msu Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    ATCC anabaena variabilis atcc 29413
    Anabaena Variabilis Atcc 29413, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 507 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anabaena variabilis atcc 29413/product/ATCC
    Average 99 stars, based on 507 article reviews
    Price from $9.99 to $1999.99
    anabaena variabilis atcc 29413 - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore monosodium urate msu
    TDM stimulation diminishes NLRP3-dependent inflammasome activation and IL-1β production. ( a , b ) WT and Mincle −/− BMDMs were stimulated with LPS or co-stimulated with LPS and TDM for 12 h or the indicated times and then treated with <t>ATP</t> for 1 h (L: LPS; T: TDM; A: ATP; UT: untreated). ( a ) Left: immunoblot analysis of proIL-1β, mature IL-1β (p17), procaspase-1 (proCasp-1) and cleaved caspase-1 (p10) in cell culture supernatants (SN) and whole-cell lysates (XT). Right: kinetic quantitative analysis of proIL-1β and mature IL-1β (p17) from the immunoblots. ( b ) Release of lactate dehydrogenase (LDH) (assessing cell death) from cells. ( c ) ELISA of released IL-1β from LPS-treated WT and Mincle −/− BMDMs, stimulated with TDM for 12 h and then treated with <t>MSU,</t> nigericin or poly(dA:dT) for 3 h. * P
    Monosodium Urate Msu, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monosodium urate msu/product/Millipore
    Average 99 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    monosodium urate msu - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    91
    InvivoGen msu crystals
    <t>MSU</t> triggered intracellular ROS is inhibited by cytochalasin B (CytB) and colchicine (col). ( A ) Neutrophils were pretreated with cytochalsin B (5 µg/mL), an inhibitor of actin polymerization, and stimulated with either MSU crystals (300 µg/mL), <t>PMA</t> (50 nM) or opsonized S. aureus and intracellular ROS production (icROS) was evaluated using luminol amplified CL. Cytochalasin B-treated cells produced lower amounts of icROS in response to MSU crystals and S. aureus as compared to untreated cells, while icROS production in response to PMA was not affected by the presence of cytochalasin B. Peak CL values are shown. ( B ) Neutrophils were pretreated with colchicine (1 µg/mL) and stimulated with either MSU crystals (300 µg/mL), PMA (50 nM) or opsonized S. aureus and icROS production was evaluated using luminol amplified CL. Colchicine decreased icROS in response to MSU crystals, while icROS production in response to PMA or S. aureus was not affected. Peak CL values are shown ( n = 7). * p ≤ 0.05 and ** p ≤ 0.01. ns = not significant.
    Msu Crystals, supplied by InvivoGen, used in various techniques. Bioz Stars score: 91/100, based on 125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/msu crystals/product/InvivoGen
    Average 91 stars, based on 125 article reviews
    Price from $9.99 to $1999.99
    msu crystals - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    89
    Crystal IS msu crystal
    Four axial spondyloarthritis (AxSpA) patients with monosodium urate <t>(MSU)</t> crystal and radiographic structural damage at the sacroiliac joint. For each set of images, panel a shows the sacroiliac joint on plain radiographs, panel b shows the three-dimensional reconstruction dual-energy computed tomography <t>(DECT)</t> images, panel c shows the corresponding coronal (patient 2 and 4) or axial (patient 1 and 3) DECT images, and panel d shows the corresponding level of computed tomography (CT) images. A large quantity of MSU crystal deposition shown as green was found at the sacroiliac joint or the surrounding area in the DECT images. Four male patients (patient 1–4), aged 36, 44, 23, and 27 years old, had serum uric acid levels of 407 μmol/L, 370 μmol/L, 572 μmol/L, and 464 μmol/L, respectively. They were graded with a scale of 0, 0, II, III at the left sacroiliac joint and 0, I, II, III at the right sacroiliac joint, respectively, on plain radiographs
    Msu Crystal, supplied by Crystal IS, used in various techniques. Bioz Stars score: 89/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/msu crystal/product/Crystal IS
    Average 89 stars, based on 65 article reviews
    Price from $9.99 to $1999.99
    msu crystal - by Bioz Stars, 2020-09
    89/100 stars
      Buy from Supplier

