msu crystals  (Thermo Fisher)


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    Structured Review

    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 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    msu crystals - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "CD44 Receptor Mediates Urate Crystal Phagocytosis by Macrophages and Regulates Inflammation in A Murine Peritoneal Model of Acute Gout"

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

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-62727-z

    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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: Expressing, Incubation, Flow Cytometry

    2) Product Images from "Monosodium urate crystals reduce osteocyte viability and indirectly promote a shift in osteocyte function towards a proinflammatory and proresorptive state"

    Article Title: Monosodium urate crystals reduce osteocyte viability and indirectly promote a shift in osteocyte function towards a proinflammatory and proresorptive state

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-018-1704-y

    Effects of COX-2 inhibition on MLO-Y4 cell responses to MSU crystal-stimulated RAW264.7 macrophage conditioned medium. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. A cyclooxygenase-2 (COX-2)-specific inhibitor (SC-236) was added to MLO-Y4 cells for 1 h prior to the addition of 40% conditioned medium for 24 h. MLO-Y4 cells were then harvested for mRNA gene expression analysis and supernatants harvested for protein quantification. a Changes in mRNA expression of inflammatory genes: tumor necrosis factor (TNF)-α, COX-2, interleukin (IL)-6, and IL-11; and bone-related genes: receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG). b Changes in TNF-α, prostaglandin E 2 (PGE 2 ), and IL-6 protein levels in MLO-Y4 cell supernatants. Data shown are pooled from five biological repeats and are presented as (SEM); one-way analysis of variance (ANOVA) with post-hoc Sidak’s test between groups as indicated. NS no significant difference
    Figure Legend Snippet: Effects of COX-2 inhibition on MLO-Y4 cell responses to MSU crystal-stimulated RAW264.7 macrophage conditioned medium. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. A cyclooxygenase-2 (COX-2)-specific inhibitor (SC-236) was added to MLO-Y4 cells for 1 h prior to the addition of 40% conditioned medium for 24 h. MLO-Y4 cells were then harvested for mRNA gene expression analysis and supernatants harvested for protein quantification. a Changes in mRNA expression of inflammatory genes: tumor necrosis factor (TNF)-α, COX-2, interleukin (IL)-6, and IL-11; and bone-related genes: receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG). b Changes in TNF-α, prostaglandin E 2 (PGE 2 ), and IL-6 protein levels in MLO-Y4 cell supernatants. Data shown are pooled from five biological repeats and are presented as (SEM); one-way analysis of variance (ANOVA) with post-hoc Sidak’s test between groups as indicated. NS no significant difference

    Techniques Used: Inhibition, Cell Culture, Expressing

    The direct effects of MSU crystals on osteocyte viability. The alamarBlue® assay was used to determine the viability of a MLO-Y4 cells and primary mouse osteocytes cultured with monosodium urate (MSU) crystals for 24 h, b MLO-Y4 cells cultured with soluble urate for 24 h, and c MLO-Y4 cells cultured with different types of crystals for 24 h. Viability was assessed 24 and 48 h after the addition of crystals or soluble urate. Data shown are pooled from three to four biological repeats and are presented as mean (SEM); by two-way ANOVA a P Interaction
    Figure Legend Snippet: The direct effects of MSU crystals on osteocyte viability. The alamarBlue® assay was used to determine the viability of a MLO-Y4 cells and primary mouse osteocytes cultured with monosodium urate (MSU) crystals for 24 h, b MLO-Y4 cells cultured with soluble urate for 24 h, and c MLO-Y4 cells cultured with different types of crystals for 24 h. Viability was assessed 24 and 48 h after the addition of crystals or soluble urate. Data shown are pooled from three to four biological repeats and are presented as mean (SEM); by two-way ANOVA a P Interaction

    Techniques Used: Alamar Blue Assay, Cell Culture

    Direct effects of MSU crystals on MLO-Y4 cell expression of bone-related or inflammatory genes. Real-time PCR was used to determine changes in the relative mRNA expression levels of a bone-related and b inflammatory genes in MLO-Y4 cells, following culture with 0.1 mg/mL monosodium urate (MSU) crystals for 0, 1, 6, and 24 h. Data shown are pooled from three biological repeats and are presented as mean (SEM); two-way ANOVA P Interaction > 0.1 for all genes. OPG osteoprotegerin, RANKL receptor activator of nuclear factor kappa-B ligand, TNF tumor necrosis factor
    Figure Legend Snippet: Direct effects of MSU crystals on MLO-Y4 cell expression of bone-related or inflammatory genes. Real-time PCR was used to determine changes in the relative mRNA expression levels of a bone-related and b inflammatory genes in MLO-Y4 cells, following culture with 0.1 mg/mL monosodium urate (MSU) crystals for 0, 1, 6, and 24 h. Data shown are pooled from three biological repeats and are presented as mean (SEM); two-way ANOVA P Interaction > 0.1 for all genes. OPG osteoprotegerin, RANKL receptor activator of nuclear factor kappa-B ligand, TNF tumor necrosis factor

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Secretion of proinflammatory mediators by MLO-Y4 cells in response to MSU crystal-stimulated RAW264.7 macrophages. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. Conditioned medium preparations were added to MLO-Y4 cells (40% final concentration in a well) for 24 h and supernatants harvested. The concentrations of a tumor necrosis factor (TNF)-α, b prostaglandin E 2 (PGE 2 ), c interleukin (IL)-6, and d osteoprotegerin (OPG) protein in the RAW264.7 macrophage conditioned medium samples (control and MSU crystal-stimulated) and the MLO-Y4 cell supernatants were measured by ELISA. Data shown are pooled from three biological repeats and are presented as mean (SEM); one-way analysis of variance (ANOVA) with post-hoc Sidak’s test between groups as indicated. NS no significant difference
    Figure Legend Snippet: Secretion of proinflammatory mediators by MLO-Y4 cells in response to MSU crystal-stimulated RAW264.7 macrophages. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. Conditioned medium preparations were added to MLO-Y4 cells (40% final concentration in a well) for 24 h and supernatants harvested. The concentrations of a tumor necrosis factor (TNF)-α, b prostaglandin E 2 (PGE 2 ), c interleukin (IL)-6, and d osteoprotegerin (OPG) protein in the RAW264.7 macrophage conditioned medium samples (control and MSU crystal-stimulated) and the MLO-Y4 cell supernatants were measured by ELISA. Data shown are pooled from three biological repeats and are presented as mean (SEM); one-way analysis of variance (ANOVA) with post-hoc Sidak’s test between groups as indicated. NS no significant difference

    Techniques Used: Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Indirect effects of MSU crystal-stimulated RAW264.7 macrophage conditioned medium on MLO-Y4 cell gene expression. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. Conditioned medium preparations were added to MLO-Y4 cells (40% final concentration in a well) for 0, 1, 6, and 24 h. MLO-Y4 cells were harvested and real-time PCR was used to determine changes in the relative mRNA expression levels of a bone-related and b inflammatory genes. Data shown are pooled from three biological repeats and are presented as mean (SEM); two-way ANOVA P Interaction = 0.007 for Tnfrsf11b , P Interaction = 0.0005 for Tnfa , P Interaction
    Figure Legend Snippet: Indirect effects of MSU crystal-stimulated RAW264.7 macrophage conditioned medium on MLO-Y4 cell gene expression. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. Conditioned medium preparations were added to MLO-Y4 cells (40% final concentration in a well) for 0, 1, 6, and 24 h. MLO-Y4 cells were harvested and real-time PCR was used to determine changes in the relative mRNA expression levels of a bone-related and b inflammatory genes. Data shown are pooled from three biological repeats and are presented as mean (SEM); two-way ANOVA P Interaction = 0.007 for Tnfrsf11b , P Interaction = 0.0005 for Tnfa , P Interaction

