoblot analysis of caspase-1 and IL-1β on cell lysate and cell-free supernatant from wild-type or Gsdmd −/− BMDMs after nigericin stimulation in the presence or absence of the caspase-1 inhibitor Ac-YVAD. ( E , F ) ELISA for released IL-1β ( E ) or LDH ( F ) from macrophages treated as in D. ( G ) Immunoblot analysis of ASC and NLRP3 on cell-free supernatant from macrophages stimulated as in D. ( H ) Yo-Pro uptake in macrophages treated as in D. Blots are representative of three to four independent experiments. Nitrocellulose membranes were cropped after protein blotting and then inmunodetection was carried out separately for each antibody. Complete blots for IL-1β and Caspase-1 are showed as supplementary. Data are presented as average ± sem from n = 3 independent experiments.

    Journal: Scientific Reports

    Article Title: Early endosome autoantigen 1 regulates IL-1β release upon caspase-1 activation independently of gasdermin D membrane permeabilization

    doi: 10.1038/s41598-019-42298-4

    Figure Lengend Snippet: Gasdermin D pore formation and pyroptosis are not involved in EEA1 release. ( A ) Immunoblot analysis of EEA1 and IL-1β on cell lysate and cell-free supernatant from LPS-primed BMDMs from wild type or Casp1/11 −/− after stimulation with nigericin, ATP, E . coli or MSU crystals as indicated. IL-1β released to the extracellular medium was detected by ELISA (bottom). ( B ) LDH release from macrophages treated as in A. ( C ) Immunoblot analysis of EEA1 on cell lysate and cell-free supernatant from BMDMs wild-type and Gsdmd −/− after nigericin stimulation. ( D ) Immunoblot analysis of caspase-1 and IL-1β on cell lysate and cell-free supernatant from wild-type or Gsdmd −/− BMDMs after nigericin stimulation in the presence or absence of the caspase-1 inhibitor Ac-YVAD. ( E , F ) ELISA for released IL-1β ( E ) or LDH ( F ) from macrophages treated as in D. ( G ) Immunoblot analysis of ASC and NLRP3 on cell-free supernatant from macrophages stimulated as in D. ( H ) Yo-Pro uptake in macrophages treated as in D. Blots are representative of three to four independent experiments. Nitrocellulose membranes were cropped after protein blotting and then inmunodetection was carried out separately for each antibody. Complete blots for IL-1β and Caspase-1 are showed as supplementary. Data are presented as average ± sem from n = 3 independent experiments.

    Article Snippet: Reagents E . coli LPS O55:B5, DAPI, ATP, triton X-100, nigericin, anti-V5 antibody, PMA and puromycin resistant MISSION® shRNA Lentiviral Transduction Particles were from Sigma-Aldrich; Recombinant human caspase-1 and caspase-1 inhibitor Ac-YVAD-AOM were from Merk-Millipore; MSU crystals were from Enzo Life Sciences; flagellin from S . typhimurium from Invivogen; the receptor-binding protein protective antigen and metalloprotease lethal factor were from List Biological; the protein-delivery reagent PULSin was from PolyPlus Transfection;Yo-Pro-1 from Life Technologies.

    Techniques: Enzyme-linked Immunosorbent Assay

    OM-85 is a priming signal for multiple inflammasomes. ( a and b ) BMDCs were primed with 10 ng/ml LPS or the indicated doses of OM-85 (referring to μg/ml) for 4 h, followed by stimulation with alum (300 μg/ml), MSU (300 μg/ml), nigericin, (0.1 μM) for 2.5 h and ATP (500 μM) for 45 min. Release of IL-1β and IL-1α was measured by ELISA. ( c and d ) BMDCs were primed as described above, followed by transfection of poly(dA:dT) (0.6 µg). 6 h after, release of IL-1β ( c ) and IL-1α ( d ) in the supernatants was measured by ELISA. Results represent mean ± SD (n = 3 technical replicates) and are representative of at least three ( a and b ) and two ( c and d ) independent experiments. us = unstimulated. Statistical differences were calculated between the condition exposed to the inflammasome activator only (Alum, MSU, ATP, or Nigericin in the absence of LPS) and the conditions treated with LPS or OM-85 and the inflammasome activator by Student’s t-test adjusted by Bonferroni correction over 4 ( a and b ). Statistical differences were calculated between the condition exposed to poly(dA:dT) in the absence of LPS and the conditions treated with LPS or OM-85 and poly(dA:dT) as well as between the condition treated by LPS and poly(dA:dT) and by OM-85 (1000 μg/ml) and poly(dA:dT) using Student’s t-test adjusted by Bonferroni correction over 5 ( c and d ). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

    Journal: Scientific Reports

    Article Title: OM-85 is an immunomodulator of interferon-β production and inflammasome activity

    doi: 10.1038/srep43844

    Figure Lengend Snippet: OM-85 is a priming signal for multiple inflammasomes. ( a and b ) BMDCs were primed with 10 ng/ml LPS or the indicated doses of OM-85 (referring to μg/ml) for 4 h, followed by stimulation with alum (300 μg/ml), MSU (300 μg/ml), nigericin, (0.1 μM) for 2.5 h and ATP (500 μM) for 45 min. Release of IL-1β and IL-1α was measured by ELISA. ( c and d ) BMDCs were primed as described above, followed by transfection of poly(dA:dT) (0.6 µg). 6 h after, release of IL-1β ( c ) and IL-1α ( d ) in the supernatants was measured by ELISA. Results represent mean ± SD (n = 3 technical replicates) and are representative of at least three ( a and b ) and two ( c and d ) independent experiments. us = unstimulated. Statistical differences were calculated between the condition exposed to the inflammasome activator only (Alum, MSU, ATP, or Nigericin in the absence of LPS) and the conditions treated with LPS or OM-85 and the inflammasome activator by Student’s t-test adjusted by Bonferroni correction over 4 ( a and b ). Statistical differences were calculated between the condition exposed to poly(dA:dT) in the absence of LPS and the conditions treated with LPS or OM-85 and poly(dA:dT) as well as between the condition treated by LPS and poly(dA:dT) and by OM-85 (1000 μg/ml) and poly(dA:dT) using Student’s t-test adjusted by Bonferroni correction over 5 ( c and d ). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

    Article Snippet: ATP (500 μM) and nigericin (0.1 μM) were from Sigma, MSU crystals (300 μg/ml) were from Adipogen, and alum (300 μg/ml) from Pierce Biochemicals (Imject-alum).

    Techniques: Enzyme-linked Immunosorbent Assay, Transfection