    91
    InvivoGen msu
    Suppression of RIPK3 and MLKL prevents crystal cytotoxicity. ( a – c ) Mouse tubular epithelial cells were transfected with specific small inhibitor (si) RNA for RIPK3 and MLKL or a control siRNA of scrambled sequence before being exposed to crystals of <t>CaOx</t> (1,000 μg ml −1 ), <t>MSU</t> (500 μg ml −1 ), CPPD (500 μg ml −1 ) and cystine (500 μg ml −1 ). Cell viability was assessed by MTT assay (a) and cell death was assessed quantifying PI positivity (b) and C shows representative images 24 h later. Original image magnification: × 200, scale bar, 100 μm. ( d ) Mouse tubular epithelial cells were pretreated with RIPK3 inhibitor dabrafenib before exposing to different type crystals. Cell viability was assessed by MTT assay 24 h later. Data are expressed as mean±s.e.m. of three independent experiments, and was analysed using Student's t -test. Baseline viability is set as 100%. * P
    Msu, supplied by InvivoGen, used in various techniques. Bioz Stars score: 91/100, based on 146 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/msu/product/InvivoGen
    Average 91 stars, based on 146 article reviews
    Price from $9.99 to $1999.99
    msu - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    91
    InvivoGen monosodium urate msu
    CAPE blocks the interaction between NLRP3 and ASC. ( A ) The chemical structure of CAPE and the proposed molecular docking model for CAPE binding to ASC. ( B ) Electrostatic surface binding model for CAPE and ASC. Red: negative charge, blue: positive charge. ( C,D ) BMDMs were primed with LPS ( C , 500 ng/ml; D , 100 ng/ml) for 4 hr. Then, the cells were treated with CAPE for 1 hr, followed by stimulation with <t>MSU</t> (500 μg/ml) for 5 hr or <t>ATP</t> (5 mM) for 1 hr. Cell lysates were immunoprepitated with anti-ASC antibody followed by immunoblotting as indicated. Representative data from at least two independent experiments are presented.
    Monosodium Urate Msu, supplied by InvivoGen, used in various techniques. Bioz Stars score: 91/100, based on 98 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monosodium urate msu/product/InvivoGen
    Average 91 stars, based on 98 article reviews
    Price from $9.99 to $1999.99
    monosodium urate msu - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    91
    InvivoGen monosodium urate msu crystals
    iNKT Cells Induce IL-1β Secretion by <t>BMDCs</t> in a CD1d-Dependent Manner (A and B) BMDCs were left unprimed or primed with Pam3CSK4 (P3C; 0.5 μg/mL) for 24 h and subsequently co-cultured with iNKT cells for 24 h in the (A) absence or (B) presence of αGalCer (50 ng/mL). IL-1β release was quantified by ELISA. As a control, BMDCs were primed with P3C for 4 h, followed by 6-h stimulation with <t>MSU</t> (100 μg/mL). The same concentrations of P3C, αGalCer, and MSU were used throughout all experiments. (C and D) P3C-primed BMDCs were cultured alone or with iNKT cells for the indicated times in the presence or absence of αGalCer. IL-1β release was quantified by ELISA (C), and cell-associated and extracellular IL-1β were analyzed by immunoblot (D). As a control, BMDCs were primed with P3C for 4 h, followed by 1-h stimulation with nigericin (10 μM). (E) P3C-primed BMDCs from WT and CD1d −/− mice were co-cultured with iNKT cells for the indicated times in the absence or presence of αGalCer. IL-1β release was quantified by ELISA. P3C-primed BMDCs stimulated with nigericin were used as a control. Error bars represent mean + SD of triplicate samples. Each panel is representative of at least three different experiments. *p
    Monosodium Urate Msu Crystals, supplied by InvivoGen, used in various techniques. Bioz Stars score: 91/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monosodium urate msu crystals/product/InvivoGen
    Average 91 stars, based on 24 article reviews
    Price from $9.99 to $1999.99
    monosodium urate msu crystals - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    91
    Enzo Biochem msu
    Extracellular succinate signals via GPR91 to stimulate macrophages to release <t>IL-1β.</t> (A) GPR91 mRNA expression in WT (Janvier C57BL/6J) inflammatory BMDMs (M-CSF + IFN-γ) ± 100 ng/ml LPS, 500 µM succinate, or 10 ng/ml IL-1β for 24 h. n = 3 of Ct values. Succinate (Succ), IL-1β, and LPS related to basal (=1). Data are representative of three experiments. (B) Succinate levels (mass spectrophotometry area ratio) in medium from cultured BMDMs. Extracellular succinate from WT (littermates; black bars) and Sucnr1 −/− (gray bars), neutral (M, M-CSF), or inflammatory (M + IFN-γ) BMDMs ± 100 ng/ml LPS for 24 h is shown. n = 6 wells. Data are representative of three experiments. (C) IL-1β in supernatants of WT (Janvier C57BL/6J) and Sucnr1 −/− neutral or inflammatory BMDMs ± 100 ng/ml LPS for 24 h. n = 3 wells and are representative of seven experiments. (D) IL-1β mRNA levels from cell lysates from WT (Janvier C57BL/6J) or Sucnr1 −/− inflammatory BMDMs ± 100 ng/ml LPS at 4 h (related to WT basal = 1). n = 2–3 of Ct values. Data are representative of two experiments. (E) IL-1β levels measured in the supernatant of WT (Janvier C57BL/6J) and Sucnr1 −/− inflammatory BMDMs stimulated with 1 ng/ml LPS and 180 µg/ml <t>MSU.</t> n = 5–6 wells. Data are representative of two experiments. (F) Western blot of HIF-1α (representative blot of two experiments) and quantification (two experiments; 100% for no stimulus, WT, and Sucnr1 −/− ) at 6 h after stimulation with 500 µM succinate, 100 ng/ml LPS, or a combination of the two in WT littermate controls and Sucnr1 −/− inflammatory BMDMs. *, P
    Msu, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 91/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/msu/product/Enzo Biochem
    Average 91 stars, based on 67 article reviews
    Price from $9.99 to $1999.99
    msu - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    90
    Biochrom msu 1 basal media
    Extracellular succinate signals via GPR91 to stimulate macrophages to release <t>IL-1β.</t> (A) GPR91 mRNA expression in WT (Janvier C57BL/6J) inflammatory BMDMs (M-CSF + IFN-γ) ± 100 ng/ml LPS, 500 µM succinate, or 10 ng/ml IL-1β for 24 h. n = 3 of Ct values. Succinate (Succ), IL-1β, and LPS related to basal (=1). Data are representative of three experiments. (B) Succinate levels (mass spectrophotometry area ratio) in medium from cultured BMDMs. Extracellular succinate from WT (littermates; black bars) and Sucnr1 −/− (gray bars), neutral (M, M-CSF), or inflammatory (M + IFN-γ) BMDMs ± 100 ng/ml LPS for 24 h is shown. n = 6 wells. Data are representative of three experiments. (C) IL-1β in supernatants of WT (Janvier C57BL/6J) and Sucnr1 −/− neutral or inflammatory BMDMs ± 100 ng/ml LPS for 24 h. n = 3 wells and are representative of seven experiments. (D) IL-1β mRNA levels from cell lysates from WT (Janvier C57BL/6J) or Sucnr1 −/− inflammatory BMDMs ± 100 ng/ml LPS at 4 h (related to WT basal = 1). n = 2–3 of Ct values. Data are representative of two experiments. (E) IL-1β levels measured in the supernatant of WT (Janvier C57BL/6J) and Sucnr1 −/− inflammatory BMDMs stimulated with 1 ng/ml LPS and 180 µg/ml <t>MSU.</t> n = 5–6 wells. Data are representative of two experiments. (F) Western blot of HIF-1α (representative blot of two experiments) and quantification (two experiments; 100% for no stimulus, WT, and Sucnr1 −/− ) at 6 h after stimulation with 500 µM succinate, 100 ng/ml LPS, or a combination of the two in WT littermate controls and Sucnr1 −/− inflammatory BMDMs. *, P
    Msu 1 Basal Media, supplied by Biochrom, used in various techniques. Bioz Stars score: 90/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/msu 1 basal media/product/Biochrom
    Average 90 stars, based on 35 article reviews
    Price from $9.99 to $1999.99
    msu 1 basal media - by Bioz Stars, 2020-09
    90/100 stars
      Buy from Supplier

    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
    https://www.bioz.com/result/msu crystals/product/Caltag-Medsystems
    Average 91 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    msu crystals - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    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
    https://www.bioz.com/result/msu crystals/product/Enzo Biochem
    Average 91 stars, based on 72 article reviews
    Price from $9.99 to $1999.99
    msu crystals - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    88
    InvivoGen msu tlrl msu
    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 Tlrl Msu, supplied by InvivoGen, used in various techniques. Bioz Stars score: 88/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/msu tlrl msu/product/InvivoGen
    Average 88 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    msu tlrl msu - by Bioz Stars, 2020-09
    88/100 stars
      Buy from Supplier

    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
    https://www.bioz.com/result/msu crystals/product/Adipogen
    Average 91 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    msu crystals - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    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
    https://www.bioz.com/result/msu crystals/product/Thermo Fisher
    Average 99 stars, based on 96 article reviews
    Price from $9.99 to $1999.99
    msu crystals - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    89
    Enzo Biochem monosodium urate msu
    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, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 89/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monosodium urate msu/product/Enzo Biochem
    Average 89 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    monosodium urate msu - by Bioz Stars, 2020-09
    89/100 stars
      Buy from Supplier

    96
    Millipore msu 1 basal media
    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 1 Basal Media, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/msu 1 basal media/product/Millipore
    Average 96 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    msu 1 basal media - by Bioz Stars, 2020-09
    96/100 stars
      Buy from Supplier

    93
    Trinity Biotech msu vdl
    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 Vdl, supplied by Trinity Biotech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/msu vdl/product/Trinity Biotech
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    msu vdl - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    93
    FUJIFILM msu
    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, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/msu/product/FUJIFILM
    Average 93 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    msu - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    Image Search Results


    TDM stimulation diminishes NLRP3-dependent inflammasome activation and IL-1β production. ( a , b ) WT and Mincle −/− BMDMs were stimulated with LPS or co-stimulated with LPS and TDM for 12 h or the indicated times and then treated with ATP for 1 h (L: LPS; T: TDM; A: ATP; UT: untreated). ( a ) Left: immunoblot analysis of proIL-1β, mature IL-1β (p17), procaspase-1 (proCasp-1) and cleaved caspase-1 (p10) in cell culture supernatants (SN) and whole-cell lysates (XT). Right: kinetic quantitative analysis of proIL-1β and mature IL-1β (p17) from the immunoblots. ( b ) Release of lactate dehydrogenase (LDH) (assessing cell death) from cells. ( c ) ELISA of released IL-1β from LPS-treated WT and Mincle −/− BMDMs, stimulated with TDM for 12 h and then treated with MSU, nigericin or poly(dA:dT) for 3 h. * P

    Journal: Nature Communications

    Article Title: Mincle-mediated translational regulation is required for strong nitric oxide production and inflammation resolution

    doi: 10.1038/ncomms11322

    Figure Lengend Snippet: TDM stimulation diminishes NLRP3-dependent inflammasome activation and IL-1β production. ( a , b ) WT and Mincle −/− BMDMs were stimulated with LPS or co-stimulated with LPS and TDM for 12 h or the indicated times and then treated with ATP for 1 h (L: LPS; T: TDM; A: ATP; UT: untreated). ( a ) Left: immunoblot analysis of proIL-1β, mature IL-1β (p17), procaspase-1 (proCasp-1) and cleaved caspase-1 (p10) in cell culture supernatants (SN) and whole-cell lysates (XT). Right: kinetic quantitative analysis of proIL-1β and mature IL-1β (p17) from the immunoblots. ( b ) Release of lactate dehydrogenase (LDH) (assessing cell death) from cells. ( c ) ELISA of released IL-1β from LPS-treated WT and Mincle −/− BMDMs, stimulated with TDM for 12 h and then treated with MSU, nigericin or poly(dA:dT) for 3 h. * P

    Article Snippet: ATP (5 mM) and monosodium urate (MSU) (300 μg ml–1 ) were from Sigma.