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

    The direct effects of MSU crystals on osteocyte viability. The alamarBlue® assay was used to determine the viability of a MLO-Y4 cells and primary mouse osteocytes cultured with monosodium urate (MSU) crystals for 24 h, b MLO-Y4 cells cultured with soluble urate for 24 h, and c MLO-Y4 cells cultured with different types of crystals for 24 h. Viability was assessed 24 and 48 h after the addition of crystals or soluble urate. Data shown are pooled from three to four biological repeats and are presented as mean (SEM); by two-way ANOVA a P Interaction
    Figure Legend Snippet: The direct effects of MSU crystals on osteocyte viability. The alamarBlue® assay was used to determine the viability of a MLO-Y4 cells and primary mouse osteocytes cultured with monosodium urate (MSU) crystals for 24 h, b MLO-Y4 cells cultured with soluble urate for 24 h, and c MLO-Y4 cells cultured with different types of crystals for 24 h. Viability was assessed 24 and 48 h after the addition of crystals or soluble urate. Data shown are pooled from three to four biological repeats and are presented as mean (SEM); by two-way ANOVA a P Interaction

    Techniques Used: Alamar Blue Assay, Cell Culture

    3) Product Images from "Bone destruction by receptor activator of nuclear factor ?B ligand-expressing T cells in chronic gouty arthritis"

    Article Title: Bone destruction by receptor activator of nuclear factor ?B ligand-expressing T cells in chronic gouty arthritis

    Journal: Arthritis Research & Therapy

    doi: 10.1186/ar3483

    Expression of pro-resorptive cytokines by monosodium urate monohydrate (MSU) crystals . (a) Freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy control subjects were cultured for the indicated times (hours) in the presence of MSU crystals (100 μg/ml). (b) PBMCs from same donors were cultured for the indicated times (that is, 4 hours for cytokines and 72 hours for receptor activator of nuclear factor κB ligand RANKL) at various MSU crystal concentration (μg/ml). (c, d) Monocytes and T cells were isolated from PBMCs by magnetic-activated cell sorting. Freshly isolated monocytes and T cells from healthy control subjects were cultured for the indicated times (hours) in the presence of MSU crystals (100 μg/ml). Total RNA was collected at each time point and at the different MSU crystal concentrations. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to determine the expressions of interleukin (IL)-1α, IL-β, IL-6, tumor necrosis factor (TNF)-α, osteoprotegerin (OPG), and RANKL. Results are representative of three independent experiments.
    Figure Legend Snippet: Expression of pro-resorptive cytokines by monosodium urate monohydrate (MSU) crystals . (a) Freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy control subjects were cultured for the indicated times (hours) in the presence of MSU crystals (100 μg/ml). (b) PBMCs from same donors were cultured for the indicated times (that is, 4 hours for cytokines and 72 hours for receptor activator of nuclear factor κB ligand RANKL) at various MSU crystal concentration (μg/ml). (c, d) Monocytes and T cells were isolated from PBMCs by magnetic-activated cell sorting. Freshly isolated monocytes and T cells from healthy control subjects were cultured for the indicated times (hours) in the presence of MSU crystals (100 μg/ml). Total RNA was collected at each time point and at the different MSU crystal concentrations. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to determine the expressions of interleukin (IL)-1α, IL-β, IL-6, tumor necrosis factor (TNF)-α, osteoprotegerin (OPG), and RANKL. Results are representative of three independent experiments.

    Techniques Used: Expressing, Isolation, Cell Culture, Concentration Assay, FACS, Reverse Transcription Polymerase Chain Reaction

    4) Product Images from "Highlight Article: Phagocytosis of monosodium urate crystals by human synoviocytes induces inflammation"

    Article Title: Highlight Article: Phagocytosis of monosodium urate crystals by human synoviocytes induces inflammation

    Journal: Experimental Biology and Medicine

    doi: 10.1177/1535370219830665

    Gene expression of HIF1A , VHL , and VEGF in FLS. The cells were treated with MSU crystals (75 µg/mL) at 24 h, and gene expression was evaluated by qRT-PCR. Each point represents the mean value ± standard deviation of at least three independent experiments. * P value ≤0.05 compared to untreated FLS.
    Figure Legend Snippet: Gene expression of HIF1A , VHL , and VEGF in FLS. The cells were treated with MSU crystals (75 µg/mL) at 24 h, and gene expression was evaluated by qRT-PCR. Each point represents the mean value ± standard deviation of at least three independent experiments. * P value ≤0.05 compared to untreated FLS.

    Techniques Used: Expressing, Quantitative RT-PCR, Standard Deviation

    VEGF protein expression in FLS. The synoviocytes were treated with MSU crystals (75 µg/mL) at 24 h, and the protein expression was evaluated by ELISA. Each point represents the mean value ± standard deviation of at least three independent experiments. * P value ≤0.05 compared to untreated FLS (control).
    Figure Legend Snippet: VEGF protein expression in FLS. The synoviocytes were treated with MSU crystals (75 µg/mL) at 24 h, and the protein expression was evaluated by ELISA. Each point represents the mean value ± standard deviation of at least three independent experiments. * P value ≤0.05 compared to untreated FLS (control).

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Assessment of MSU-crystal phagocytosis. Rounded typical morphology of the adherent cells when it detached and suspended. (a) Unstimulated FLS. (b) The arrows point to two engulfed MSU crystal in a FLS. Representative images of > 20 fields from five independent experiments are shown. Original magnification ×40, polarized light microscopy. (A color version of this figure is available in the online journal).
    Figure Legend Snippet: Assessment of MSU-crystal phagocytosis. Rounded typical morphology of the adherent cells when it detached and suspended. (a) Unstimulated FLS. (b) The arrows point to two engulfed MSU crystal in a FLS. Representative images of > 20 fields from five independent experiments are shown. Original magnification ×40, polarized light microscopy. (A color version of this figure is available in the online journal).

    Techniques Used: Light Microscopy

    IL-1β production in FLS exposed to MSU-crystals. Columns show quantification of IL-1β secretion on FLS treated with MSU at different time. Quantification of IL-1β production was by microplate reader. Values are expressed as the mean ± standard deviation of at least three independent experiments. * P value ≤ 0.05 vs. control.
    Figure Legend Snippet: IL-1β production in FLS exposed to MSU-crystals. Columns show quantification of IL-1β secretion on FLS treated with MSU at different time. Quantification of IL-1β production was by microplate reader. Values are expressed as the mean ± standard deviation of at least three independent experiments. * P value ≤ 0.05 vs. control.

    Techniques Used: Standard Deviation

    Phagocytosis of MSU crystals by FLS. (a) Scanning electron micrograph of untreated FLS exhibiting cell nuclear (N), and vesicular structures (yellow arrows). (b) FLS treated with MSU crystals exhibiting swollen vesicular structures (yellow arrows), MSU crystal cavity (yellow asterisk) and cell nuclear (N) structures. Images are representative of one of five separate experiments with FLS from different patients. Phagocytosis analysis by flow cytometry. (c) Forward scatter, (d) Side scatter. Each bar represents the mean value ± standard deviation of at least three independent experiments. (A color version of this figure is available in the online journal).
    Figure Legend Snippet: Phagocytosis of MSU crystals by FLS. (a) Scanning electron micrograph of untreated FLS exhibiting cell nuclear (N), and vesicular structures (yellow arrows). (b) FLS treated with MSU crystals exhibiting swollen vesicular structures (yellow arrows), MSU crystal cavity (yellow asterisk) and cell nuclear (N) structures. Images are representative of one of five separate experiments with FLS from different patients. Phagocytosis analysis by flow cytometry. (c) Forward scatter, (d) Side scatter. Each bar represents the mean value ± standard deviation of at least three independent experiments. (A color version of this figure is available in the online journal).

    Techniques Used: Flow Cytometry, Standard Deviation

    Production of cytokines on FLS treated with MSU crystals. Cytokines were evaluated by the Milliplex Human Adipocyte Magnetic Panel. (a) Levels of IL-6, (b) TNF-α, (c) HGF, (d) IL-8, (e) MCP-1, (f) NGF. Each point represents the mean value ± standard deviation of at least three independent experiments. * P value ≤0.05 in comparison to control.
    Figure Legend Snippet: Production of cytokines on FLS treated with MSU crystals. Cytokines were evaluated by the Milliplex Human Adipocyte Magnetic Panel. (a) Levels of IL-6, (b) TNF-α, (c) HGF, (d) IL-8, (e) MCP-1, (f) NGF. Each point represents the mean value ± standard deviation of at least three independent experiments. * P value ≤0.05 in comparison to control.

