msu  (InvivoGen)

 
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  • 99
    Name:
    MSU Crystals
    Description:
    Monosodium Urate Crystals
    Catalog Number:
    tlrl-msu-25
    Price:
    None
    Size:
    25 mg
    Category:
    MSU Crystals Inflammasome Inducers PRR Ligands
    Buy from Supplier


    Structured Review

    InvivoGen msu
    Suppression of RIPK3 and MLKL prevents crystal cytotoxicity. ( a – c ) Mouse tubular epithelial cells were transfected with specific small inhibitor (si) RNA for RIPK3 and MLKL or a control siRNA of scrambled sequence before being exposed to crystals of <t>CaOx</t> (1,000 μg ml −1 ), <t>MSU</t> (500 μg ml −1 ), CPPD (500 μg ml −1 ) and cystine (500 μg ml −1 ). Cell viability was assessed by MTT assay (a) and cell death was assessed quantifying PI positivity (b) and C shows representative images 24 h later. Original image magnification: × 200, scale bar, 100 μm. ( d ) Mouse tubular epithelial cells were pretreated with RIPK3 inhibitor dabrafenib before exposing to different type crystals. Cell viability was assessed by MTT assay 24 h later. Data are expressed as mean±s.e.m. of three independent experiments, and was analysed using Student's t -test. Baseline viability is set as 100%. * P
    Monosodium Urate Crystals
    https://www.bioz.com/result/msu/product/InvivoGen
    Average 99 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    msu - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Cytotoxicity of crystals involves RIPK3-MLKL-mediated necroptosis"

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

    Journal: Nature Communications

    doi: 10.1038/ncomms10274

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

    Techniques Used: Transfection, Sequencing, MTT Assay

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

    Techniques Used: MTT Assay

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

    Techniques Used: Expressing, Western Blot, Isolation, MTT Assay

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

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

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

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

    2) Product Images from "Caspase-11 Mediates Neutrophil Chemotaxis and Extracellular Trap Formation During Acute Gouty Arthritis Through Alteration of Cofilin Phosphorylation"

    Article Title: Caspase-11 Mediates Neutrophil Chemotaxis and Extracellular Trap Formation During Acute Gouty Arthritis Through Alteration of Cofilin Phosphorylation

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.02519

    Caspase-11 −/− neutrophils produce less NETs independently of Rip3 phosphorylation and associated with altered cofilin phosphorylation in response to MSU in vitro . (A,B) Immunofluorescence assay of WT and caspase-11 −/− neutrophils treated with MSU, MSU + ATP, IL-1β, IL-1β + MSU, and PMA for 3 h. Cells were stained with neutrophil elastase (NE) and DAPI (DNA) to visualize NET formation, n = 3. (C) Quantification of NET formation in vitro . Response measured from WT and caspase-11 −/− neutrophils stimulated with MSU, ATP, IL-1β, combinations of treatments, and PMA. Cells were stained with Sytox green, n = 2 independent experiments (3 mice per group, total 6 mice), * p
    Figure Legend Snippet: Caspase-11 −/− neutrophils produce less NETs independently of Rip3 phosphorylation and associated with altered cofilin phosphorylation in response to MSU in vitro . (A,B) Immunofluorescence assay of WT and caspase-11 −/− neutrophils treated with MSU, MSU + ATP, IL-1β, IL-1β + MSU, and PMA for 3 h. Cells were stained with neutrophil elastase (NE) and DAPI (DNA) to visualize NET formation, n = 3. (C) Quantification of NET formation in vitro . Response measured from WT and caspase-11 −/− neutrophils stimulated with MSU, ATP, IL-1β, combinations of treatments, and PMA. Cells were stained with Sytox green, n = 2 independent experiments (3 mice per group, total 6 mice), * p

    Techniques Used: In Vitro, Immunofluorescence, Staining, Mouse Assay

    3) Product Images from "Caspase-11 Mediates Neutrophil Chemotaxis and Extracellular Trap Formation During Acute Gouty Arthritis Through Alteration of Cofilin Phosphorylation"

    Article Title: Caspase-11 Mediates Neutrophil Chemotaxis and Extracellular Trap Formation During Acute Gouty Arthritis Through Alteration of Cofilin Phosphorylation

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.02519

    Caspase-11 −/− neutrophils produce less NETs independently of Rip3 phosphorylation and associated with altered cofilin phosphorylation in response to MSU in vitro . (A,B) Immunofluorescence assay of WT and caspase-11 −/− neutrophils treated with MSU, MSU + ATP, IL-1β, IL-1β + MSU, and PMA for 3 h. Cells were stained with neutrophil elastase (NE) and DAPI (DNA) to visualize NET formation, n = 3. (C) Quantification of NET formation in vitro . Response measured from WT and caspase-11 −/− neutrophils stimulated with MSU, ATP, IL-1β, combinations of treatments, and PMA. Cells were stained with Sytox green, n = 2 independent experiments (3 mice per group, total 6 mice), * p
    Figure Legend Snippet: Caspase-11 −/− neutrophils produce less NETs independently of Rip3 phosphorylation and associated with altered cofilin phosphorylation in response to MSU in vitro . (A,B) Immunofluorescence assay of WT and caspase-11 −/− neutrophils treated with MSU, MSU + ATP, IL-1β, IL-1β + MSU, and PMA for 3 h. Cells were stained with neutrophil elastase (NE) and DAPI (DNA) to visualize NET formation, n = 3. (C) Quantification of NET formation in vitro . Response measured from WT and caspase-11 −/− neutrophils stimulated with MSU, ATP, IL-1β, combinations of treatments, and PMA. Cells were stained with Sytox green, n = 2 independent experiments (3 mice per group, total 6 mice), * p

    Techniques Used: In Vitro, Immunofluorescence, Staining, Mouse Assay

    4) Product Images from "Specific calpain inhibition protects kidney against inflammaging"