    Techniques: Activation Assay, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay

    Airway administration of MSU crystals induces IL-33 and TSLP production in the lungs and trigger innate type 2 responses. (A and B) Naïve WT BALB/c mice were untreated or administered once i.n. with MSU crystals (1 mg/dose) or PBS. After 3 hr,

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Airway uric acid is a sensor of inhaled protease allergens and initiates type 2 immune responses in respiratory mucosa

    doi: 10.4049/jimmunol.1400110

    Figure Lengend Snippet: Airway administration of MSU crystals induces IL-33 and TSLP production in the lungs and trigger innate type 2 responses. (A and B) Naïve WT BALB/c mice were untreated or administered once i.n. with MSU crystals (1 mg/dose) or PBS. After 3 hr,

    Article Snippet: Monosodium urate (MSU) crystals were purchased from Sigma-Aldrich, suspended in PBS at 20 mg/ml, and sonicated for 20 min in an ultrasonic cleaner (BRANSON 2200, Branson Ultrasonics, Danbury, CT) before use.

    Techniques: Mouse Assay

    MSU triggered intracellular ROS is inhibited by cytochalasin B (CytB) and colchicine (col). ( A ) Neutrophils were pretreated with cytochalsin B (5 µg/mL), an inhibitor of actin polymerization, and stimulated with either MSU crystals (300 µg/mL), PMA (50 nM) or opsonized S. aureus and intracellular ROS production (icROS) was evaluated using luminol amplified CL. Cytochalasin B-treated cells produced lower amounts of icROS in response to MSU crystals and S. aureus as compared to untreated cells, while icROS production in response to PMA was not affected by the presence of cytochalasin B. Peak CL values are shown. ( B ) Neutrophils were pretreated with colchicine (1 µg/mL) and stimulated with either MSU crystals (300 µg/mL), PMA (50 nM) or opsonized S. aureus and icROS production was evaluated using luminol amplified CL. Colchicine decreased icROS in response to MSU crystals, while icROS production in response to PMA or S. aureus was not affected. Peak CL values are shown ( n = 7). * p ≤ 0.05 and ** p ≤ 0.01. ns = not significant.

    Journal: International Journal of Molecular Sciences

    Article Title: In Vivo Transmigrated Human Neutrophils Are Highly Primed for Intracellular Radical Production Induced by Monosodium Urate Crystals

    doi: 10.3390/ijms21113750

    Figure Lengend Snippet: MSU triggered intracellular ROS is inhibited by cytochalasin B (CytB) and colchicine (col). ( A ) Neutrophils were pretreated with cytochalsin B (5 µg/mL), an inhibitor of actin polymerization, and stimulated with either MSU crystals (300 µg/mL), PMA (50 nM) or opsonized S. aureus and intracellular ROS production (icROS) was evaluated using luminol amplified CL. Cytochalasin B-treated cells produced lower amounts of icROS in response to MSU crystals and S. aureus as compared to untreated cells, while icROS production in response to PMA was not affected by the presence of cytochalasin B. Peak CL values are shown. ( B ) Neutrophils were pretreated with colchicine (1 µg/mL) and stimulated with either MSU crystals (300 µg/mL), PMA (50 nM) or opsonized S. aureus and icROS production was evaluated using luminol amplified CL. Colchicine decreased icROS in response to MSU crystals, while icROS production in response to PMA or S. aureus was not affected. Peak CL values are shown ( n = 7). * p ≤ 0.05 and ** p ≤ 0.01. ns = not significant.

    Article Snippet: After 5 min equilibration at 37 °C, cells were stimulated with MSU crystals (300 µg/mL, unless otherwise stated), PMA (50 nM), or serum-opsonized bacteria (Staphylococcus aureus opsonized with 10% human serum for 20 min at 37 °C) added at a multiplicity of infection (MOI) of 10, in the presence or absence of the following inhibitors or drugs; DPI (10 µM), GSK (20 µM) or cytochalasin B (5 µg/mL) at 37 °C for 5 min prior to use, or colchicine (1 µg/mL) at 37 °C for 20 min prior to use.

    Techniques: Amplification, Produced

    MSU crystals induce neutrophil extracellular trap (NET) formation. ( A ) Neutrophils stimulated with MSU crystals formed NETs as confirmed by DNA- (blue) and MPO- (green) labelled micrographs of neutrophils 3 h after stimulation with MSU crystals (300 µg/mL). Scale bar = 10 µm. ( B ) NET formation as measured with Sytox Green fluorescence over 3 h. To the left, a representative kinetic curve of neutrophils stimulated with buffer (black squares, broken line), MSU crystals (300 µg/mL, triangles, dotted line), or PMA (50 nM, black dots, solid line). MSU crystals (300 µg/mL) triggered a statistically significant DNA release ( p = 0.0005 compared to buffer treated cells after 180 min, n = 12). To the right, NET formation evaluated after 3 h with different concentrations of MSU crystals, mean fluorescence +/− SEM are shown ( n = 3). Statistical significance was calculated by the use of the Wilcoxon matched-pairs signed rank test.

    Journal: International Journal of Molecular Sciences

    Article Title: In Vivo Transmigrated Human Neutrophils Are Highly Primed for Intracellular Radical Production Induced by Monosodium Urate Crystals

    doi: 10.3390/ijms21113750

    Figure Lengend Snippet: MSU crystals induce neutrophil extracellular trap (NET) formation. ( A ) Neutrophils stimulated with MSU crystals formed NETs as confirmed by DNA- (blue) and MPO- (green) labelled micrographs of neutrophils 3 h after stimulation with MSU crystals (300 µg/mL). Scale bar = 10 µm. ( B ) NET formation as measured with Sytox Green fluorescence over 3 h. To the left, a representative kinetic curve of neutrophils stimulated with buffer (black squares, broken line), MSU crystals (300 µg/mL, triangles, dotted line), or PMA (50 nM, black dots, solid line). MSU crystals (300 µg/mL) triggered a statistically significant DNA release ( p = 0.0005 compared to buffer treated cells after 180 min, n = 12). To the right, NET formation evaluated after 3 h with different concentrations of MSU crystals, mean fluorescence +/− SEM are shown ( n = 3). Statistical significance was calculated by the use of the Wilcoxon matched-pairs signed rank test.

    Article Snippet: After 5 min equilibration at 37 °C, cells were stimulated with MSU crystals (300 µg/mL, unless otherwise stated), PMA (50 nM), or serum-opsonized bacteria (Staphylococcus aureus opsonized with 10% human serum for 20 min at 37 °C) added at a multiplicity of infection (MOI) of 10, in the presence or absence of the following inhibitors or drugs; DPI (10 µM), GSK (20 µM) or cytochalasin B (5 µg/mL) at 37 °C for 5 min prior to use, or colchicine (1 µg/mL) at 37 °C for 20 min prior to use.