    Techniques Used: Standard Deviation

    Dose response curve of PhIx of MSU crystal by FLS. Quantification of cells with internalized MSU crystals was determined by polarized light microscopy. Data are presented as mean value ± standard deviation of at least three independent experiments. * P value ≤0.05 in comparison to zero time. PhIx: phagocytic index.
    Figure Legend Snippet: Dose response curve of PhIx of MSU crystal by FLS. Quantification of cells with internalized MSU crystals was determined by polarized light microscopy. Data are presented as mean value ± standard deviation of at least three independent experiments. * P value ≤0.05 in comparison to zero time. PhIx: phagocytic index.

    Techniques Used: Light Microscopy, Standard Deviation

    5) Product Images from "CD44 Receptor Mediates Urate Crystal Phagocytosis by Macrophages and Regulates Inflammation in A Murine Peritoneal Model of Acute Gout"

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

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-62727-z

    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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: Flow Cytometry, Enzyme-linked Immunosorbent Assay

    6) Product Images from "Monosodium urate crystals reduce osteocyte viability and indirectly promote a shift in osteocyte function towards a proinflammatory and proresorptive state"

    Article Title: Monosodium urate crystals reduce osteocyte viability and indirectly promote a shift in osteocyte function towards a proinflammatory and proresorptive state

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-018-1704-y

    Effects of COX-2 inhibition on MLO-Y4 cell responses to MSU crystal-stimulated RAW264.7 macrophage conditioned medium. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. A cyclooxygenase-2 (COX-2)-specific inhibitor (SC-236) was added to MLO-Y4 cells for 1 h prior to the addition of 40% conditioned medium for 24 h. MLO-Y4 cells were then harvested for mRNA gene expression analysis and supernatants harvested for protein quantification. a Changes in mRNA expression of inflammatory genes: tumor necrosis factor (TNF)-α, COX-2, interleukin (IL)-6, and IL-11; and bone-related genes: receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG). b Changes in TNF-α, prostaglandin E 2 (PGE 2 ), and IL-6 protein levels in MLO-Y4 cell supernatants. Data shown are pooled from five biological repeats and are presented as (SEM); one-way analysis of variance (ANOVA) with post-hoc Sidak’s test between groups as indicated. NS no significant difference
    Figure Legend Snippet: Effects of COX-2 inhibition on MLO-Y4 cell responses to MSU crystal-stimulated RAW264.7 macrophage conditioned medium. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. A cyclooxygenase-2 (COX-2)-specific inhibitor (SC-236) was added to MLO-Y4 cells for 1 h prior to the addition of 40% conditioned medium for 24 h. MLO-Y4 cells were then harvested for mRNA gene expression analysis and supernatants harvested for protein quantification. a Changes in mRNA expression of inflammatory genes: tumor necrosis factor (TNF)-α, COX-2, interleukin (IL)-6, and IL-11; and bone-related genes: receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG). b Changes in TNF-α, prostaglandin E 2 (PGE 2 ), and IL-6 protein levels in MLO-Y4 cell supernatants. Data shown are pooled from five biological repeats and are presented as (SEM); one-way analysis of variance (ANOVA) with post-hoc Sidak’s test between groups as indicated. NS no significant difference

    Techniques Used: Inhibition, Cell Culture, Expressing

    The direct effects of MSU crystals on osteocyte viability. The alamarBlue® assay was used to determine the viability of a MLO-Y4 cells and primary mouse osteocytes cultured with monosodium urate (MSU) crystals for 24 h, b MLO-Y4 cells cultured with soluble urate for 24 h, and c MLO-Y4 cells cultured with different types of crystals for 24 h. Viability was assessed 24 and 48 h after the addition of crystals or soluble urate. Data shown are pooled from three to four biological repeats and are presented as mean (SEM); by two-way ANOVA a P Interaction
    Figure Legend Snippet: The direct effects of MSU crystals on osteocyte viability. The alamarBlue® assay was used to determine the viability of a MLO-Y4 cells and primary mouse osteocytes cultured with monosodium urate (MSU) crystals for 24 h, b MLO-Y4 cells cultured with soluble urate for 24 h, and c MLO-Y4 cells cultured with different types of crystals for 24 h. Viability was assessed 24 and 48 h after the addition of crystals or soluble urate. Data shown are pooled from three to four biological repeats and are presented as mean (SEM); by two-way ANOVA a P Interaction

    Techniques Used: Alamar Blue Assay, Cell Culture

    Direct effects of MSU crystals on MLO-Y4 cell expression of bone-related or inflammatory genes. Real-time PCR was used to determine changes in the relative mRNA expression levels of a bone-related and b inflammatory genes in MLO-Y4 cells, following culture with 0.1 mg/mL monosodium urate (MSU) crystals for 0, 1, 6, and 24 h. Data shown are pooled from three biological repeats and are presented as mean (SEM); two-way ANOVA P Interaction > 0.1 for all genes. OPG osteoprotegerin, RANKL receptor activator of nuclear factor kappa-B ligand, TNF tumor necrosis factor
    Figure Legend Snippet: Direct effects of MSU crystals on MLO-Y4 cell expression of bone-related or inflammatory genes. Real-time PCR was used to determine changes in the relative mRNA expression levels of a bone-related and b inflammatory genes in MLO-Y4 cells, following culture with 0.1 mg/mL monosodium urate (MSU) crystals for 0, 1, 6, and 24 h. Data shown are pooled from three biological repeats and are presented as mean (SEM); two-way ANOVA P Interaction > 0.1 for all genes. OPG osteoprotegerin, RANKL receptor activator of nuclear factor kappa-B ligand, TNF tumor necrosis factor

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Secretion of proinflammatory mediators by MLO-Y4 cells in response to MSU crystal-stimulated RAW264.7 macrophages. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. Conditioned medium preparations were added to MLO-Y4 cells (40% final concentration in a well) for 24 h and supernatants harvested. The concentrations of a tumor necrosis factor (TNF)-α, b prostaglandin E 2 (PGE 2 ), c interleukin (IL)-6, and d osteoprotegerin (OPG) protein in the RAW264.7 macrophage conditioned medium samples (control and MSU crystal-stimulated) and the MLO-Y4 cell supernatants were measured by ELISA. Data shown are pooled from three biological repeats and are presented as mean (SEM); one-way analysis of variance (ANOVA) with post-hoc Sidak’s test between groups as indicated. NS no significant difference
    Figure Legend Snippet: Secretion of proinflammatory mediators by MLO-Y4 cells in response to MSU crystal-stimulated RAW264.7 macrophages. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. Conditioned medium preparations were added to MLO-Y4 cells (40% final concentration in a well) for 24 h and supernatants harvested. The concentrations of a tumor necrosis factor (TNF)-α, b prostaglandin E 2 (PGE 2 ), c interleukin (IL)-6, and d osteoprotegerin (OPG) protein in the RAW264.7 macrophage conditioned medium samples (control and MSU crystal-stimulated) and the MLO-Y4 cell supernatants were measured by ELISA. Data shown are pooled from three biological repeats and are presented as mean (SEM); one-way analysis of variance (ANOVA) with post-hoc Sidak’s test between groups as indicated. NS no significant difference

    Techniques Used: Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Indirect effects of MSU crystal-stimulated RAW264.7 macrophage conditioned medium on MLO-Y4 cell gene expression. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. Conditioned medium preparations were added to MLO-Y4 cells (40% final concentration in a well) for 0, 1, 6, and 24 h. MLO-Y4 cells were harvested and real-time PCR was used to determine changes in the relative mRNA expression levels of a bone-related and b inflammatory genes. Data shown are pooled from three biological repeats and are presented as mean (SEM); two-way ANOVA P Interaction = 0.007 for Tnfrsf11b , P Interaction = 0.0005 for Tnfa , P Interaction
    Figure Legend Snippet: Indirect effects of MSU crystal-stimulated RAW264.7 macrophage conditioned medium on MLO-Y4 cell gene expression. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. Conditioned medium preparations were added to MLO-Y4 cells (40% final concentration in a well) for 0, 1, 6, and 24 h. MLO-Y4 cells were harvested and real-time PCR was used to determine changes in the relative mRNA expression levels of a bone-related and b inflammatory genes. Data shown are pooled from three biological repeats and are presented as mean (SEM); two-way ANOVA P Interaction = 0.007 for Tnfrsf11b , P Interaction = 0.0005 for Tnfa , P Interaction

