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  • 99
    ATCC anabaena variabilis atcc 29413
    Anabaena Variabilis Atcc 29413, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 579 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    InvivoGen msu crystals
    Msu Crystals, supplied by InvivoGen, used in various techniques. Bioz Stars score: 99/100, based on 120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Crystal IS msu crystal
    ( a ) Optimization of the orientation angle of the linear polarizer ( γ ). The normalized output amplitude is plotted as a function of the sample fast-axis orientation, <t>α</t> , for different linear polarizer orientations ( γ = 50°, 55°, 65°, 75°). ( b ) The simulated normalized output images of <t>MSU</t> crystals at varying orientations, using γ = 65°. The MSU crystals are simulated as cylinders with a birefringence of | Δ n| = 0.1, diameter of 0.5 μm, length of 10 μm, and the fast axis is along the long axis of the crystals.
    Msu Crystal, supplied by Crystal IS, used in various techniques. Bioz Stars score: 89/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    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
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    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
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    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
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    91
    Caltag-Medsystems msu crystals
    <t>IL-1β</t> secretion by PBMCs following exposure to <t>MSU</t> crystals or SNPs. Secretion of IL-1β by PBMCs with (main figure) or without (inset) initial exposure to 10 ng/mL LPS for 3 h followed by exposure to MSU crystals (100 μg/mL) or SNPs (100 μg/mL) for a further 3 h. The supernatant was either collected immediately for analysis (3 h; open bars) or following a further 21 h of cell incubation in fresh (i.e. without added particles or MAMPs) TCM (3 + 21 h; black bars). All data are expressed as mean ± SEM ( n = 4). Results were analysed by paired T test in comparison to baseline (B), i.e. non-particle-exposed cells; * p
    Msu Crystals, supplied by Caltag-Medsystems, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Enzo Biochem msu crystals
    Gasdermin D pore formation and pyroptosis are not involved in EEA1 release. ( A ) Immunoblot analysis of EEA1 and IL-1β on cell lysate and cell-free supernatant from LPS-primed BMDMs from wild type or Casp1/11 −/− after stimulation with nigericin, ATP, E . coli or <t>MSU</t> crystals as indicated. IL-1β released to the extracellular medium was detected by ELISA (bottom). ( B ) LDH release from macrophages treated as in A. ( C ) Immunoblot analysis of EEA1 on cell lysate and cell-free supernatant from BMDMs wild-type and Gsdmd −/− after nigericin stimulation. ( D ) Immunoblot analysis of <t>caspase-1</t> and IL-1β on cell lysate and cell-free supernatant from wild-type or Gsdmd −/− BMDMs after nigericin stimulation in the presence or absence of the caspase-1 inhibitor Ac-YVAD. ( E , F ) ELISA for released IL-1β ( E ) or LDH ( F ) from macrophages treated as in D. ( G ) Immunoblot analysis of ASC and NLRP3 on cell-free supernatant from macrophages stimulated as in D. ( H ) Yo-Pro uptake in macrophages treated as in D. Blots are representative of three to four independent experiments. Nitrocellulose membranes were cropped after protein blotting and then inmunodetection was carried out separately for each antibody. Complete blots for IL-1β and Caspase-1 are showed as supplementary. Data are presented as average ± sem from n = 3 independent experiments.
    Msu Crystals, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 91/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    Image Search Results


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

    Journal: Scientific Reports

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

    doi: 10.1038/srep28793

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

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

    Techniques:

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

    Journal: Scientific Reports

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

    doi: 10.1038/srep28793

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

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

    Techniques:

    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

    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