    Techniques: Fluorescence

    MSU crystal induced NET formation is independent of ROS. ( A ) Neutrophils were pre-treated with the NADPH-oxidase inhibitors DPI (10 μM) or GSK (20 µM) and thereafter stimulated with MSU crystals (300 μg/mL) or PMA (50 nM) before NET formation was evaluated by Sytox green measurement after 3 h. As opposed to PMA stimulated neutrophils, MSU treated neutrophils formed NETs also in the presence of the inhibitors. Mean +/− SEM of three independent experiments are shown, a paired t -test was used for statistical comparisons. ( B ) Neutrophils from one patient with CGD (left) as well as from one individual with complete MPO-deficiency (MPO-, right) formed NETs in response to MSU (300 μg/mL), dotted lines, but not to PMA (50 nM, solid lines). * p ≤ 0.05 and ** p ≤ 0.01. ns = not significant.

    Journal: International Journal of Molecular Sciences

    Article Title: In Vivo Transmigrated Human Neutrophils Are Highly Primed for Intracellular Radical Production Induced by Monosodium Urate Crystals

    doi: 10.3390/ijms21113750

    Figure Lengend Snippet: MSU crystal induced NET formation is independent of ROS. ( A ) Neutrophils were pre-treated with the NADPH-oxidase inhibitors DPI (10 μM) or GSK (20 µM) and thereafter stimulated with MSU crystals (300 μg/mL) or PMA (50 nM) before NET formation was evaluated by Sytox green measurement after 3 h. As opposed to PMA stimulated neutrophils, MSU treated neutrophils formed NETs also in the presence of the inhibitors. Mean +/− SEM of three independent experiments are shown, a paired t -test was used for statistical comparisons. ( B ) Neutrophils from one patient with CGD (left) as well as from one individual with complete MPO-deficiency (MPO-, right) formed NETs in response to MSU (300 μg/mL), dotted lines, but not to PMA (50 nM, solid lines). * p ≤ 0.05 and ** p ≤ 0.01. ns = not significant.

    Article Snippet: After 5 min equilibration at 37 °C, cells were stimulated with MSU crystals (300 µg/mL, unless otherwise stated), PMA (50 nM), or serum-opsonized bacteria (Staphylococcus aureus opsonized with 10% human serum for 20 min at 37 °C) added at a multiplicity of infection (MOI) of 10, in the presence or absence of the following inhibitors or drugs; DPI (10 µM), GSK (20 µM) or cytochalasin B (5 µg/mL) at 37 °C for 5 min prior to use, or colchicine (1 µg/mL) at 37 °C for 20 min prior to use.

    Techniques:

    Effect of APO on cell viability and IL-1 β secretion in LPS-primed BMDMs. (a) BMDMs were treated with the indicated concentration of APO for 24 h. Cell viability was measured by the LDH assay. (b–f) LPS-primed BMDMs were pretreated with APO or zVAD (20 μ M), and IL-1 β secretion was determined by ELISA upon stimulation with (b) 150 μ g/mL silica for 3 h, (c) 10 μ M nigericin for 1 h, (d) 5 mM ATP for 1 h, (e) 2 μ g/mL poly (dA:dT) for 1 h, and (f) 1.5 μ g/mL flagellin for 3 h. (g) TNF- α release from LPS-primed BMDMs was determined upon stimulation with 10 μ M nigericin for 1 h. (h) The impact of APO on MSU-induced IL-1 β production in mice. Data were expressed as the mean ± SEM ( n = 3). Statistical analysis was performed using Student's t -test. ∗ p

    Journal: Mediators of Inflammation

    Article Title: Artemisia Extract Suppresses NLRP3 and AIM2 Inflammasome Activation by Inhibition of ASC Phosphorylation

    doi: 10.1155/2018/6054069

    Figure Lengend Snippet: Effect of APO on cell viability and IL-1 β secretion in LPS-primed BMDMs. (a) BMDMs were treated with the indicated concentration of APO for 24 h. Cell viability was measured by the LDH assay. (b–f) LPS-primed BMDMs were pretreated with APO or zVAD (20 μ M), and IL-1 β secretion was determined by ELISA upon stimulation with (b) 150 μ g/mL silica for 3 h, (c) 10 μ M nigericin for 1 h, (d) 5 mM ATP for 1 h, (e) 2 μ g/mL poly (dA:dT) for 1 h, and (f) 1.5 μ g/mL flagellin for 3 h. (g) TNF- α release from LPS-primed BMDMs was determined upon stimulation with 10 μ M nigericin for 1 h. (h) The impact of APO on MSU-induced IL-1 β production in mice. Data were expressed as the mean ± SEM ( n = 3). Statistical analysis was performed using Student's t -test. ∗ p

    Article Snippet: Silica crystals (tlrl-sio), nigericin (tlrl-nig), ultrapure flagellin from Salmonella typhimurium (tlrl-epstfla), poly(deoxyadenylic-deoxythymidylic) acid (poly dA:dT, tlrl-patn), monosodium urate (MSU) crystal (tlrl-msu), and carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (zVAD-FMK, tlrl-vad) were purchased from InvivoGen (San Diego, CA, USA).

    Techniques: Concentration Assay, Lactate Dehydrogenase Assay, Enzyme-linked Immunosorbent Assay, Mouse Assay

    Effects of GW-A2 on NLRP3 inflammasome activation. ( A and B ) J774A.1 macrophages were incubated for 30 min with or without 2 μM of GW-A2, for 5.5 h with or without 0.1 μg/ml of E . coli LPS, and then for 30 min with or without 5 mM of ATP ( A ) or 10 μM of nigericin ( B ). The levels of IL-1β in the culture medium and activated caspase-1 (p10) in the cells were measured by ELISA and Western blotting, respectively. ( C — F ) J774A.1 macrophages were incubated for 5.5 h with 0.1 μg/ml of E . coli LPS, for 30 min with or without 2 μM of GW-A2, and then for 30 min with or without 5 mM of ATP ( C and E ), 10 μM of nigericin ( D ) or 100 μg/ml of MSU ( F ). The levels of IL-1β and TNF-α in the culture medium and activated caspase-1 (p10) in the cells were measured by ELISA and Western blotting, respectively. The data are expressed as the mean ± SD of three independent experiments. The Western blotting results shown are a representative experiment of three independent experiments. *, ** and *** indicate significant differences, representing p

    Journal: PLoS ONE

    Article Title: A synthetic cationic antimicrobial peptide inhibits inflammatory response and the NLRP3 inflammasome by neutralizing LPS and ATP

    doi: 10.1371/journal.pone.0182057

    Figure Lengend Snippet: Effects of GW-A2 on NLRP3 inflammasome activation. ( A and B ) J774A.1 macrophages were incubated for 30 min with or without 2 μM of GW-A2, for 5.5 h with or without 0.1 μg/ml of E . coli LPS, and then for 30 min with or without 5 mM of ATP ( A ) or 10 μM of nigericin ( B ). The levels of IL-1β in the culture medium and activated caspase-1 (p10) in the cells were measured by ELISA and Western blotting, respectively. ( C — F ) J774A.1 macrophages were incubated for 5.5 h with 0.1 μg/ml of E . coli LPS, for 30 min with or without 2 μM of GW-A2, and then for 30 min with or without 5 mM of ATP ( C and E ), 10 μM of nigericin ( D ) or 100 μg/ml of MSU ( F ). The levels of IL-1β and TNF-α in the culture medium and activated caspase-1 (p10) in the cells were measured by ELISA and Western blotting, respectively. The data are expressed as the mean ± SD of three independent experiments. The Western blotting results shown are a representative experiment of three independent experiments. *, ** and *** indicate significant differences, representing p

    Article Snippet: Pam3CSK4 (tlrl-pms), ATP (tlrl-atp), nigericin (tlrl-nig) and monosodium urate crystal (MSU) (tlrl-msu) were purchased from InvivoGen (San Diego, CA, USA).