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

    7) Product Images from "Monosodium urate crystals reduce osteocyte viability and indirectly promote a shift in osteocyte function towards a proinflammatory and proresorptive state"

    Article Title: Monosodium urate crystals reduce osteocyte viability and indirectly promote a shift in osteocyte function towards a proinflammatory and proresorptive state

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-018-1704-y

    Effects of COX-2 inhibition on MLO-Y4 cell responses to MSU crystal-stimulated RAW264.7 macrophage conditioned medium. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. A cyclooxygenase-2 (COX-2)-specific inhibitor (SC-236) was added to MLO-Y4 cells for 1 h prior to the addition of 40% conditioned medium for 24 h. MLO-Y4 cells were then harvested for mRNA gene expression analysis and supernatants harvested for protein quantification. a Changes in mRNA expression of inflammatory genes: tumor necrosis factor (TNF)-α, COX-2, interleukin (IL)-6, and IL-11; and bone-related genes: receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG). b Changes in TNF-α, prostaglandin E 2 (PGE 2 ), and IL-6 protein levels in MLO-Y4 cell supernatants. Data shown are pooled from five biological repeats and are presented as (SEM); one-way analysis of variance (ANOVA) with post-hoc Sidak’s test between groups as indicated. NS no significant difference
    Figure Legend Snippet: Effects of COX-2 inhibition on MLO-Y4 cell responses to MSU crystal-stimulated RAW264.7 macrophage conditioned medium. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. A cyclooxygenase-2 (COX-2)-specific inhibitor (SC-236) was added to MLO-Y4 cells for 1 h prior to the addition of 40% conditioned medium for 24 h. MLO-Y4 cells were then harvested for mRNA gene expression analysis and supernatants harvested for protein quantification. a Changes in mRNA expression of inflammatory genes: tumor necrosis factor (TNF)-α, COX-2, interleukin (IL)-6, and IL-11; and bone-related genes: receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG). b Changes in TNF-α, prostaglandin E 2 (PGE 2 ), and IL-6 protein levels in MLO-Y4 cell supernatants. Data shown are pooled from five biological repeats and are presented as (SEM); one-way analysis of variance (ANOVA) with post-hoc Sidak’s test between groups as indicated. NS no significant difference

    Techniques Used: Inhibition, Cell Culture, Expressing

    The direct effects of MSU crystals on osteocyte viability. The alamarBlue® assay was used to determine the viability of a MLO-Y4 cells and primary mouse osteocytes cultured with monosodium urate (MSU) crystals for 24 h, b MLO-Y4 cells cultured with soluble urate for 24 h, and c MLO-Y4 cells cultured with different types of crystals for 24 h. Viability was assessed 24 and 48 h after the addition of crystals or soluble urate. Data shown are pooled from three to four biological repeats and are presented as mean (SEM); by two-way ANOVA a P Interaction
    Figure Legend Snippet: The direct effects of MSU crystals on osteocyte viability. The alamarBlue® assay was used to determine the viability of a MLO-Y4 cells and primary mouse osteocytes cultured with monosodium urate (MSU) crystals for 24 h, b MLO-Y4 cells cultured with soluble urate for 24 h, and c MLO-Y4 cells cultured with different types of crystals for 24 h. Viability was assessed 24 and 48 h after the addition of crystals or soluble urate. Data shown are pooled from three to four biological repeats and are presented as mean (SEM); by two-way ANOVA a P Interaction

    Techniques Used: Alamar Blue Assay, Cell Culture

    Direct effects of MSU crystals on MLO-Y4 cell expression of bone-related or inflammatory genes. Real-time PCR was used to determine changes in the relative mRNA expression levels of a bone-related and b inflammatory genes in MLO-Y4 cells, following culture with 0.1 mg/mL monosodium urate (MSU) crystals for 0, 1, 6, and 24 h. Data shown are pooled from three biological repeats and are presented as mean (SEM); two-way ANOVA P Interaction > 0.1 for all genes. OPG osteoprotegerin, RANKL receptor activator of nuclear factor kappa-B ligand, TNF tumor necrosis factor
    Figure Legend Snippet: Direct effects of MSU crystals on MLO-Y4 cell expression of bone-related or inflammatory genes. Real-time PCR was used to determine changes in the relative mRNA expression levels of a bone-related and b inflammatory genes in MLO-Y4 cells, following culture with 0.1 mg/mL monosodium urate (MSU) crystals for 0, 1, 6, and 24 h. Data shown are pooled from three biological repeats and are presented as mean (SEM); two-way ANOVA P Interaction > 0.1 for all genes. OPG osteoprotegerin, RANKL receptor activator of nuclear factor kappa-B ligand, TNF tumor necrosis factor

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Secretion of proinflammatory mediators by MLO-Y4 cells in response to MSU crystal-stimulated RAW264.7 macrophages. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. Conditioned medium preparations were added to MLO-Y4 cells (40% final concentration in a well) for 24 h and supernatants harvested. The concentrations of a tumor necrosis factor (TNF)-α, b prostaglandin E 2 (PGE 2 ), c interleukin (IL)-6, and d osteoprotegerin (OPG) protein in the RAW264.7 macrophage conditioned medium samples (control and MSU crystal-stimulated) and the MLO-Y4 cell supernatants were measured by ELISA. Data shown are pooled from three biological repeats and are presented as mean (SEM); one-way analysis of variance (ANOVA) with post-hoc Sidak’s test between groups as indicated. NS no significant difference
    Figure Legend Snippet: Secretion of proinflammatory mediators by MLO-Y4 cells in response to MSU crystal-stimulated RAW264.7 macrophages. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. Conditioned medium preparations were added to MLO-Y4 cells (40% final concentration in a well) for 24 h and supernatants harvested. The concentrations of a tumor necrosis factor (TNF)-α, b prostaglandin E 2 (PGE 2 ), c interleukin (IL)-6, and d osteoprotegerin (OPG) protein in the RAW264.7 macrophage conditioned medium samples (control and MSU crystal-stimulated) and the MLO-Y4 cell supernatants were measured by ELISA. Data shown are pooled from three biological repeats and are presented as mean (SEM); one-way analysis of variance (ANOVA) with post-hoc Sidak’s test between groups as indicated. NS no significant difference

    Techniques Used: Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Indirect effects of MSU crystal-stimulated RAW264.7 macrophage conditioned medium on MLO-Y4 cell gene expression. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. Conditioned medium preparations were added to MLO-Y4 cells (40% final concentration in a well) for 0, 1, 6, and 24 h. MLO-Y4 cells were harvested and real-time PCR was used to determine changes in the relative mRNA expression levels of a bone-related and b inflammatory genes. Data shown are pooled from three biological repeats and are presented as mean (SEM); two-way ANOVA P Interaction = 0.007 for Tnfrsf11b , P Interaction = 0.0005 for Tnfa , P Interaction
    Figure Legend Snippet: Indirect effects of MSU crystal-stimulated RAW264.7 macrophage conditioned medium on MLO-Y4 cell gene expression. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. Conditioned medium preparations were added to MLO-Y4 cells (40% final concentration in a well) for 0, 1, 6, and 24 h. MLO-Y4 cells were harvested and real-time PCR was used to determine changes in the relative mRNA expression levels of a bone-related and b inflammatory genes. Data shown are pooled from three biological repeats and are presented as mean (SEM); two-way ANOVA P Interaction = 0.007 for Tnfrsf11b , P Interaction = 0.0005 for Tnfa , P Interaction