    Article Title: Specific calpain inhibition protects kidney against inflammaging

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-07922-1

    Calpastatin overexpression inhibits IL-1α and Il-1β synthesis in vitro and in vivo . In vitro , macrophages isolated from WT and CalpTG (TG) mice bones have been exposed to sodium urate crystals (MSU), Silica crystals (Si.), Adenosine TriPhosphate (ATP) or Nigericin (Niger.), with or without priming by Lipopolysaccharides (LPS). IL-1α and IL-1β expression at the mRNA level was globally reduced (ANOVA) by calpastatin overexpression ( A,B , n = 4/condition). Similarly, IL-1α and IL-1β cytokine production by macrophages was also globally reduced by calpastatin overexpression, especially IL-1α synthesis after exposure to particulate inflammasome activators MSU and Si. ( C,D , *p
    Figure Legend Snippet: Calpastatin overexpression inhibits IL-1α and Il-1β synthesis in vitro and in vivo . In vitro , macrophages isolated from WT and CalpTG (TG) mice bones have been exposed to sodium urate crystals (MSU), Silica crystals (Si.), Adenosine TriPhosphate (ATP) or Nigericin (Niger.), with or without priming by Lipopolysaccharides (LPS). IL-1α and IL-1β expression at the mRNA level was globally reduced (ANOVA) by calpastatin overexpression ( A,B , n = 4/condition). Similarly, IL-1α and IL-1β cytokine production by macrophages was also globally reduced by calpastatin overexpression, especially IL-1α synthesis after exposure to particulate inflammasome activators MSU and Si. ( C,D , *p

    Techniques Used: Over Expression, In Vitro, In Vivo, Isolation, Mouse Assay, Expressing

    5) Product Images from "Elderly Patients Exhibit Stronger Inflammatory Responses during Gout Attacks"

    Article Title: Elderly Patients Exhibit Stronger Inflammatory Responses during Gout Attacks

    Journal: Journal of Korean Medical Science

    doi: 10.3346/jkms.2017.32.12.1967

    Age-dependent IL-1β production by monocytes following activation with urate crystal. ( A ) Monocytes from 10 consecutive gout patients were stimulated with MSU crystals or LPS as described in Materials and Methods. MSU induced the monocytes to preferentially secrete IL-1β as compared to LPS. The bars indicate the mean, and the error bars indicate standard error. ( B ) The IL-1β production but not that of IL-6 or TNF-α correlated with patients' age. Correlation between cytokine production and age was assessed using Spearman's correlation. MSU = monosodium urate, LPS = lipopolysaccharide, IL = interleukin, TNF = tumor necrosis factor.
    Figure Legend Snippet: Age-dependent IL-1β production by monocytes following activation with urate crystal. ( A ) Monocytes from 10 consecutive gout patients were stimulated with MSU crystals or LPS as described in Materials and Methods. MSU induced the monocytes to preferentially secrete IL-1β as compared to LPS. The bars indicate the mean, and the error bars indicate standard error. ( B ) The IL-1β production but not that of IL-6 or TNF-α correlated with patients' age. Correlation between cytokine production and age was assessed using Spearman's correlation. MSU = monosodium urate, LPS = lipopolysaccharide, IL = interleukin, TNF = tumor necrosis factor.

    Techniques Used: Activation Assay

    6) Product Images from "The PYRIN domain-only protein POP3 inhibits AIM2-like receptor inflammasomes and regulates responses to DNA virus infections"

    Article Title: The PYRIN domain-only protein POP3 inhibits AIM2-like receptor inflammasomes and regulates responses to DNA virus infections

    Journal: Nature immunology

    doi: 10.1038/ni.2829

    POP3 expression in BMDM inhibits AIM2 and IFI16 inflammasome-mediated cytokine release (a–h) BMDM were infected with MVA, MCMV or KSHV, treated with LPS/ATP, MDP, MSU or transfected with poly(dA:dT) or flagellin for 16 h and analyzed for a–c, g, mature IL-1β, d, mature IL-18, e, IL-6 and f, TNF, h, IFN-β by ELISA, as indicated (n = 3 [a, b, d–h], n = 6 [c] ± s.e.m.). Data are representative of three (a–g), two (c, h) experiments. (a) * P =0.0064, ** P =0.0188; (b) * P =0.0015, ** P =0.0026; (c) * P =0.0020; (d) * P
    Figure Legend Snippet: POP3 expression in BMDM inhibits AIM2 and IFI16 inflammasome-mediated cytokine release (a–h) BMDM were infected with MVA, MCMV or KSHV, treated with LPS/ATP, MDP, MSU or transfected with poly(dA:dT) or flagellin for 16 h and analyzed for a–c, g, mature IL-1β, d, mature IL-18, e, IL-6 and f, TNF, h, IFN-β by ELISA, as indicated (n = 3 [a, b, d–h], n = 6 [c] ± s.e.m.). Data are representative of three (a–g), two (c, h) experiments. (a) * P =0.0064, ** P =0.0188; (b) * P =0.0015, ** P =0.0026; (c) * P =0.0020; (d) * P

    Techniques Used: Expressing, Infection, Transfection, Enzyme-linked Immunosorbent Assay