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

    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

    Four axial spondyloarthritis (AxSpA) patients with monosodium urate (MSU) crystal and radiographic structural damage at the sacroiliac joint. For each set of images, panel a shows the sacroiliac joint on plain radiographs, panel b shows the three-dimensional reconstruction dual-energy computed tomography (DECT) images, panel c shows the corresponding coronal (patient 2 and 4) or axial (patient 1 and 3) DECT images, and panel d shows the corresponding level of computed tomography (CT) images. A large quantity of MSU crystal deposition shown as green was found at the sacroiliac joint or the surrounding area in the DECT images. Four male patients (patient 1–4), aged 36, 44, 23, and 27 years old, had serum uric acid levels of 407 μmol/L, 370 μmol/L, 572 μmol/L, and 464 μmol/L, respectively. They were graded with a scale of 0, 0, II, III at the left sacroiliac joint and 0, I, II, III at the right sacroiliac joint, respectively, on plain radiographs

    Journal: Arthritis Research & Therapy

    Article Title: Monosodium urate crystal deposition associated with the progress of radiographic grade at the sacroiliac joint in axial SpA: a dual-energy CT study

    doi: 10.1186/s13075-017-1286-0

    Figure Lengend Snippet: Four axial spondyloarthritis (AxSpA) patients with monosodium urate (MSU) crystal and radiographic structural damage at the sacroiliac joint. For each set of images, panel a shows the sacroiliac joint on plain radiographs, panel b shows the three-dimensional reconstruction dual-energy computed tomography (DECT) images, panel c shows the corresponding coronal (patient 2 and 4) or axial (patient 1 and 3) DECT images, and panel d shows the corresponding level of computed tomography (CT) images. A large quantity of MSU crystal deposition shown as green was found at the sacroiliac joint or the surrounding area in the DECT images. Four male patients (patient 1–4), aged 36, 44, 23, and 27 years old, had serum uric acid levels of 407 μmol/L, 370 μmol/L, 572 μmol/L, and 464 μmol/L, respectively. They were graded with a scale of 0, 0, II, III at the left sacroiliac joint and 0, I, II, III at the right sacroiliac joint, respectively, on plain radiographs

    Article Snippet: Reproducibility of DECT MSU crystal volume measurement and correlation between the average MSU crystal volume and serum uric acid Of the patients who showed the presence of MSU crystal deposition, 57 showed deposition at the left sacroiliac joint, 54 at the right sacroiliac joint, and 154 at the pelvis.

    Techniques: Computed Tomography

    Bland-Altman plots for interobserver reproducibility analysis. a The dual-energy computed tomography (DECT) monosodium urate (MSU) crystal volume at the left sacroiliac joint. b The DECT MSU crystal volume at the right sacroiliac joint. c The DECT MSU crystal volume at the pelvis. Solid line shows bias and dashed lines show the 95% limits of agreement

    Journal: Arthritis Research & Therapy

    Article Title: Monosodium urate crystal deposition associated with the progress of radiographic grade at the sacroiliac joint in axial SpA: a dual-energy CT study

    doi: 10.1186/s13075-017-1286-0

    Figure Lengend Snippet: Bland-Altman plots for interobserver reproducibility analysis. a The dual-energy computed tomography (DECT) monosodium urate (MSU) crystal volume at the left sacroiliac joint. b The DECT MSU crystal volume at the right sacroiliac joint. c The DECT MSU crystal volume at the pelvis. Solid line shows bias and dashed lines show the 95% limits of agreement

    Article Snippet: Reproducibility of DECT MSU crystal volume measurement and correlation between the average MSU crystal volume and serum uric acid Of the patients who showed the presence of MSU crystal deposition, 57 showed deposition at the left sacroiliac joint, 54 at the right sacroiliac joint, and 154 at the pelvis.

    Techniques: Computed Tomography

    Suppression of RIPK3 and MLKL prevents crystal cytotoxicity. ( a – c ) Mouse tubular epithelial cells were transfected with specific small inhibitor (si) RNA for RIPK3 and MLKL or a control siRNA of scrambled sequence before being exposed to crystals of CaOx (1,000 μg ml −1 ), MSU (500 μg ml −1 ), CPPD (500 μg ml −1 ) and cystine (500 μg ml −1 ). Cell viability was assessed by MTT assay (a) and cell death was assessed quantifying PI positivity (b) and C shows representative images 24 h later. Original image magnification: × 200, scale bar, 100 μm. ( d ) Mouse tubular epithelial cells were pretreated with RIPK3 inhibitor dabrafenib before exposing to different type crystals. Cell viability was assessed by MTT assay 24 h later. Data are expressed as mean±s.e.m. of three independent experiments, and was analysed using Student's t -test. Baseline viability is set as 100%. * P

    Journal: Nature Communications

    Article Title: Cytotoxicity of crystals involves RIPK3-MLKL-mediated necroptosis

    doi: 10.1038/ncomms10274

    Figure Lengend Snippet: Suppression of RIPK3 and MLKL prevents crystal cytotoxicity. ( a – c ) Mouse tubular epithelial cells were transfected with specific small inhibitor (si) RNA for RIPK3 and MLKL or a control siRNA of scrambled sequence before being exposed to crystals of CaOx (1,000 μg ml −1 ), MSU (500 μg ml −1 ), CPPD (500 μg ml −1 ) and cystine (500 μg ml −1 ). Cell viability was assessed by MTT assay (a) and cell death was assessed quantifying PI positivity (b) and C shows representative images 24 h later. Original image magnification: × 200, scale bar, 100 μm. ( d ) Mouse tubular epithelial cells were pretreated with RIPK3 inhibitor dabrafenib before exposing to different type crystals. Cell viability was assessed by MTT assay 24 h later. Data are expressed as mean±s.e.m. of three independent experiments, and was analysed using Student's t -test. Baseline viability is set as 100%. * P

    Article Snippet: All cells were stimulated with different doses of crystals of CaOx (1–2 μm size; Alfa aesar, Germany), MSU (25–125 nm size; Invivogen, Germany), CPPD (25–125 nm size; Invivogen, Germany) and cystine (1–2 μm size; Sigma Aldrich, Germany) in different experiments.

    Techniques: Transfection, Sequencing, MTT Assay

    Necroptosis is involved in human acute oxalate nephropathy. ( a – c ) Primary renal human progenitor cells were pretreated with either ZVAD–FMK (10 μM) and Nec-1 (100 μM) or NSA (1 μM) before being exposed to CaOx (1000 μg ml −1 ), MSU (500 μg ml −1 ), CPPD (500 μg ml −1 ) and cystine (500 μg ml −1 ). Cell viability was assessed by MTT assay ( a and b ) and cell death was assessed quantifying PI positivity ( c ) 24 h later. Data are expressed as mean±s.e.m. of three independent experiments. Baseline viability is set as 100%. Data were analysed using Student's t -test. * P

    Journal: Nature Communications

    Article Title: Cytotoxicity of crystals involves RIPK3-MLKL-mediated necroptosis

    doi: 10.1038/ncomms10274

    Figure Lengend Snippet: Necroptosis is involved in human acute oxalate nephropathy. ( a – c ) Primary renal human progenitor cells were pretreated with either ZVAD–FMK (10 μM) and Nec-1 (100 μM) or NSA (1 μM) before being exposed to CaOx (1000 μg ml −1 ), MSU (500 μg ml −1 ), CPPD (500 μg ml −1 ) and cystine (500 μg ml −1 ). Cell viability was assessed by MTT assay ( a and b ) and cell death was assessed quantifying PI positivity ( c ) 24 h later. Data are expressed as mean±s.e.m. of three independent experiments. Baseline viability is set as 100%. Data were analysed using Student's t -test. * P

    Article Snippet: All cells were stimulated with different doses of crystals of CaOx (1–2 μm size; Alfa aesar, Germany), MSU (25–125 nm size; Invivogen, Germany), CPPD (25–125 nm size; Invivogen, Germany) and cystine (1–2 μm size; Sigma Aldrich, Germany) in different experiments.