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

    The direct effects of MSU crystals on osteocyte viability. The alamarBlue® assay was used to determine the viability of a MLO-Y4 cells and primary mouse osteocytes cultured with monosodium urate (MSU) crystals for 24 h, b MLO-Y4 cells cultured with soluble urate for 24 h, and c MLO-Y4 cells cultured with different types of crystals for 24 h. Viability was assessed 24 and 48 h after the addition of crystals or soluble urate. Data shown are pooled from three to four biological repeats and are presented as mean (SEM); by two-way ANOVA a P Interaction
    Figure Legend Snippet: The direct effects of MSU crystals on osteocyte viability. The alamarBlue® assay was used to determine the viability of a MLO-Y4 cells and primary mouse osteocytes cultured with monosodium urate (MSU) crystals for 24 h, b MLO-Y4 cells cultured with soluble urate for 24 h, and c MLO-Y4 cells cultured with different types of crystals for 24 h. Viability was assessed 24 and 48 h after the addition of crystals or soluble urate. Data shown are pooled from three to four biological repeats and are presented as mean (SEM); by two-way ANOVA a P Interaction

    Techniques Used: Alamar Blue Assay, Cell Culture

    8) Product Images from "Monosodium Urate in the Presence of RANKL Promotes Osteoclast Formation through Activation of c-Jun N-Terminal Kinase"

    Article Title: Monosodium Urate in the Presence of RANKL Promotes Osteoclast Formation through Activation of c-Jun N-Terminal Kinase

    Journal: Mediators of Inflammation

    doi: 10.1155/2015/597512

    Induction of osteoclast differentiation through activation of TRAF-6 and JNK. (a) The protein expression of cytoplasmic molecules of the RANKL-RANK signaling pathway was assessed in RAW 264.7 cells alone (controls) and experimental cells incubated with MSU crystals alone (0.1 mg/mL), RANKL alone (100 ng/mL), or both for 1 h. (b) Phospho-c-Jun and NFATc1 production was measured in RAW 264.7 cells alone (controls) and experimental cells incubated with MSU crystals alone (0.1 mg/mL), RANKL alone (100 ng/mL), or both for 24 h. (c) The levels of phospho-c-Jun and NFATc1 protein were measured using a JNK inhibitor (SP600125), p38 inhibitor (SB203580), and ERK inhibitor (U0126) at dosages of 10 and 20 μ M. (d) TRAF-6 mRNA expression was assessed in RAW 264.7 cells alone (controls) and cells transfected with negative siRNA and TRAF-6 siRNA at different dosages (0, 30, 50, and 100 ng/mL) and incubation times (24, 48, and 72 h) in the presence and absence of both MSU crystals (0.1 mg/mL) and RANKL (100 ng/mL). ∗ p
    Figure Legend Snippet: Induction of osteoclast differentiation through activation of TRAF-6 and JNK. (a) The protein expression of cytoplasmic molecules of the RANKL-RANK signaling pathway was assessed in RAW 264.7 cells alone (controls) and experimental cells incubated with MSU crystals alone (0.1 mg/mL), RANKL alone (100 ng/mL), or both for 1 h. (b) Phospho-c-Jun and NFATc1 production was measured in RAW 264.7 cells alone (controls) and experimental cells incubated with MSU crystals alone (0.1 mg/mL), RANKL alone (100 ng/mL), or both for 24 h. (c) The levels of phospho-c-Jun and NFATc1 protein were measured using a JNK inhibitor (SP600125), p38 inhibitor (SB203580), and ERK inhibitor (U0126) at dosages of 10 and 20 μ M. (d) TRAF-6 mRNA expression was assessed in RAW 264.7 cells alone (controls) and cells transfected with negative siRNA and TRAF-6 siRNA at different dosages (0, 30, 50, and 100 ng/mL) and incubation times (24, 48, and 72 h) in the presence and absence of both MSU crystals (0.1 mg/mL) and RANKL (100 ng/mL). ∗ p

    Techniques Used: Activation Assay, Expressing, Incubation, Transfection

    9) Product Images from "Morin, a Bioflavonoid Suppresses Monosodium Urate Crystal-Induced Inflammatory Immune Response in RAW 264.7 Macrophages through the Inhibition of Inflammatory Mediators, Intracellular ROS Levels and NF-κB Activation"

    Article Title: Morin, a Bioflavonoid Suppresses Monosodium Urate Crystal-Induced Inflammatory Immune Response in RAW 264.7 Macrophages through the Inhibition of Inflammatory Mediators, Intracellular ROS Levels and NF-κB Activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0145093

    Effect of morin on MSU crystal induced activation of NF-κBp65, COX-2 and TNF-α in RAW 264.7 macrophage cells. RAW 264.7 macrophage cells were treated with varying concentrations of morin (100–300 μM/ml) for 24 h and BAY 11–7028 (10 μM) for 1 h prior to the stimulation of MSU (1 mg/ml) for 24 h. Western blotting analysis was performed as described in materials and methods (i) The expression and translocation of NF-κBp65 protein were evaluated by measuring protein levels in the cytosolic and nuclear cell fractions (ii) COX-2 and TNF-α protein expression were evaluated in the whole cell lysates. β-actin and Lamin A were used as an internal loading control. The values are expressed as mean ± SEM of three independent experiments. Comparisons were made as follows: a—Control versus MSU (1mg/ml) stimulated, MSU+ morin treated groups (100–300 μM), MSU + Colchicine (1 μM); b—MSU stimulated versus MSU + morin (100–300 μM), MSU + Colchicine (1μM). * P
    Figure Legend Snippet: Effect of morin on MSU crystal induced activation of NF-κBp65, COX-2 and TNF-α in RAW 264.7 macrophage cells. RAW 264.7 macrophage cells were treated with varying concentrations of morin (100–300 μM/ml) for 24 h and BAY 11–7028 (10 μM) for 1 h prior to the stimulation of MSU (1 mg/ml) for 24 h. Western blotting analysis was performed as described in materials and methods (i) The expression and translocation of NF-κBp65 protein were evaluated by measuring protein levels in the cytosolic and nuclear cell fractions (ii) COX-2 and TNF-α protein expression were evaluated in the whole cell lysates. β-actin and Lamin A were used as an internal loading control. The values are expressed as mean ± SEM of three independent experiments. Comparisons were made as follows: a—Control versus MSU (1mg/ml) stimulated, MSU+ morin treated groups (100–300 μM), MSU + Colchicine (1 μM); b—MSU stimulated versus MSU + morin (100–300 μM), MSU + Colchicine (1μM). * P

    Techniques Used: Activation Assay, Western Blot, Expressing, Translocation Assay

    10) Product Images from "Monosodium urate crystals reduce osteocyte viability and indirectly promote a shift in osteocyte function towards a proinflammatory and proresorptive state"

    Article Title: Monosodium urate crystals reduce osteocyte viability and indirectly promote a shift in osteocyte function towards a proinflammatory and proresorptive state

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-018-1704-y

    Effects of COX-2 inhibition on MLO-Y4 cell responses to MSU crystal-stimulated RAW264.7 macrophage conditioned medium. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. A cyclooxygenase-2 (COX-2)-specific inhibitor (SC-236) was added to MLO-Y4 cells for 1 h prior to the addition of 40% conditioned medium for 24 h. MLO-Y4 cells were then harvested for mRNA gene expression analysis and supernatants harvested for protein quantification. a Changes in mRNA expression of inflammatory genes: tumor necrosis factor (TNF)-α, COX-2, interleukin (IL)-6, and IL-11; and bone-related genes: receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG). b Changes in TNF-α, prostaglandin E 2 (PGE 2 ), and IL-6 protein levels in MLO-Y4 cell supernatants. Data shown are pooled from five biological repeats and are presented as (SEM); one-way analysis of variance (ANOVA) with post-hoc Sidak’s test between groups as indicated. NS no significant difference
    Figure Legend Snippet: Effects of COX-2 inhibition on MLO-Y4 cell responses to MSU crystal-stimulated RAW264.7 macrophage conditioned medium. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. A cyclooxygenase-2 (COX-2)-specific inhibitor (SC-236) was added to MLO-Y4 cells for 1 h prior to the addition of 40% conditioned medium for 24 h. MLO-Y4 cells were then harvested for mRNA gene expression analysis and supernatants harvested for protein quantification. a Changes in mRNA expression of inflammatory genes: tumor necrosis factor (TNF)-α, COX-2, interleukin (IL)-6, and IL-11; and bone-related genes: receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG). b Changes in TNF-α, prostaglandin E 2 (PGE 2 ), and IL-6 protein levels in MLO-Y4 cell supernatants. Data shown are pooled from five biological repeats and are presented as (SEM); one-way analysis of variance (ANOVA) with post-hoc Sidak’s test between groups as indicated. NS no significant difference