    CD68-POP3 TG mice are impaired in AIM2-dependent and viral DNA-induced host defense in vivo (a) WT and CD68-POP3 TG mice were infected with MCMV and serum levels of IL-18, IFN-γ and TNF were determined by ELISA 36 h after infection. (b) Spleens were isolated from above mice and analyzed for weight and number of splenocytes by flow cytometry. (c) Immunophenotyping of splenocytes after 36 h of MCMV infection. (d, e) Intracellular expression of IFN-γ by splenic NK cells 36 h after MCMV infection, cultured for 4 h ex vivo in medium (Med) alone or in the presence of PMA and ionomycin (P+I) and gated for CD11b + NK1.1 + cells, d, showing the result from a representative mouse with the percentage of IFN-γ + Ly49H + and Ly49H − NK cells indicated in the quadrants and e, results from 4–6 mice/group. (f) Mice were infected with MCMV as above and serum was analyzed for IFN-β by ELISA 11 and 36 h after infection. (g) Splenic viral titers 36 h after MCMV infection (n=6 per group). (h, i) WT and CD68-POP3 TG mice were i.p. injected with MSU crystals or PBS and h, IL-1β levels were determined by peritoneal lavage 7 h after injection by ELISA and i, mice were imaged for myeloperoxidase (MPO) activity in vivo and quantified after 5 h. Data are representative of two (a–h) and one (i) experiments. All data show the mean value. (a) * P =0.0451; (e) * P =0.0457, ** P =0.0447; (f) * P =0.0003; (g) * P =0.0028.
    Figure Legend Snippet: CD68-POP3 TG mice are impaired in AIM2-dependent and viral DNA-induced host defense in vivo (a) WT and CD68-POP3 TG mice were infected with MCMV and serum levels of IL-18, IFN-γ and TNF were determined by ELISA 36 h after infection. (b) Spleens were isolated from above mice and analyzed for weight and number of splenocytes by flow cytometry. (c) Immunophenotyping of splenocytes after 36 h of MCMV infection. (d, e) Intracellular expression of IFN-γ by splenic NK cells 36 h after MCMV infection, cultured for 4 h ex vivo in medium (Med) alone or in the presence of PMA and ionomycin (P+I) and gated for CD11b + NK1.1 + cells, d, showing the result from a representative mouse with the percentage of IFN-γ + Ly49H + and Ly49H − NK cells indicated in the quadrants and e, results from 4–6 mice/group. (f) Mice were infected with MCMV as above and serum was analyzed for IFN-β by ELISA 11 and 36 h after infection. (g) Splenic viral titers 36 h after MCMV infection (n=6 per group). (h, i) WT and CD68-POP3 TG mice were i.p. injected with MSU crystals or PBS and h, IL-1β levels were determined by peritoneal lavage 7 h after injection by ELISA and i, mice were imaged for myeloperoxidase (MPO) activity in vivo and quantified after 5 h. Data are representative of two (a–h) and one (i) experiments. All data show the mean value. (a) * P =0.0451; (e) * P =0.0457, ** P =0.0447; (f) * P =0.0003; (g) * P =0.0028.

    Techniques Used: Mouse Assay, In Vivo, Infection, Enzyme-linked Immunosorbent Assay, Isolation, Flow Cytometry, Cytometry, Expressing, Cell Culture, Ex Vivo, Injection, Activity Assay

    Silencing of POP3 in hMΦ enhances ALR-mediated IL-1β and IL-18 release (a–f, j, k) hMΦ were transfected with either control or POP3 siRNAs, confirmed for POP3 silencing by RT-PCR ( a, insert), and treated with LPS, MDP, MSU or SiO 2 , transfected with poly(dA:dT), LeTx, or flagellin, or infected with MVA as indicated for 16 h and analyzed a–c, for mature IL-1β; d, mature IL-18; e, TNF and k, IFN-β by ELISA (n = 3 ± s.e.m.). (f) POP3 silencing and ASC , AIM2 and IFI16 expression were determined by Real-Time PCR. (g–i) Stable THP-1 (GFP) or THP-1 (GFP-POP3) cells were analyzed for secretion of g, IL-1β and h, IL-18 in response to MVA and MCMV infection, transfection of poly(dA:dT) and treatment with MSU by ELISA (n = 3 ± s.e.m.). i, THP-1 (GFP) or THP-1 (GFP-POP3) cells were treated with IFN-β for the indicated times and expression of GFP and POP3 was analyzed by immunoblot and compared to β-tubulin. (j, k) hMΦ were transfected with siRNAs as above, j, transduced with either control GFP or GFP-POP3 expressing AdV, and either mock or MVA infected and analyzed for mature IL-1β by ELISA (n = 3 ± s.e.m.) or k, analyzed for secretion of IFN-β by ELISA (n = 3 ± s.e.m.). Data are representative of 3 experiments (a–f) and 2 experiments (g, h, j, k) or one experiment (i). (a) * P
    Figure Legend Snippet: Silencing of POP3 in hMΦ enhances ALR-mediated IL-1β and IL-18 release (a–f, j, k) hMΦ were transfected with either control or POP3 siRNAs, confirmed for POP3 silencing by RT-PCR ( a, insert), and treated with LPS, MDP, MSU or SiO 2 , transfected with poly(dA:dT), LeTx, or flagellin, or infected with MVA as indicated for 16 h and analyzed a–c, for mature IL-1β; d, mature IL-18; e, TNF and k, IFN-β by ELISA (n = 3 ± s.e.m.). (f) POP3 silencing and ASC , AIM2 and IFI16 expression were determined by Real-Time PCR. (g–i) Stable THP-1 (GFP) or THP-1 (GFP-POP3) cells were analyzed for secretion of g, IL-1β and h, IL-18 in response to MVA and MCMV infection, transfection of poly(dA:dT) and treatment with MSU by ELISA (n = 3 ± s.e.m.). i, THP-1 (GFP) or THP-1 (GFP-POP3) cells were treated with IFN-β for the indicated times and expression of GFP and POP3 was analyzed by immunoblot and compared to β-tubulin. (j, k) hMΦ were transfected with siRNAs as above, j, transduced with either control GFP or GFP-POP3 expressing AdV, and either mock or MVA infected and analyzed for mature IL-1β by ELISA (n = 3 ± s.e.m.) or k, analyzed for secretion of IFN-β by ELISA (n = 3 ± s.e.m.). Data are representative of 3 experiments (a–f) and 2 experiments (g, h, j, k) or one experiment (i). (a) * P