    Techniques: MTT Assay

    Crystal cytotoxicity involves the necroptosis pathway. ( a ): Protein expression of TNFR1, RIPK1 and RIPK3 was determined by western blot from total proteins isolated 18 h after stimulation of mouse tubular epithelial cells with crystals of CaOx (1,000 μg ml −1 ), MSU (500 μg ml −1 ), CPPD (500 μg ml −1 ) and cystine (500 μg ml −1 ). β-actin was used as loading control. ( b ) Mouse tubular epithelial cells were exposed to different concentrations of CaOx, MSU, CPPD or cystine crystals as indicated in the presence or absence of necrostatin (Nec)-1 (100 μM) together with the pan-caspase inhibitor ZVAD–FMK–FMK (10 μM). Cell viability was assessed by MTT assay 24 h later. Data are expressed as mean cell viability±s.e.m. of three independent experiments. Baseline viability is set as 100%. ( c ) In similar experiments necrotic tubular epithelial cells were identified via propidium iodide positivity and the results were expressed as mean fluorescent intensity on digital analysis of pictures taken from culture dishes. Representative images are shown at an original magnification of × 200, scale bar, 40 μm. Data were analysed using Student's t -test. * P

    Journal: Nature Communications

    Article Title: Cytotoxicity of crystals involves RIPK3-MLKL-mediated necroptosis

    doi: 10.1038/ncomms10274

    Figure Lengend Snippet: Crystal cytotoxicity involves the necroptosis pathway. ( a ): Protein expression of TNFR1, RIPK1 and RIPK3 was determined by western blot from total proteins isolated 18 h after stimulation of mouse tubular epithelial cells with crystals of CaOx (1,000 μg ml −1 ), MSU (500 μg ml −1 ), CPPD (500 μg ml −1 ) and cystine (500 μg ml −1 ). β-actin was used as loading control. ( b ) Mouse tubular epithelial cells were exposed to different concentrations of CaOx, MSU, CPPD or cystine crystals as indicated in the presence or absence of necrostatin (Nec)-1 (100 μM) together with the pan-caspase inhibitor ZVAD–FMK–FMK (10 μM). Cell viability was assessed by MTT assay 24 h later. Data are expressed as mean cell viability±s.e.m. of three independent experiments. Baseline viability is set as 100%. ( c ) In similar experiments necrotic tubular epithelial cells were identified via propidium iodide positivity and the results were expressed as mean fluorescent intensity on digital analysis of pictures taken from culture dishes. Representative images are shown at an original magnification of × 200, scale bar, 40 μm. Data were analysed using Student's t -test. * P

    Article Snippet: All cells were stimulated with different doses of crystals of CaOx (1–2 μm size; Alfa aesar, Germany), MSU (25–125 nm size; Invivogen, Germany), CPPD (25–125 nm size; Invivogen, Germany) and cystine (1–2 μm size; Sigma Aldrich, Germany) in different experiments.

    Techniques: Expressing, Western Blot, Isolation, MTT Assay

    Necrostatin-1 and neutrophil recruitment. ( a , b ): i.p. injection of CaOx, MSU, CPPD or cystine crystals into C57BL/6 mice ( n =4 in each group) induces neutrophil recruitment to the peritoneal cavity as determined by flow cytometry of peritoneal lavage fluids. This process is not affected by concomitant necrostatin-1 treatment. A shows representative flow cytometry data for each crystal presented as mean fluorescence intensity for the neutrophil marker. ( b ) Shows data of each mouse with the mean of wild-type mice set as 100%. NS, not significant. ( c ) Similar experiments using the air pouch model of neutrophil recruitment gave identical results ( n =5 in each group) and are expressed in the same manner. ( d – f ) In vivo microscopy studies of the postischemic musculus cremaster were performed as a second model of injury-associated and ROS-dependent neutrophil recruitment in C57BL/6 mice. Necrostatin-1 significantly reduced the microvascular leakage of FITC-labelled dextran particles as illustrated by representative images in D at an original magnification of × 400, scale bar, 100 μm and quantitatively in ( e ). In the same experiment leukocyte transmigration from the microvasculature was quantified by counting from video recordings taken at baseline and at 60 and 120 min. Data are means±s.e.m. from seven mice in each group. Data were analysed using one-way ANOVA with post hoc Bonferroni's correction. * P

    Journal: Nature Communications

    Article Title: Cytotoxicity of crystals involves RIPK3-MLKL-mediated necroptosis

    doi: 10.1038/ncomms10274

    Figure Lengend Snippet: Necrostatin-1 and neutrophil recruitment. ( a , b ): i.p. injection of CaOx, MSU, CPPD or cystine crystals into C57BL/6 mice ( n =4 in each group) induces neutrophil recruitment to the peritoneal cavity as determined by flow cytometry of peritoneal lavage fluids. This process is not affected by concomitant necrostatin-1 treatment. A shows representative flow cytometry data for each crystal presented as mean fluorescence intensity for the neutrophil marker. ( b ) Shows data of each mouse with the mean of wild-type mice set as 100%. NS, not significant. ( c ) Similar experiments using the air pouch model of neutrophil recruitment gave identical results ( n =5 in each group) and are expressed in the same manner. ( d – f ) In vivo microscopy studies of the postischemic musculus cremaster were performed as a second model of injury-associated and ROS-dependent neutrophil recruitment in C57BL/6 mice. Necrostatin-1 significantly reduced the microvascular leakage of FITC-labelled dextran particles as illustrated by representative images in D at an original magnification of × 400, scale bar, 100 μm and quantitatively in ( e ). In the same experiment leukocyte transmigration from the microvasculature was quantified by counting from video recordings taken at baseline and at 60 and 120 min. Data are means±s.e.m. from seven mice in each group. Data were analysed using one-way ANOVA with post hoc Bonferroni's correction. * P

    Article Snippet: All cells were stimulated with different doses of crystals of CaOx (1–2 μm size; Alfa aesar, Germany), MSU (25–125 nm size; Invivogen, Germany), CPPD (25–125 nm size; Invivogen, Germany) and cystine (1–2 μm size; Sigma Aldrich, Germany) in different experiments.

    Techniques: Injection, Mouse Assay, Flow Cytometry, Cytometry, Fluorescence, Marker, In Vivo, Microscopy, Transmigration Assay

    Crystals induce primary cell necrosis. ( a ): Various crystals were incubated with tubular epithelial cells as indicated. Images show the crystal shapes at a magnification of × 1,000 (left), TEM images shows that these crystals induce necrosis of tubular epithelial cells as indicated by ruptured plasma membranes (middle, × 2,000), scale bar, 2 μm. The images on the right show the same rhodamine-labelled monolayers (red) 24 h later. When Sytox green is added to the medium cells with permeable plasma membranes turn green indicating cell death (× 200), scale bar, 40 μm. ( b ) Flow cytometry was used to define the type and stage of tubular cell (MTC) death on CaOx crystal exposure or ultraviolet light type B for 90 s (s) over a period of 50 h as described in methods. Data for viable cells, apoptotic cells and necrotic cells are expressed as the percentage of viable cells±s.e.m. of all cells for each time point. ( c ): The graphs show a quantitative analysis of the same experiment displaying all different phenotypes of tubular epithelial cells. Flow cytometry cell death definitions are described in the methods section. ( d ) Mouse tubular epithelial cell viability on crystal exposure by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay with and without the pan-caspase inhibitor ZVAD–FMK–FMK. All data are mean±s.e.m. of at least three independent experiments. CaOx, MSU, CPPD, NS, not significant. * P