    Techniques Used: Inhibition, Cell Culture, Expressing

    The direct effects of MSU crystals on osteocyte viability. The alamarBlue® assay was used to determine the viability of a MLO-Y4 cells and primary mouse osteocytes cultured with monosodium urate (MSU) crystals for 24 h, b MLO-Y4 cells cultured with soluble urate for 24 h, and c MLO-Y4 cells cultured with different types of crystals for 24 h. Viability was assessed 24 and 48 h after the addition of crystals or soluble urate. Data shown are pooled from three to four biological repeats and are presented as mean (SEM); by two-way ANOVA a P Interaction
    Figure Legend Snippet: The direct effects of MSU crystals on osteocyte viability. The alamarBlue® assay was used to determine the viability of a MLO-Y4 cells and primary mouse osteocytes cultured with monosodium urate (MSU) crystals for 24 h, b MLO-Y4 cells cultured with soluble urate for 24 h, and c MLO-Y4 cells cultured with different types of crystals for 24 h. Viability was assessed 24 and 48 h after the addition of crystals or soluble urate. Data shown are pooled from three to four biological repeats and are presented as mean (SEM); by two-way ANOVA a P Interaction

    Techniques Used: Alamar Blue Assay, Cell Culture

    Direct effects of MSU crystals on MLO-Y4 cell expression of bone-related or inflammatory genes. Real-time PCR was used to determine changes in the relative mRNA expression levels of a bone-related and b inflammatory genes in MLO-Y4 cells, following culture with 0.1 mg/mL monosodium urate (MSU) crystals for 0, 1, 6, and 24 h. Data shown are pooled from three biological repeats and are presented as mean (SEM); two-way ANOVA P Interaction > 0.1 for all genes. OPG osteoprotegerin, RANKL receptor activator of nuclear factor kappa-B ligand, TNF tumor necrosis factor
    Figure Legend Snippet: Direct effects of MSU crystals on MLO-Y4 cell expression of bone-related or inflammatory genes. Real-time PCR was used to determine changes in the relative mRNA expression levels of a bone-related and b inflammatory genes in MLO-Y4 cells, following culture with 0.1 mg/mL monosodium urate (MSU) crystals for 0, 1, 6, and 24 h. Data shown are pooled from three biological repeats and are presented as mean (SEM); two-way ANOVA P Interaction > 0.1 for all genes. OPG osteoprotegerin, RANKL receptor activator of nuclear factor kappa-B ligand, TNF tumor necrosis factor

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Secretion of proinflammatory mediators by MLO-Y4 cells in response to MSU crystal-stimulated RAW264.7 macrophages. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. Conditioned medium preparations were added to MLO-Y4 cells (40% final concentration in a well) for 24 h and supernatants harvested. The concentrations of a tumor necrosis factor (TNF)-α, b prostaglandin E 2 (PGE 2 ), c interleukin (IL)-6, and d osteoprotegerin (OPG) protein in the RAW264.7 macrophage conditioned medium samples (control and MSU crystal-stimulated) and the MLO-Y4 cell supernatants were measured by ELISA. Data shown are pooled from three biological repeats and are presented as mean (SEM); one-way analysis of variance (ANOVA) with post-hoc Sidak’s test between groups as indicated. NS no significant difference
    Figure Legend Snippet: Secretion of proinflammatory mediators by MLO-Y4 cells in response to MSU crystal-stimulated RAW264.7 macrophages. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. Conditioned medium preparations were added to MLO-Y4 cells (40% final concentration in a well) for 24 h and supernatants harvested. The concentrations of a tumor necrosis factor (TNF)-α, b prostaglandin E 2 (PGE 2 ), c interleukin (IL)-6, and d osteoprotegerin (OPG) protein in the RAW264.7 macrophage conditioned medium samples (control and MSU crystal-stimulated) and the MLO-Y4 cell supernatants were measured by ELISA. Data shown are pooled from three biological repeats and are presented as mean (SEM); one-way analysis of variance (ANOVA) with post-hoc Sidak’s test between groups as indicated. NS no significant difference

    Techniques Used: Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Indirect effects of MSU crystal-stimulated RAW264.7 macrophage conditioned medium on MLO-Y4 cell gene expression. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. Conditioned medium preparations were added to MLO-Y4 cells (40% final concentration in a well) for 0, 1, 6, and 24 h. MLO-Y4 cells were harvested and real-time PCR was used to determine changes in the relative mRNA expression levels of a bone-related and b inflammatory genes. Data shown are pooled from three biological repeats and are presented as mean (SEM); two-way ANOVA P Interaction = 0.007 for Tnfrsf11b , P Interaction = 0.0005 for Tnfa , P Interaction
    Figure Legend Snippet: Indirect effects of MSU crystal-stimulated RAW264.7 macrophage conditioned medium on MLO-Y4 cell gene expression. RAW264.7 macrophages were cultured with or without 0.5 mg/mL monosodium urate (MSU) crystals for 24 h for preparation of MSU crystal-stimulated conditioned medium and control conditioned medium, respectively. Conditioned medium preparations were added to MLO-Y4 cells (40% final concentration in a well) for 0, 1, 6, and 24 h. MLO-Y4 cells were harvested and real-time PCR was used to determine changes in the relative mRNA expression levels of a bone-related and b inflammatory genes. Data shown are pooled from three biological repeats and are presented as mean (SEM); two-way ANOVA P Interaction = 0.007 for Tnfrsf11b , P Interaction = 0.0005 for Tnfa , P Interaction

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

    11) Product Images from "The metastatic tumor antigen 1-transglutaminase-2 pathway is involved in self-limitation of monosodium urate crystal-induced inflammation by upregulating TGF-β1"

    Article Title: The metastatic tumor antigen 1-transglutaminase-2 pathway is involved in self-limitation of monosodium urate crystal-induced inflammation by upregulating TGF-β1

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-015-0592-7

    Effect of metastatic tumor antigen 1 (MTA1) knockdown on monosodium urate (MSU) crystal-induced cytokine production in RAW264.7 cells. (A) Time course of the MSU crystal (1 mg/ml) induced increase in MTA1 and TG2 mRNA expression in RAW264.7 detected by RT-PCR. Quantification of RT-PCR analysis of MTA1 and TG2 mRNA expressions is also shown. (B) RT-PCR analysis of RAW264.7 cells transfected with MTA1 specific small interfering RNAs (siRNAs) for 48 h and stimulated then with or without MSU crystals (1 mg/ml) for 4 h. Quantification of RT-PCR analysis of MTA1, TG2, TGF-β1, IL-1β and TNF-α mRNA expressions. (C) Cells were stimulated with or without MSU crystals (1 mg/ml) for 36 h. The levels of TGF-β1, IL-1β and TNF-α in supernatants were collected and evaluated by ELISA. (D) In addition, MTA1 knockdown macrophages were also treated by MSU crystals (1 mg/ml) in the presence of recombinant TG2 (10 ng/ml) or IgG (10 ng/ml) for 36 h. The levels of TGF-β1, IL-1β and TNF-α in supernatants were collected and evaluated by ELISA. The results are expressed as the means ± SEM from three independent experiments (n ≧ 3, * = P
    Figure Legend Snippet: Effect of metastatic tumor antigen 1 (MTA1) knockdown on monosodium urate (MSU) crystal-induced cytokine production in RAW264.7 cells. (A) Time course of the MSU crystal (1 mg/ml) induced increase in MTA1 and TG2 mRNA expression in RAW264.7 detected by RT-PCR. Quantification of RT-PCR analysis of MTA1 and TG2 mRNA expressions is also shown. (B) RT-PCR analysis of RAW264.7 cells transfected with MTA1 specific small interfering RNAs (siRNAs) for 48 h and stimulated then with or without MSU crystals (1 mg/ml) for 4 h. Quantification of RT-PCR analysis of MTA1, TG2, TGF-β1, IL-1β and TNF-α mRNA expressions. (C) Cells were stimulated with or without MSU crystals (1 mg/ml) for 36 h. The levels of TGF-β1, IL-1β and TNF-α in supernatants were collected and evaluated by ELISA. (D) In addition, MTA1 knockdown macrophages were also treated by MSU crystals (1 mg/ml) in the presence of recombinant TG2 (10 ng/ml) or IgG (10 ng/ml) for 36 h. The levels of TGF-β1, IL-1β and TNF-α in supernatants were collected and evaluated by ELISA. The results are expressed as the means ± SEM from three independent experiments (n ≧ 3, * = P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Enzyme-linked Immunosorbent Assay, Recombinant