    Techniques Used: Transfection, Reverse Transcription Polymerase Chain Reaction, Infection, Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction, Transduction

    7) Product Images from "PTPN22 regulates NLRP3-mediated IL1B secretion in an autophagy-dependent manner"

    Article Title: PTPN22 regulates NLRP3-mediated IL1B secretion in an autophagy-dependent manner

    Journal: Autophagy

    doi: 10.1080/15548627.2017.1341453

    PYCARD is required for recruitment of NLRP3 into phagophores. BMDC from WT or pycard −/− cells were primed for 16 h with upLPS to induce NLRP3 and IL1B expression, before activation with MSU (150 μg/ml) for 6 h. (A) confocal microscopy of LC3B (red) and NLRP3 (green) immunostained cells; blue: DNA stained with DAPI, scale bar: 10 μm. (B) Western blots from autophagosome-enriched fractions and lysates were probed for the indicated proteins. (C) NLRP3 or SQSTM1 were precipitated from the lysate or from autophagosome-enriched fractions and probed for the indicated proteins. P-Tyr stands for phospho-tyrosine. Data are representative for 1 out of 3 independent experiments with 3 replicates each (n = 3), except for confocal microscopy where the experiment has been performed only once with 3 replicates. Images are representative for at least 5 scanned areas for each depicted condition. Numbers below the western blot pictures show results of densitometric measurements.
    Figure Legend Snippet: PYCARD is required for recruitment of NLRP3 into phagophores. BMDC from WT or pycard −/− cells were primed for 16 h with upLPS to induce NLRP3 and IL1B expression, before activation with MSU (150 μg/ml) for 6 h. (A) confocal microscopy of LC3B (red) and NLRP3 (green) immunostained cells; blue: DNA stained with DAPI, scale bar: 10 μm. (B) Western blots from autophagosome-enriched fractions and lysates were probed for the indicated proteins. (C) NLRP3 or SQSTM1 were precipitated from the lysate or from autophagosome-enriched fractions and probed for the indicated proteins. P-Tyr stands for phospho-tyrosine. Data are representative for 1 out of 3 independent experiments with 3 replicates each (n = 3), except for confocal microscopy where the experiment has been performed only once with 3 replicates. Images are representative for at least 5 scanned areas for each depicted condition. Numbers below the western blot pictures show results of densitometric measurements.

    Techniques Used: Expressing, Activation Assay, Confocal Microscopy, Staining, Western Blot

    NLRP3 lacking the phosphorylation site is not recruited to phagophores. BMDC from nlrp3 −/− mice were transfected with a wild-type (WT) Nlrp3 expression vector, or a Nlrp3 construct where Tyr859 in NLRP3 is replaced with a phenylalanine (Y859F; Y > F) to abolish NLRP3 phosphorylation. The cells were primed for 16 h with upLPS to induce NLRP3 and IL1B expression, before activation with MSU (150 µg/ml) for 6 h. Shown is (A) representative western blot pictures from cell lysates and autophagosome-enriched fractions, (B) confocal microscopy of LC3B (red) and NLRP3 (green) immunostained cells; blue: DNA stained with DAPI, scale bar: 10 μm; and (C) IL1B in the cell culture supernatant. Data are representative for 1 out of 3 independent experiments with 3 replicates each (n = 3), except for confocal microscopy where the experiment has been performed only twice with 3 replicates. Images are representative for at least 5 scanned areas for each depicted condition. Numbers below the western blot pictures show results of densitometric measurements. * = p
    Figure Legend Snippet: NLRP3 lacking the phosphorylation site is not recruited to phagophores. BMDC from nlrp3 −/− mice were transfected with a wild-type (WT) Nlrp3 expression vector, or a Nlrp3 construct where Tyr859 in NLRP3 is replaced with a phenylalanine (Y859F; Y > F) to abolish NLRP3 phosphorylation. The cells were primed for 16 h with upLPS to induce NLRP3 and IL1B expression, before activation with MSU (150 µg/ml) for 6 h. Shown is (A) representative western blot pictures from cell lysates and autophagosome-enriched fractions, (B) confocal microscopy of LC3B (red) and NLRP3 (green) immunostained cells; blue: DNA stained with DAPI, scale bar: 10 μm; and (C) IL1B in the cell culture supernatant. Data are representative for 1 out of 3 independent experiments with 3 replicates each (n = 3), except for confocal microscopy where the experiment has been performed only twice with 3 replicates. Images are representative for at least 5 scanned areas for each depicted condition. Numbers below the western blot pictures show results of densitometric measurements. * = p

    Techniques Used: Mouse Assay, Transfection, Expressing, Plasmid Preparation, Construct, Activation Assay, Western Blot, Confocal Microscopy, Staining, Cell Culture