    Journal: Nature Communications

    Article Title: Cytotoxicity of crystals involves RIPK3-MLKL-mediated necroptosis

    doi: 10.1038/ncomms10274

    Figure Lengend Snippet: Crystals induce primary cell necrosis. ( a ): Various crystals were incubated with tubular epithelial cells as indicated. Images show the crystal shapes at a magnification of × 1,000 (left), TEM images shows that these crystals induce necrosis of tubular epithelial cells as indicated by ruptured plasma membranes (middle, × 2,000), scale bar, 2 μm. The images on the right show the same rhodamine-labelled monolayers (red) 24 h later. When Sytox green is added to the medium cells with permeable plasma membranes turn green indicating cell death (× 200), scale bar, 40 μm. ( b ) Flow cytometry was used to define the type and stage of tubular cell (MTC) death on CaOx crystal exposure or ultraviolet light type B for 90 s (s) over a period of 50 h as described in methods. Data for viable cells, apoptotic cells and necrotic cells are expressed as the percentage of viable cells±s.e.m. of all cells for each time point. ( c ): The graphs show a quantitative analysis of the same experiment displaying all different phenotypes of tubular epithelial cells. Flow cytometry cell death definitions are described in the methods section. ( d ) Mouse tubular epithelial cell viability on crystal exposure by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay with and without the pan-caspase inhibitor ZVAD–FMK–FMK. All data are mean±s.e.m. of at least three independent experiments. CaOx, MSU, CPPD, NS, not significant. * P

    Article Snippet: All cells were stimulated with different doses of crystals of CaOx (1–2 μm size; Alfa aesar, Germany), MSU (25–125 nm size; Invivogen, Germany), CPPD (25–125 nm size; Invivogen, Germany) and cystine (1–2 μm size; Sigma Aldrich, Germany) in different experiments.

    Techniques: Incubation, Transmission Electron Microscopy, Flow Cytometry, Cytometry

    CAPE blocks the interaction between NLRP3 and ASC. ( A ) The chemical structure of CAPE and the proposed molecular docking model for CAPE binding to ASC. ( B ) Electrostatic surface binding model for CAPE and ASC. Red: negative charge, blue: positive charge. ( C,D ) BMDMs were primed with LPS ( C , 500 ng/ml; D , 100 ng/ml) for 4 hr. Then, the cells were treated with CAPE for 1 hr, followed by stimulation with MSU (500 μg/ml) for 5 hr or ATP (5 mM) for 1 hr. Cell lysates were immunoprepitated with anti-ASC antibody followed by immunoblotting as indicated. Representative data from at least two independent experiments are presented.

    Journal: Scientific Reports

    Article Title: Targeting ASC in NLRP3 inflammasome by caffeic acid phenethyl ester: a novel strategy to treat acute gout

    doi: 10.1038/srep38622

    Figure Lengend Snippet: CAPE blocks the interaction between NLRP3 and ASC. ( A ) The chemical structure of CAPE and the proposed molecular docking model for CAPE binding to ASC. ( B ) Electrostatic surface binding model for CAPE and ASC. Red: negative charge, blue: positive charge. ( C,D ) BMDMs were primed with LPS ( C , 500 ng/ml; D , 100 ng/ml) for 4 hr. Then, the cells were treated with CAPE for 1 hr, followed by stimulation with MSU (500 μg/ml) for 5 hr or ATP (5 mM) for 1 hr. Cell lysates were immunoprepitated with anti-ASC antibody followed by immunoblotting as indicated. Representative data from at least two independent experiments are presented.

    Article Snippet: Monosodium urate (MSU) and ATP were purchased from Invivogen (Carlsbad, CA).

    Techniques: Binding Assay

    iNKT Cells Induce IL-1β Secretion by BMDCs in a CD1d-Dependent Manner (A and B) BMDCs were left unprimed or primed with Pam3CSK4 (P3C; 0.5 μg/mL) for 24 h and subsequently co-cultured with iNKT cells for 24 h in the (A) absence or (B) presence of αGalCer (50 ng/mL). IL-1β release was quantified by ELISA. As a control, BMDCs were primed with P3C for 4 h, followed by 6-h stimulation with MSU (100 μg/mL). The same concentrations of P3C, αGalCer, and MSU were used throughout all experiments. (C and D) P3C-primed BMDCs were cultured alone or with iNKT cells for the indicated times in the presence or absence of αGalCer. IL-1β release was quantified by ELISA (C), and cell-associated and extracellular IL-1β were analyzed by immunoblot (D). As a control, BMDCs were primed with P3C for 4 h, followed by 1-h stimulation with nigericin (10 μM). (E) P3C-primed BMDCs from WT and CD1d −/− mice were co-cultured with iNKT cells for the indicated times in the absence or presence of αGalCer. IL-1β release was quantified by ELISA. P3C-primed BMDCs stimulated with nigericin were used as a control. Error bars represent mean + SD of triplicate samples. Each panel is representative of at least three different experiments. *p

    Journal: Cell reports

    Article Title: A Two-Cell Model for IL-1β Release Mediated by Death-Receptor Signaling

    doi: 10.1016/j.celrep.2020.03.030

    Figure Lengend Snippet: iNKT Cells Induce IL-1β Secretion by BMDCs in a CD1d-Dependent Manner (A and B) BMDCs were left unprimed or primed with Pam3CSK4 (P3C; 0.5 μg/mL) for 24 h and subsequently co-cultured with iNKT cells for 24 h in the (A) absence or (B) presence of αGalCer (50 ng/mL). IL-1β release was quantified by ELISA. As a control, BMDCs were primed with P3C for 4 h, followed by 6-h stimulation with MSU (100 μg/mL). The same concentrations of P3C, αGalCer, and MSU were used throughout all experiments. (C and D) P3C-primed BMDCs were cultured alone or with iNKT cells for the indicated times in the presence or absence of αGalCer. IL-1β release was quantified by ELISA (C), and cell-associated and extracellular IL-1β were analyzed by immunoblot (D). As a control, BMDCs were primed with P3C for 4 h, followed by 1-h stimulation with nigericin (10 μM). (E) P3C-primed BMDCs from WT and CD1d −/− mice were co-cultured with iNKT cells for the indicated times in the absence or presence of αGalCer. IL-1β release was quantified by ELISA. P3C-primed BMDCs stimulated with nigericin were used as a control. Error bars represent mean + SD of triplicate samples. Each panel is representative of at least three different experiments. *p

    Article Snippet: For inflammasome activation controls, BMDCs were primed for 4 hours and treated with monosodium urate (MSU) crystals (Invivogen, 100 μg/ml) for 6 hours or nigericin (Invivogen, 10 μM) for 1 hour.

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

    iNKT Cells Employ the Death Receptor Ligand FasL to Instruct TLR-Primed BMDCs to Release IL-1β (A) iNKT cells were cultured in the presence of increasing concentrations of plate-bound anti-CD3 for 24 h, after which their cell-free Supes were used to stimulate P3C-primed BMDCs for 24 h. IL-1β (left) and interferon γ (IFN-γ; right) in the Supes were quantified by ELISA. Nigericin-stimulated BMDCs were used as a control. (B) The ImmGen database was mined to compare the expression of cell-surface proteins (Gene Ontology term “cell surface,” GO0009986) expressed by iNKT cells versus other αβ T cells. The red dots represent genes expressed by iNKT cells with a fold change of greater than 5 compared with all other αβ T cells. Expression values comparing class means for specific T cell populations are shown in the right panel. (C) P3C-primed BMDCs were co-cultured with iNKT cells for 24 h in the presence of a FasL-blocking antibody (aFasL mAb) or an isotype control. IL-1β release was quantified by ELISA. MSU-treated BMDCs were used as a control. (D) P3C-primed BMDCs from WT and Fas −/− mice were co-cultured with iNKT cells for 24 h. IL-1β release was quantified by ELISA. MSU-treated BMDCs were used as a control. (E) Resting iNKT cells were stained with a FasL-specific antibody or an isotype control and subsequently analyzed by flow cytometry. The left panel represents surface staining, whereas the right panel represents permeabilized iNKT cells. (F) P3C-primed BMDCs from WT and CD1d −/− mice were co-cultured with CellTrace Violet-labeled-iNKT cells for the indicated times in the presence or absence of αGalCer, and the expression of FasL on the surface of iNKT cells was analyzed by flow cytometry. Numbers on the right of each histogram represent mean fluorescence intensity values. Error bars represent mean + SD of triplicate samples. Each panel is representative of at least three different experiments.