    Related Articles

    Flow Cytometry:

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    Article Snippet: .. Flow cytometry For analysis of surface markers, cells were stained in PBS containing 2% (wt/vol) BSA, with anti-CD4 (RM4-5), anti-CD8α (53-6.7), anti-TCRβ (H57-597), anti-CD69 (H1.2F3), anti-CD25 (PC61.5), anti-CD44 (1M7), anti-CD62L (MEL-14), anti-CD45.1 (A20), anti-CD45.2 (104), anti-PD-1 (J43), anti-GL7 (GL-7), anti-CD95 (15A7), anti-ICOS (C398.4A), anti-GITR (DTA-1), anti-CD19 (1D3), anti-CXCR3 (CXCR3-173), anti-MHCII (M5/114.15.2), anti-CD11b (M1/70), anti-CD11c (N418), anti-Ly6G (RB6-8C5; all from eBioscience). .. CXCR5 was stained with biotinylated anti-CXCR5 (clone 2G8) and streptavid in-conjugated PE (both from BD Biosciences).

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    Article Snippet: .. Alternatively, CD3+ cells were isolated from spleen by a negative selection kit (Invitrogen Dynal) and stained with the Db -GagL tetramers and anti-CD8, anti-CD62L (Becton Dickinson), anti-CD44, anti-CD127 and anti-CD25 mAbs (eBioscience) and analyzed by flow cytometry. .. IFN-γ production in CD3+ cells cultured in the presence of PMA-ionomycin and brefeldin A was measured by using the intracellular staining kit (Cytofix/Cytoperm Fixation/Permeabilization, Becton Dickinson) according to the supplier's protocol.

    Selection:

    Article Title: A Crucial Role for Infected-Cell/Antibody Immune Complexes in the Enhancement of Endogenous Antiviral Immunity by Short Passive Immunotherapy
    Article Snippet: .. Alternatively, CD3+ cells were isolated from spleen by a negative selection kit (Invitrogen Dynal) and stained with the Db -GagL tetramers and anti-CD8, anti-CD62L (Becton Dickinson), anti-CD44, anti-CD127 and anti-CD25 mAbs (eBioscience) and analyzed by flow cytometry. .. IFN-γ production in CD3+ cells cultured in the presence of PMA-ionomycin and brefeldin A was measured by using the intracellular staining kit (Cytofix/Cytoperm Fixation/Permeabilization, Becton Dickinson) according to the supplier's protocol.

    In Vitro:

    Article Title: Transforming growth factor-? signaling to T cells inhibits autoimmunity during lymphopenia-driven proliferation
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    Isolation:

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    Article Title: A Crucial Role for Infected-Cell/Antibody Immune Complexes in the Enhancement of Endogenous Antiviral Immunity by Short Passive Immunotherapy
    Article Snippet: .. Alternatively, CD3+ cells were isolated from spleen by a negative selection kit (Invitrogen Dynal) and stained with the Db -GagL tetramers and anti-CD8, anti-CD62L (Becton Dickinson), anti-CD44, anti-CD127 and anti-CD25 mAbs (eBioscience) and analyzed by flow cytometry. .. IFN-γ production in CD3+ cells cultured in the presence of PMA-ionomycin and brefeldin A was measured by using the intracellular staining kit (Cytofix/Cytoperm Fixation/Permeabilization, Becton Dickinson) according to the supplier's protocol.

    Cytometry:

    Article Title: Regulatory T cells require the phosphatase PTEN to restrain type 1 and follicular helper T-cell responses
    Article Snippet: .. Flow cytometry For analysis of surface markers, cells were stained in PBS containing 2% (wt/vol) BSA, with anti-CD4 (RM4-5), anti-CD8α (53-6.7), anti-TCRβ (H57-597), anti-CD69 (H1.2F3), anti-CD25 (PC61.5), anti-CD44 (1M7), anti-CD62L (MEL-14), anti-CD45.1 (A20), anti-CD45.2 (104), anti-PD-1 (J43), anti-GL7 (GL-7), anti-CD95 (15A7), anti-ICOS (C398.4A), anti-GITR (DTA-1), anti-CD19 (1D3), anti-CXCR3 (CXCR3-173), anti-MHCII (M5/114.15.2), anti-CD11b (M1/70), anti-CD11c (N418), anti-Ly6G (RB6-8C5; all from eBioscience). .. CXCR5 was stained with biotinylated anti-CXCR5 (clone 2G8) and streptavid in-conjugated PE (both from BD Biosciences).

    Article Title: A Crucial Role for Infected-Cell/Antibody Immune Complexes in the Enhancement of Endogenous Antiviral Immunity by Short Passive Immunotherapy
    Article Snippet: .. Alternatively, CD3+ cells were isolated from spleen by a negative selection kit (Invitrogen Dynal) and stained with the Db -GagL tetramers and anti-CD8, anti-CD62L (Becton Dickinson), anti-CD44, anti-CD127 and anti-CD25 mAbs (eBioscience) and analyzed by flow cytometry. .. IFN-γ production in CD3+ cells cultured in the presence of PMA-ionomycin and brefeldin A was measured by using the intracellular staining kit (Cytofix/Cytoperm Fixation/Permeabilization, Becton Dickinson) according to the supplier's protocol.

    Labeling:

    Article Title: Transforming growth factor-? signaling to T cells inhibits autoimmunity during lymphopenia-driven proliferation
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    Purification:

    Article Title: Transforming growth factor-? signaling to T cells inhibits autoimmunity during lymphopenia-driven proliferation
    Article Snippet: .. In vitro proliferation Naive OT-1 T cells were purified using a CD8 isolation kit (Miltenyi) with supplemental anti-CD44, and labeled with 5 μM CFSE (Invitrogen). .. T cell and NK cell depleted splenocytes were purified by a similar procedure as OT-1 T cells with an antibody mixture containing biotin-αTCRβ (H57-597), biotin-αTCRγδ (eBioGL3) and biotin-αNK1.1 (pk136) and used as APCs.

    other:

    Article Title: Osteopontin and iCD8α cells promote intestinal intraepithelial lymphocyte homeostasis
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    Cell Culture:

    Article Title: Osteopontin and iCD8α cells promote intestinal intraepithelial lymphocyte homeostasis
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    Staining:

    Article Title: Regulatory T cells require the phosphatase PTEN to restrain type 1 and follicular helper T-cell responses
    Article Snippet: .. Flow cytometry For analysis of surface markers, cells were stained in PBS containing 2% (wt/vol) BSA, with anti-CD4 (RM4-5), anti-CD8α (53-6.7), anti-TCRβ (H57-597), anti-CD69 (H1.2F3), anti-CD25 (PC61.5), anti-CD44 (1M7), anti-CD62L (MEL-14), anti-CD45.1 (A20), anti-CD45.2 (104), anti-PD-1 (J43), anti-GL7 (GL-7), anti-CD95 (15A7), anti-ICOS (C398.4A), anti-GITR (DTA-1), anti-CD19 (1D3), anti-CXCR3 (CXCR3-173), anti-MHCII (M5/114.15.2), anti-CD11b (M1/70), anti-CD11c (N418), anti-Ly6G (RB6-8C5; all from eBioscience). .. CXCR5 was stained with biotinylated anti-CXCR5 (clone 2G8) and streptavid in-conjugated PE (both from BD Biosciences).