    Phosphorylated NLRP3 is found in autophagosomes. (A) BMDC were primed for 16 h with upLPS to induce induction of NLRP3 and IL1B expression, before treatment with MSU for 6 h. The pictures show representative western blots from the indicated proteins in cell lysates and autophagosome-enriched fractions as indicated. (B) Confocal microscopy of THP-1 cells treated with LPS, or LPS and MSU, immunostained for NLRP3 (green) and LC3B (red; left side of the figure) or immunostained for NLRP3 (green) and the lysosome marker LAMP1 (red; right side of the figure); below and beside the image: z-stack at position of the arrows. scale bars: 10 μm. P-Tyr stands for phospho-tyrosine. (C) BMDC from WT mice, ptpn22 −/− mice or mice expressing a gain-of-function variant in Ptpn22 (619W) were treated as in (A) and immunostained for NLRP3 (green) and LC3B (red). (D) BMDC from WT or ptpn22 −/− mice were treated as in (A) and NLRP3 precipitated from either cell lysates or autophagosome-enriched fractions. The pictures show representative western blots for phospho-tyrosine (p-Tyr) and NLRP3 run in conventional SDS-PAGE. (E) control-transfected WT BMDC, control-transfected ptpn22 −/− BMDC, or ptpn22 −/− BMDC transfected with a vector expressing a loss-of-function variant in ptpn22 (263Q) were treated as in (A) and lysates run on Phos-tag gels, which allow the separation of phosphorylated from nonphosphorylated proteins due to slower migration of the phosphorylated forms. Data are representative for 1 out of 3 independent experiments with 3 replicates each (n = 3), except for confocal microscopy, where the experiment has been performed only twice with 3 replicates. Images are representative for at least 5 scanned areas for each depicted condition. Scale bars: 10 μm. P-Tyr stands for phospho-tyrosine. Numbers below the western blot pictures show results of densitometric measurements.
    Figure Legend Snippet: Phosphorylated NLRP3 is found in autophagosomes. (A) BMDC were primed for 16 h with upLPS to induce induction of NLRP3 and IL1B expression, before treatment with MSU for 6 h. The pictures show representative western blots from the indicated proteins in cell lysates and autophagosome-enriched fractions as indicated. (B) Confocal microscopy of THP-1 cells treated with LPS, or LPS and MSU, immunostained for NLRP3 (green) and LC3B (red; left side of the figure) or immunostained for NLRP3 (green) and the lysosome marker LAMP1 (red; right side of the figure); below and beside the image: z-stack at position of the arrows. scale bars: 10 μm. P-Tyr stands for phospho-tyrosine. (C) BMDC from WT mice, ptpn22 −/− mice or mice expressing a gain-of-function variant in Ptpn22 (619W) were treated as in (A) and immunostained for NLRP3 (green) and LC3B (red). (D) BMDC from WT or ptpn22 −/− mice were treated as in (A) and NLRP3 precipitated from either cell lysates or autophagosome-enriched fractions. The pictures show representative western blots for phospho-tyrosine (p-Tyr) and NLRP3 run in conventional SDS-PAGE. (E) control-transfected WT BMDC, control-transfected ptpn22 −/− BMDC, or ptpn22 −/− BMDC transfected with a vector expressing a loss-of-function variant in ptpn22 (263Q) were treated as in (A) and lysates run on Phos-tag gels, which allow the separation of phosphorylated from nonphosphorylated proteins due to slower migration of the phosphorylated forms. Data are representative for 1 out of 3 independent experiments with 3 replicates each (n = 3), except for confocal microscopy, where the experiment has been performed only twice with 3 replicates. Images are representative for at least 5 scanned areas for each depicted condition. Scale bars: 10 μm. P-Tyr stands for phospho-tyrosine. Numbers below the western blot pictures show results of densitometric measurements.

    Techniques Used: Expressing, Western Blot, Confocal Microscopy, Marker, Mouse Assay, Variant Assay, SDS Page, Transfection, Plasmid Preparation, Migration

    NLRP3 interacts with SQSTM1 upon its activation. (A) BMDC from nlrp3 −/− mice were transfected with a wild-type (WT) Nlrp3 expression vector, a Nlrp3 construct where Tyr859 in NLRP3 is replaced with a phenylalanine (Y859F; Y > F) to abolish NLRP3 phosphorylation, or with a phsopho-mimetic NLRP3, where Tyr859 was replaced with a glutamate (Y859E; Y > E). The cells were primed for 16 h with upLPS to induce NLRP3 and IL1B expression, before activation with MSU (150 µg/ml) for 6 h. NLRP3 or SQSTM1 were precipitated from the lysate and probed for the indicated proteins. (B) BMDC from WT or ptpn22 −/− cells were primed for 16 h with upLPS to induce NLRP3 and IL1B expression, before activation with MSU (150 µg/ml) for 6 h. NLRP3 or SQSTM1 were precipitated from the lysate and probed for the indicated proteins. Data is representative for 1 out of 3 independent experiments with 3 replicates each (n = 3). Numbers below the western blot pictures show results of densitometric measurements.
    Figure Legend Snippet: NLRP3 interacts with SQSTM1 upon its activation. (A) BMDC from nlrp3 −/− mice were transfected with a wild-type (WT) Nlrp3 expression vector, a Nlrp3 construct where Tyr859 in NLRP3 is replaced with a phenylalanine (Y859F; Y > F) to abolish NLRP3 phosphorylation, or with a phsopho-mimetic NLRP3, where Tyr859 was replaced with a glutamate (Y859E; Y > E). The cells were primed for 16 h with upLPS to induce NLRP3 and IL1B expression, before activation with MSU (150 µg/ml) for 6 h. NLRP3 or SQSTM1 were precipitated from the lysate and probed for the indicated proteins. (B) BMDC from WT or ptpn22 −/− cells were primed for 16 h with upLPS to induce NLRP3 and IL1B expression, before activation with MSU (150 µg/ml) for 6 h. NLRP3 or SQSTM1 were precipitated from the lysate and probed for the indicated proteins. Data is representative for 1 out of 3 independent experiments with 3 replicates each (n = 3). Numbers below the western blot pictures show results of densitometric measurements.