    Journal: Cell reports

    Article Title: A Two-Cell Model for IL-1β Release Mediated by Death-Receptor Signaling

    doi: 10.1016/j.celrep.2020.03.030

    Figure Lengend Snippet: iNKT Cells Employ the Death Receptor Ligand FasL to Instruct TLR-Primed BMDCs to Release IL-1β (A) iNKT cells were cultured in the presence of increasing concentrations of plate-bound anti-CD3 for 24 h, after which their cell-free Supes were used to stimulate P3C-primed BMDCs for 24 h. IL-1β (left) and interferon γ (IFN-γ; right) in the Supes were quantified by ELISA. Nigericin-stimulated BMDCs were used as a control. (B) The ImmGen database was mined to compare the expression of cell-surface proteins (Gene Ontology term “cell surface,” GO0009986) expressed by iNKT cells versus other αβ T cells. The red dots represent genes expressed by iNKT cells with a fold change of greater than 5 compared with all other αβ T cells. Expression values comparing class means for specific T cell populations are shown in the right panel. (C) P3C-primed BMDCs were co-cultured with iNKT cells for 24 h in the presence of a FasL-blocking antibody (aFasL mAb) or an isotype control. IL-1β release was quantified by ELISA. MSU-treated BMDCs were used as a control. (D) P3C-primed BMDCs from WT and Fas −/− mice were co-cultured with iNKT cells for 24 h. IL-1β release was quantified by ELISA. MSU-treated BMDCs were used as a control. (E) Resting iNKT cells were stained with a FasL-specific antibody or an isotype control and subsequently analyzed by flow cytometry. The left panel represents surface staining, whereas the right panel represents permeabilized iNKT cells. (F) P3C-primed BMDCs from WT and CD1d −/− mice were co-cultured with CellTrace Violet-labeled-iNKT cells for the indicated times in the presence or absence of αGalCer, and the expression of FasL on the surface of iNKT cells was analyzed by flow cytometry. Numbers on the right of each histogram represent mean fluorescence intensity values. Error bars represent mean + SD of triplicate samples. Each panel is representative of at least three different experiments.

    Article Snippet: For inflammasome activation controls, BMDCs were primed for 4 hours and treated with monosodium urate (MSU) crystals (Invivogen, 100 μg/ml) for 6 hours or nigericin (Invivogen, 10 μM) for 1 hour.

    Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Blocking Assay, Mouse Assay, Staining, Flow Cytometry, Labeling, Fluorescence

    Extracellular succinate signals via GPR91 to stimulate macrophages to release IL-1β. (A) GPR91 mRNA expression in WT (Janvier C57BL/6J) inflammatory BMDMs (M-CSF + IFN-γ) ± 100 ng/ml LPS, 500 µM succinate, or 10 ng/ml IL-1β for 24 h. n = 3 of Ct values. Succinate (Succ), IL-1β, and LPS related to basal (=1). Data are representative of three experiments. (B) Succinate levels (mass spectrophotometry area ratio) in medium from cultured BMDMs. Extracellular succinate from WT (littermates; black bars) and Sucnr1 −/− (gray bars), neutral (M, M-CSF), or inflammatory (M + IFN-γ) BMDMs ± 100 ng/ml LPS for 24 h is shown. n = 6 wells. Data are representative of three experiments. (C) IL-1β in supernatants of WT (Janvier C57BL/6J) and Sucnr1 −/− neutral or inflammatory BMDMs ± 100 ng/ml LPS for 24 h. n = 3 wells and are representative of seven experiments. (D) IL-1β mRNA levels from cell lysates from WT (Janvier C57BL/6J) or Sucnr1 −/− inflammatory BMDMs ± 100 ng/ml LPS at 4 h (related to WT basal = 1). n = 2–3 of Ct values. Data are representative of two experiments. (E) IL-1β levels measured in the supernatant of WT (Janvier C57BL/6J) and Sucnr1 −/− inflammatory BMDMs stimulated with 1 ng/ml LPS and 180 µg/ml MSU. n = 5–6 wells. Data are representative of two experiments. (F) Western blot of HIF-1α (representative blot of two experiments) and quantification (two experiments; 100% for no stimulus, WT, and Sucnr1 −/− ) at 6 h after stimulation with 500 µM succinate, 100 ng/ml LPS, or a combination of the two in WT littermate controls and Sucnr1 −/− inflammatory BMDMs. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: GPR91 senses extracellular succinate released from inflammatory macrophages and exacerbates rheumatoid arthritis

    doi: 10.1084/jem.20160061

    Figure Lengend Snippet: Extracellular succinate signals via GPR91 to stimulate macrophages to release IL-1β. (A) GPR91 mRNA expression in WT (Janvier C57BL/6J) inflammatory BMDMs (M-CSF + IFN-γ) ± 100 ng/ml LPS, 500 µM succinate, or 10 ng/ml IL-1β for 24 h. n = 3 of Ct values. Succinate (Succ), IL-1β, and LPS related to basal (=1). Data are representative of three experiments. (B) Succinate levels (mass spectrophotometry area ratio) in medium from cultured BMDMs. Extracellular succinate from WT (littermates; black bars) and Sucnr1 −/− (gray bars), neutral (M, M-CSF), or inflammatory (M + IFN-γ) BMDMs ± 100 ng/ml LPS for 24 h is shown. n = 6 wells. Data are representative of three experiments. (C) IL-1β in supernatants of WT (Janvier C57BL/6J) and Sucnr1 −/− neutral or inflammatory BMDMs ± 100 ng/ml LPS for 24 h. n = 3 wells and are representative of seven experiments. (D) IL-1β mRNA levels from cell lysates from WT (Janvier C57BL/6J) or Sucnr1 −/− inflammatory BMDMs ± 100 ng/ml LPS at 4 h (related to WT basal = 1). n = 2–3 of Ct values. Data are representative of two experiments. (E) IL-1β levels measured in the supernatant of WT (Janvier C57BL/6J) and Sucnr1 −/− inflammatory BMDMs stimulated with 1 ng/ml LPS and 180 µg/ml MSU. n = 5–6 wells. Data are representative of two experiments. (F) Western blot of HIF-1α (representative blot of two experiments) and quantification (two experiments; 100% for no stimulus, WT, and Sucnr1 −/− ) at 6 h after stimulation with 500 µM succinate, 100 ng/ml LPS, or a combination of the two in WT littermate controls and Sucnr1 −/− inflammatory BMDMs. *, P

    Article Snippet: In vitro stimulation of mouse macrophages or U937 cells U937 or mouse macrophages derived from 7–9-wk-old age-matched animals were incubated for up to 24 h in 96-well flat-bottom plates at a density of 105 cells per well in RPMI-1640 containing 1 or 100 ng/ml LPS (InvivoGen), 500 µM succinate (sodium succinate dibasic hexahydrate; Sigma-Aldrich), 10 ng/ml mouse IL-1β (PeproTech), 180 µg/ml MSU (Enzo Life Sciences), 5 µM GPR91 antagonist GPR91A1 (synthesized in house from ), and 10% human RA SFs (Asterand).

    Techniques: Expressing, Spectrophotometry, Cell Culture, Western Blot

    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

    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