    Article Title: A Crucial Role for Infected-Cell/Antibody Immune Complexes in the Enhancement of Endogenous Antiviral Immunity by Short Passive Immunotherapy
    Article Snippet: .. Alternatively, CD3+ cells were isolated from spleen by a negative selection kit (Invitrogen Dynal) and stained with the Db -GagL tetramers and anti-CD8, anti-CD62L (Becton Dickinson), anti-CD44, anti-CD127 and anti-CD25 mAbs (eBioscience) and analyzed by flow cytometry. .. IFN-γ production in CD3+ cells cultured in the presence of PMA-ionomycin and brefeldin A was measured by using the intracellular staining kit (Cytofix/Cytoperm Fixation/Permeabilization, Becton Dickinson) according to the supplier's protocol.

    FACS:

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    Article Snippet: .. Flow cytometric cell sorting of the polyclonal T cell subsets was then performed using GFP as well as anti-CD44, -CD73, -FR4, and -CD25 antibodies, together with other non-T cell markers (B220, F4/80, CD8α, NK1.1, CD11c, and CD11b) as ‘dump’ reagents (eBiosciences, Inc, San Diego, CA). .. Cells were physically sorted on a BD FACSAria (BD Biosciences, San Jose, CA) at the University of Minnesota Flow Cytometry Core Facility.

    Mouse Assay:

    Article Title: Osteopontin and iCD8α cells promote intestinal intraepithelial lymphocyte homeostasis
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    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 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 (500,000 cells per well) were incubated with MSU crystals (100 μg/mL) for 3, 6, 12 and 24 hours followed by media collections and cell harvest to perform CD44 gene expression studies as described above using commercially available primers and probes for CD44 (Hs01075864_m1) and GAPDH (Hs03929097_g1) (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 (500,000 cells per well) were incubated with MSU crystals (100 μg/mL) for 3, 6, 12 and 24 hours followed by media collections and cell harvest to perform CD44 gene expression studies as described above using commercially available primers and probes for CD44 (Hs01075864_m1) and GAPDH (Hs03929097_g1) (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 (500,000 cells per well) were incubated with MSU crystals (100 μg/mL) for 3, 6, 12 and 24 hours followed by media collections and cell harvest to perform CD44 gene expression studies as described above using commercially available primers and probes for CD44 (Hs01075864_m1) and GAPDH (Hs03929097_g1) (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

    Induction of osteoclast differentiation through activation of TRAF-6 and JNK. (a) The protein expression of cytoplasmic molecules of the RANKL-RANK signaling pathway was assessed in RAW 264.7 cells alone (controls) and experimental cells incubated with MSU crystals alone (0.1 mg/mL), RANKL alone (100 ng/mL), or both for 1 h. (b) Phospho-c-Jun and NFATc1 production was measured in RAW 264.7 cells alone (controls) and experimental cells incubated with MSU crystals alone (0.1 mg/mL), RANKL alone (100 ng/mL), or both for 24 h. (c) The levels of phospho-c-Jun and NFATc1 protein were measured using a JNK inhibitor (SP600125), p38 inhibitor (SB203580), and ERK inhibitor (U0126) at dosages of 10 and 20 μ M. (d) TRAF-6 mRNA expression was assessed in RAW 264.7 cells alone (controls) and cells transfected with negative siRNA and TRAF-6 siRNA at different dosages (0, 30, 50, and 100 ng/mL) and incubation times (24, 48, and 72 h) in the presence and absence of both MSU crystals (0.1 mg/mL) and RANKL (100 ng/mL). ∗ p

    Journal: Mediators of Inflammation

    Article Title: Monosodium Urate in the Presence of RANKL Promotes Osteoclast Formation through Activation of c-Jun N-Terminal Kinase

    doi: 10.1155/2015/597512

    Figure Lengend Snippet: Induction of osteoclast differentiation through activation of TRAF-6 and JNK. (a) The protein expression of cytoplasmic molecules of the RANKL-RANK signaling pathway was assessed in RAW 264.7 cells alone (controls) and experimental cells incubated with MSU crystals alone (0.1 mg/mL), RANKL alone (100 ng/mL), or both for 1 h. (b) Phospho-c-Jun and NFATc1 production was measured in RAW 264.7 cells alone (controls) and experimental cells incubated with MSU crystals alone (0.1 mg/mL), RANKL alone (100 ng/mL), or both for 24 h. (c) The levels of phospho-c-Jun and NFATc1 protein were measured using a JNK inhibitor (SP600125), p38 inhibitor (SB203580), and ERK inhibitor (U0126) at dosages of 10 and 20 μ M. (d) TRAF-6 mRNA expression was assessed in RAW 264.7 cells alone (controls) and cells transfected with negative siRNA and TRAF-6 siRNA at different dosages (0, 30, 50, and 100 ng/mL) and incubation times (24, 48, and 72 h) in the presence and absence of both MSU crystals (0.1 mg/mL) and RANKL (100 ng/mL). ∗ p

    Article Snippet: Cells (4 × 106 ) pretreated with specific mitogen-activated protein kinase (MAPK) inhibitors such as U0126 for ERK, SP600125 for JNK, and SB203580 for p38 (Sigma, St. Louis, MO, USA) for 1 h were incubated with MSU crystals (0.1 mg/mL) alone, RNAKL (100 ng/mL) alone, or both for 72 h. Proteins were detected with the SuperSignal West Pico chemiluminescent kit (Thermo Scientific, Rockford, IL, USA).

    Techniques: Activation Assay, Expressing, Incubation, Transfection

    Effect of morin on MSU crystal induced activation of NF-κBp65, COX-2 and TNF-α in RAW 264.7 macrophage cells. RAW 264.7 macrophage cells were treated with varying concentrations of morin (100–300 μM/ml) for 24 h and BAY 11–7028 (10 μM) for 1 h prior to the stimulation of MSU (1 mg/ml) for 24 h. Western blotting analysis was performed as described in materials and methods (i) The expression and translocation of NF-κBp65 protein were evaluated by measuring protein levels in the cytosolic and nuclear cell fractions (ii) COX-2 and TNF-α protein expression were evaluated in the whole cell lysates. β-actin and Lamin A were used as an internal loading control. The values are expressed as mean ± SEM of three independent experiments. Comparisons were made as follows: a—Control versus MSU (1mg/ml) stimulated, MSU+ morin treated groups (100–300 μM), MSU + Colchicine (1 μM); b—MSU stimulated versus MSU + morin (100–300 μM), MSU + Colchicine (1μM). * P

    Journal: PLoS ONE

    Article Title: Morin, a Bioflavonoid Suppresses Monosodium Urate Crystal-Induced Inflammatory Immune Response in RAW 264.7 Macrophages through the Inhibition of Inflammatory Mediators, Intracellular ROS Levels and NF-κB Activation

    doi: 10.1371/journal.pone.0145093

    Figure Lengend Snippet: Effect of morin on MSU crystal induced activation of NF-κBp65, COX-2 and TNF-α in RAW 264.7 macrophage cells. RAW 264.7 macrophage cells were treated with varying concentrations of morin (100–300 μM/ml) for 24 h and BAY 11–7028 (10 μM) for 1 h prior to the stimulation of MSU (1 mg/ml) for 24 h. Western blotting analysis was performed as described in materials and methods (i) The expression and translocation of NF-κBp65 protein were evaluated by measuring protein levels in the cytosolic and nuclear cell fractions (ii) COX-2 and TNF-α protein expression were evaluated in the whole cell lysates. β-actin and Lamin A were used as an internal loading control. The values are expressed as mean ± SEM of three independent experiments. Comparisons were made as follows: a—Control versus MSU (1mg/ml) stimulated, MSU+ morin treated groups (100–300 μM), MSU + Colchicine (1 μM); b—MSU stimulated versus MSU + morin (100–300 μM), MSU + Colchicine (1μM). * P

    Article Snippet: Western Blotting Analysis RAW 264.7 macrophage cells were pre-treated with the various concentrations of morin (100–300 μM) or colchicine (1 μM) for 24 h or BAY 11–7082 (10 μM) 1 h prior to the stimulation of MSU crystals (1mg/ml) for the next 24 h. Cytosolic and nuclear extracts were prepared with NE-PER nuclear and cytosolic extraction reagent kit (Thermo-Fisher Scientific, Waltham, Ma, USA) for the detection of NF-κBp65 protein expression,.

    Techniques: Activation Assay, Western Blot, Expressing, Translocation Assay