    Techniques Used: Activation Assay, Mouse Assay, Transfection, Expressing, Plasmid Preparation, Construct, Western Blot

    8) Product Images from "Calcium Pyrophosphate And Monosodium Urate Activate The NLRP3 Inflammasome Within Bladder Urothelium Via Reactive Oxygen Species And TXNIP"

    Article Title: Calcium Pyrophosphate And Monosodium Urate Activate The NLRP3 Inflammasome Within Bladder Urothelium Via Reactive Oxygen Species And TXNIP

    Journal: Research and Reports in Urology

    doi: 10.2147/RRU.S225767

    The stone DAMPs CPPD and MSU activate caspase-1 in a dose-dependent manner. Urothelial cells were incubated overnight prior to treatment with either CPPD ( A ) or MSU ( B ) for 24 hrs. Additional wells were treated with 1.25 mM ATP for 1 hr to indicate maximal caspase-1 activation and DAMP-treated wells were normalized to these ATP-treated wells. n=9 for all doses of CPPD and MSU. *p
    Figure Legend Snippet: The stone DAMPs CPPD and MSU activate caspase-1 in a dose-dependent manner. Urothelial cells were incubated overnight prior to treatment with either CPPD ( A ) or MSU ( B ) for 24 hrs. Additional wells were treated with 1.25 mM ATP for 1 hr to indicate maximal caspase-1 activation and DAMP-treated wells were normalized to these ATP-treated wells. n=9 for all doses of CPPD and MSU. *p

    Techniques Used: Incubation, Activation Assay

    Verapamil suppresses caspase-1 activation. Urothelial cells were incubated overnight and then treated with decreasing concentrations of Verapamil for 4 hrs before treatment with 62.5 μg/mL CPPD ( A ) or 1.25 μg/mL MSU ( B ) for 24 hrs. The caspase-1 assay was then performed as described in the Materials and Methods section. Both CPPD and MSU-treated cells had the same IC 50 (100 μM). n= 10 for all doses of Verapamil and CPPD treated wells; n= 5 for all doses of Verapamil and MSU treated wells. *p
    Figure Legend Snippet: Verapamil suppresses caspase-1 activation. Urothelial cells were incubated overnight and then treated with decreasing concentrations of Verapamil for 4 hrs before treatment with 62.5 μg/mL CPPD ( A ) or 1.25 μg/mL MSU ( B ) for 24 hrs. The caspase-1 assay was then performed as described in the Materials and Methods section. Both CPPD and MSU-treated cells had the same IC 50 (100 μM). n= 10 for all doses of Verapamil and CPPD treated wells; n= 5 for all doses of Verapamil and MSU treated wells. *p

    Techniques Used: Activation Assay, Incubation

    NAC inhibits caspase-1 activation in cells treated with CPPD or MSU. Urothelial cells were incubated overnight and then treated with decreasing concentrations of NAC for 1 hr before treatment with 62.5 μg/mL CPPD ( A ) or 1.25 μg/mL MSU ( B ) for 24 hrs. The caspase-1 assay was then performed as described in the Materials and Methods section. CPPD-treated cells had a higher IC 50 (625 μM) versus MSU-treated cells (IC 50 = 31.25 μM). n=9 for all NAC treatment doses for both CPPD and MSU. *p
    Figure Legend Snippet: NAC inhibits caspase-1 activation in cells treated with CPPD or MSU. Urothelial cells were incubated overnight and then treated with decreasing concentrations of NAC for 1 hr before treatment with 62.5 μg/mL CPPD ( A ) or 1.25 μg/mL MSU ( B ) for 24 hrs. The caspase-1 assay was then performed as described in the Materials and Methods section. CPPD-treated cells had a higher IC 50 (625 μM) versus MSU-treated cells (IC 50 = 31.25 μM). n=9 for all NAC treatment doses for both CPPD and MSU. *p

    Techniques Used: Activation Assay, Incubation

    9) Product Images from "IKKα negatively regulates ASC-dependent inflammasome activation"

    Article Title: IKKα negatively regulates ASC-dependent inflammasome activation

    Journal: Nature communications

    doi: 10.1038/ncomms5977

    IKKα is a critical regulator of NLRP3 inflammasome activation in vivo ( a , b ) Irradiated WT mice received bone marrow from either WT or IKKα K44A mice. After six weeks of engraftment, chimeric mice received 30 mg/kg Ultrapure LPS in 100 μl PBS (i.p). Serum was collected after 2 hours and levels of IL-1β and TNF-α were measured by ELISA ( a ). Splenocytes were collected and cultured for 2 hours with or without re-stimulation with LPS (1μg/ml). Cell-free supernatants were collected and levels of IL-1β and TNF-α were measured by ELISA ( b ). ( c ) Chimeric mice as described in ( a ) received 1 mg MSU in 100 μl PBS (i.p.). Five hours later, peritoneal lavage was performed and infiltrating neutrophils were quantified ( left panel ). Serum was collected and IL-1β levels were determined by ELISA ( right panel ). ( d , e ) Chimeric mice as described in ( a ) received 1 mg sterilized silica by intra-tracheal instillation. Thity-six hours later, lungs were harvested and lung sections were stained with hematoxylin and eosin ( upper panels ) or with anti-neutrophil antibody (NIMP-R14) ( lower panels ); scale bar represents 50 μm ( d ). Broncho-alveolar lavage (BAL) was performed and IL-1β levels in cell-free BAL fluid was assessed by ELISA ( e ). N.D. indicates not detectable, and “UN” indicates left untreated. Data are representative of two independent experiments. n = 5 mice per group in each experiment. Error bars represent s.e.m. of biological replicates. * P
    Figure Legend Snippet: IKKα is a critical regulator of NLRP3 inflammasome activation in vivo ( a , b ) Irradiated WT mice received bone marrow from either WT or IKKα K44A mice. After six weeks of engraftment, chimeric mice received 30 mg/kg Ultrapure LPS in 100 μl PBS (i.p). Serum was collected after 2 hours and levels of IL-1β and TNF-α were measured by ELISA ( a ). Splenocytes were collected and cultured for 2 hours with or without re-stimulation with LPS (1μg/ml). Cell-free supernatants were collected and levels of IL-1β and TNF-α were measured by ELISA ( b ). ( c ) Chimeric mice as described in ( a ) received 1 mg MSU in 100 μl PBS (i.p.). Five hours later, peritoneal lavage was performed and infiltrating neutrophils were quantified ( left panel ). Serum was collected and IL-1β levels were determined by ELISA ( right panel ). ( d , e ) Chimeric mice as described in ( a ) received 1 mg sterilized silica by intra-tracheal instillation. Thity-six hours later, lungs were harvested and lung sections were stained with hematoxylin and eosin ( upper panels ) or with anti-neutrophil antibody (NIMP-R14) ( lower panels ); scale bar represents 50 μm ( d ). Broncho-alveolar lavage (BAL) was performed and IL-1β levels in cell-free BAL fluid was assessed by ELISA ( e ). N.D. indicates not detectable, and “UN” indicates left untreated. Data are representative of two independent experiments. n = 5 mice per group in each experiment. Error bars represent s.e.m. of biological replicates. * P

    Techniques Used: Activation Assay, In Vivo, Irradiation, Mouse Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Staining

    Related Articles

    Produced:

    Article Title: Recombinant human proteoglycan-4 reduces phagocytosis of urate crystals and downstream nuclear factor kappa B and inflammasome activation and production of cytokines and chemokines in human and murine macrophages
    Article Snippet: .. THP-1 macrophages were treated with endotoxin-free MSU crystals (100μg/ml; Invivogen, USA) ± bovine submaxillary mucin (BSM; molecular mass > 1000 KDa) (Sigma Aldrich) (25 μg/ml) or rhPRG4 (molecular mass is approximately 240 KDa) (100 μg/ml) for 2 and 4 h at 37 °C. rhPRG4 is an endotoxin-free full-length product produced by CHO-M cells (Lubris, Framingham, MA, USA) [ ]. ..

    Marker:

    Article Title: Uric acid crystals induces placental inflammation and alters trophoblast function via an IL-1-dependent pathway: implication for FGR
    Article Snippet: .. Both MSU crystals and IL-1β significantly induced cell death in cytotrophoblast cultures, as seen by the 3.3-fold and 2.7-fold increase, respectively, in the percentage of cells positive for the apoptotic marker M30 at 48h ( ). .. Treatment of MSU crystals-exposed cytotrophoblasts with IL-1Ra was protective, with decreased percentage of M30+ apoptotic cells ( ).

    Injection:

    Article Title: The Immunomodulatory Metabolite Itaconate Modifies NLRP3 and Inhibits Inflammasome Activation
    Article Snippet: .. MSU-Induced Peritonitis Model 6-week old female C57BL/6J mice were injected intraperitoneally with a mixture of 4-OI (50 mg/kg) in 60% cyclodextrin in PBS and MSU crystals (30mg/kg, Invivogen) suspended in PBS for 6 h. Mice were euthanized in a CO2 chamber and peritoneal lavage was performed using 2.5 mL PBS. .. The cells in the lavage fluid were pelleted and the supernatant was removed and analyzed by ELISA for IL-1β and IL-6 concentration.

    other:

    Article Title: Uric acid crystals induces placental inflammation and alters trophoblast function via an IL-1-dependent pathway: implication for FGR
    Article Snippet: Since IL-1β is a well-known mediator of MSU crystals actions in immune cells, and it was strongly induced in term cytotrophoblasts in response to MSU crystals, we addressed the mechanisms of IL-1β production.

    Article Title: Uric acid crystals induces placental inflammation and alters trophoblast function via an IL-1-dependent pathway: implication for FGR
    Article Snippet: Consistent with these findings in cytotrophoblast, treatment with MSU crystals or IL-1β induced apoptosis in term placental explants, ( ) which was mainly observed in cytotrophoblast cells (arrowheads in ).

    Article Title: Uric acid crystals induces placental inflammation and alters trophoblast function via an IL-1-dependent pathway: implication for FGR
    Article Snippet: This was predominantly observed within the junctional zone, where all doses of MSU crystals led to a significant elevation of monocytes/macrophages (CD68+ cells) and within the labyrinth (fetal side), where only the highest dose of MSU crystals induced a significant increased in CD68+ monocytes/macrophages ( ).

    Article Title: MicroRNA-488 and -920 regulate the production of proinflammatory cytokines in acute gouty arthritis
    Article Snippet: MSU crystals can induce a variety of cytokines, including IL-1β, IL-6, IL-8, and TNF-α [ ].

    Article Title: Uric acid crystals induces placental inflammation and alters trophoblast function via an IL-1-dependent pathway: implication for FGR
    Article Snippet: The secretion of IL-1β induced by MSU crystals in cytotrophoblasts was dependent on the common processor of pro-IL-1β, caspase-1, which was ascertained using a caspase-1 inhibitor ( ).

    Mouse Assay:

    Article Title: The Immunomodulatory Metabolite Itaconate Modifies NLRP3 and Inhibits Inflammasome Activation
    Article Snippet: .. MSU-Induced Peritonitis Model 6-week old female C57BL/6J mice were injected intraperitoneally with a mixture of 4-OI (50 mg/kg) in 60% cyclodextrin in PBS and MSU crystals (30mg/kg, Invivogen) suspended in PBS for 6 h. Mice were euthanized in a CO2 chamber and peritoneal lavage was performed using 2.5 mL PBS. .. The cells in the lavage fluid were pelleted and the supernatant was removed and analyzed by ELISA for IL-1β and IL-6 concentration.

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    InvivoGen msu
    Suppression of RIPK3 and MLKL prevents crystal cytotoxicity. ( a – c ) Mouse tubular epithelial cells were transfected with specific small inhibitor (si) RNA for RIPK3 and MLKL or a control siRNA of scrambled sequence before being exposed to crystals of <t>CaOx</t> (1,000 μg ml −1 ), <t>MSU</t> (500 μg ml −1 ), CPPD (500 μg ml −1 ) and cystine (500 μg ml −1 ). Cell viability was assessed by MTT assay (a) and cell death was assessed quantifying PI positivity (b) and C shows representative images 24 h later. Original image magnification: × 200, scale bar, 100 μm. ( d ) Mouse tubular epithelial cells were pretreated with RIPK3 inhibitor dabrafenib before exposing to different type crystals. Cell viability was assessed by MTT assay 24 h later. Data are expressed as mean±s.e.m. of three independent experiments, and was analysed using Student's t -test. Baseline viability is set as 100%. * P
    Msu, supplied by InvivoGen, used in various techniques. Bioz Stars score: 91/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    InvivoGen msu crystals
    <t>MSU</t> triggered intracellular ROS is inhibited by cytochalasin B (CytB) and colchicine (col). ( A ) Neutrophils were pretreated with cytochalsin B (5 µg/mL), an inhibitor of actin polymerization, and stimulated with either MSU crystals (300 µg/mL), <t>PMA</t> (50 nM) or opsonized S. aureus and intracellular ROS production (icROS) was evaluated using luminol amplified CL. Cytochalasin B-treated cells produced lower amounts of icROS in response to MSU crystals and S. aureus as compared to untreated cells, while icROS production in response to PMA was not affected by the presence of cytochalasin B. Peak CL values are shown. ( B ) Neutrophils were pretreated with colchicine (1 µg/mL) and stimulated with either MSU crystals (300 µg/mL), PMA (50 nM) or opsonized S. aureus and icROS production was evaluated using luminol amplified CL. Colchicine decreased icROS in response to MSU crystals, while icROS production in response to PMA or S. aureus was not affected. Peak CL values are shown ( n = 7). * p ≤ 0.05 and ** p ≤ 0.01. ns = not significant.
    Msu Crystals, supplied by InvivoGen, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Suppression of RIPK3 and MLKL prevents crystal cytotoxicity. ( a – c ) Mouse tubular epithelial cells were transfected with specific small inhibitor (si) RNA for RIPK3 and MLKL or a control siRNA of scrambled sequence before being exposed to crystals of CaOx (1,000 μg ml −1 ), MSU (500 μg ml −1 ), CPPD (500 μg ml −1 ) and cystine (500 μg ml −1 ). Cell viability was assessed by MTT assay (a) and cell death was assessed quantifying PI positivity (b) and C shows representative images 24 h later. Original image magnification: × 200, scale bar, 100 μm. ( d ) Mouse tubular epithelial cells were pretreated with RIPK3 inhibitor dabrafenib before exposing to different type crystals. Cell viability was assessed by MTT assay 24 h later. Data are expressed as mean±s.e.m. of three independent experiments, and was analysed using Student's t -test. Baseline viability is set as 100%. * P

    Journal: Nature Communications

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

    doi: 10.1038/ncomms10274

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

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

    Techniques: Transfection, Sequencing, MTT Assay

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

    Journal: Nature Communications

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

    doi: 10.1038/ncomms10274

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

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

    Techniques: MTT Assay

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

    Journal: Nature Communications

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

    doi: 10.1038/ncomms10274

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

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

    Techniques: Expressing, Western Blot, Isolation, MTT Assay

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

    Journal: Nature Communications

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

    doi: 10.1038/ncomms10274

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

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

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

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

    Journal: Nature Communications

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

    doi: 10.1038/ncomms10274

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

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

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

    Caspase-11 −/− neutrophils produce less NETs independently of Rip3 phosphorylation and associated with altered cofilin phosphorylation in response to MSU in vitro . (A,B) Immunofluorescence assay of WT and caspase-11 −/− neutrophils treated with MSU, MSU + ATP, IL-1β, IL-1β + MSU, and PMA for 3 h. Cells were stained with neutrophil elastase (NE) and DAPI (DNA) to visualize NET formation, n = 3. (C) Quantification of NET formation in vitro . Response measured from WT and caspase-11 −/− neutrophils stimulated with MSU, ATP, IL-1β, combinations of treatments, and PMA. Cells were stained with Sytox green, n = 2 independent experiments (3 mice per group, total 6 mice), * p

    Journal: Frontiers in Immunology

    Article Title: Caspase-11 Mediates Neutrophil Chemotaxis and Extracellular Trap Formation During Acute Gouty Arthritis Through Alteration of Cofilin Phosphorylation

    doi: 10.3389/fimmu.2019.02519

    Figure Lengend Snippet: Caspase-11 −/− neutrophils produce less NETs independently of Rip3 phosphorylation and associated with altered cofilin phosphorylation in response to MSU in vitro . (A,B) Immunofluorescence assay of WT and caspase-11 −/− neutrophils treated with MSU, MSU + ATP, IL-1β, IL-1β + MSU, and PMA for 3 h. Cells were stained with neutrophil elastase (NE) and DAPI (DNA) to visualize NET formation, n = 3. (C) Quantification of NET formation in vitro . Response measured from WT and caspase-11 −/− neutrophils stimulated with MSU, ATP, IL-1β, combinations of treatments, and PMA. Cells were stained with Sytox green, n = 2 independent experiments (3 mice per group, total 6 mice), * p

    Article Snippet: PMNs were stimulated for 3 h with 100 μg/ml MSU (Invivogen, tlrl-msu), 100 μM ATP (Roche, 10 519 987 001), 25 ng/ml IL-1β (R & D systems, 401-ML-005/CF), and 100 nM PMA (Sigma-Aldrich, #P8139-10MG).

    Techniques: In Vitro, Immunofluorescence, Staining, Mouse Assay

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

    Journal: International Journal of Molecular Sciences

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

    doi: 10.3390/ijms21113750

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

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

    Techniques: Amplification, Produced

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

    Journal: International Journal of Molecular Sciences

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

    doi: 10.3390/ijms21113750

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

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

    Techniques: Fluorescence

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

    Journal: International Journal of Molecular Sciences

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

    doi: 10.3390/ijms21113750

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

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

    Techniques: