ethylenediaminetetraacetic acid  (Millipore)


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    Structured Review

    Millipore ethylenediaminetetraacetic acid
    Matrix metalloproteinase (MMP) gene expression and protein activity. (A) RNA-seq showed MMP2 was not differentially regulated but MMP14 was significantly upregulated. (B) On zymography, bands corresponding to pro MMP2 and active MMP2 were seen. The Std lane corresponds to recombinant MMP2. Inhibition with <t>ethylenediaminetetraacetic</t> acid shows absence of activity. (C)
    Ethylenediaminetetraacetic Acid, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ethylenediaminetetraacetic acid/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ethylenediaminetetraacetic acid - by Bioz Stars, 2020-04
    99/100 stars

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    1) Product Images from "Mechanical and Biochemical Effects of Progesterone on Engineered Cervical Tissue"

    Article Title: Mechanical and Biochemical Effects of Progesterone on Engineered Cervical Tissue

    Journal: Tissue Engineering. Part A

    doi: 10.1089/ten.tea.2018.0036

    Matrix metalloproteinase (MMP) gene expression and protein activity. (A) RNA-seq showed MMP2 was not differentially regulated but MMP14 was significantly upregulated. (B) On zymography, bands corresponding to pro MMP2 and active MMP2 were seen. The Std lane corresponds to recombinant MMP2. Inhibition with ethylenediaminetetraacetic acid shows absence of activity. (C)
    Figure Legend Snippet: Matrix metalloproteinase (MMP) gene expression and protein activity. (A) RNA-seq showed MMP2 was not differentially regulated but MMP14 was significantly upregulated. (B) On zymography, bands corresponding to pro MMP2 and active MMP2 were seen. The Std lane corresponds to recombinant MMP2. Inhibition with ethylenediaminetetraacetic acid shows absence of activity. (C)

    Techniques Used: Expressing, Activity Assay, RNA Sequencing Assay, Zymography, Recombinant, Inhibition

    Related Articles

    Chemiluminescent ELISA:

    Article Title: TNF Signaling Impacts Glucagon-Like Peptide-1 Expression and Secretion
    Article Snippet: Plasma were obtained in the presence of EDTA (10% of blood), DPP4 inhibitor (10 μl/ml blood, Millipore), and protease and phosphatase inhibitor mini table (0.00987 g/mouse, Thermo Fisher). .. GLP-1 (High Sensitivity GLP-1 Active Chemiluminescent ELISA Kit, EMD Millipore Corporation), and GIP (RAT/MOUSE GIP (TOTAL) ELISA KIT, EMD Millipore Corporation) levels in these plasmas were determined per manufacturer’s instructions.

    Zymography:

    Article Title: Mechanical and Biochemical Effects of Progesterone on Engineered Cervical Tissue
    Article Snippet: Paragraph title: Gelatin zymography ... Ethylenediaminetetraacetic acid (15 mM) was used for positive control of MMP inhibition and recombinant MMP2 (MilliporeSigma, Burlington, MA) was used as a positive control.

    Positive Control:

    Article Title: Mechanical and Biochemical Effects of Progesterone on Engineered Cervical Tissue
    Article Snippet: .. Ethylenediaminetetraacetic acid (15 mM) was used for positive control of MMP inhibition and recombinant MMP2 (MilliporeSigma, Burlington, MA) was used as a positive control. ..

    Enzyme-linked Immunosorbent Assay:

    Article Title: TNF Signaling Impacts Glucagon-Like Peptide-1 Expression and Secretion
    Article Snippet: When performing IPGTT and OGTT, sera were harvested at 0, 15, and 30 minutes after intraperitoneal injection or oral gavage of glucose, and insulin levels in these sera were determined using Ultra Sensitive Mouse Insulin ELISA Kit (Crystal Chemical) per manufacturer’s instructions. .. Plasma were obtained in the presence of EDTA (10% of blood), DPP4 inhibitor (10 μl/ml blood, Millipore), and protease and phosphatase inhibitor mini table (0.00987 g/mouse, Thermo Fisher).

    Incubation:

    Article Title: Viability Quantitative PCR Utilizing Propidium Monoazide, Spheroplast Formation, and Campylobacter coli as a Bacterial Model
    Article Snippet: In brief, 0.5 ml of a hyperosmotic buffer (S1) containing PBS, sucrose, EDTA, and lysozyme (all reagents from Sigma-Aldrich, Steinheim, Germany) was added to each C. coli sample at a ratio of 1:1 in a microcentrifuge tube (2.0 ml; Kisker Biotech GmbH & Co., Steinfurt, Germany). .. C. coli cell suspensions were incubated with the S1 buffer at 37°C in order to attain optimum lysozyme activity according to the manufacturer’s instructions.

    Article Title: Expression and oncogenic properties of membranous Notch1 in oral leukoplakia and oral squamous cell carcinoma
    Article Snippet: .. For the EDTA (Sigma-Aldrich; Merck KGaA, Taufkirchen, Bayern, Germany) activation assay, WSU-HN4 cells were washed twice in phosphate-buffered saline (PBS) and incubated in PBS, 2.5 mM EDTA or 2.5 mM CaCl2 (Sigma-Aldrich; Merck KGaA) for 15 min at 37°C. .. The cells were then recovered in DMEM for 4 h and subjected to western blot analysis or immunofluorescence staining.

    Article Title: Influence of PEGylation of Vitamin-K-Loaded Mixed Micelles on the Uptake by and Transport through Caco-2 Cells
    Article Snippet: After incubation for 2 h at 4 and 37 °C, respectively, the medium was removed, and the cells were washed three times with PBS. .. Subsequently, 90 μL of solution of 0.02% EDTA and 0.05% trypsin solutions (Sigma) were added to detach the cells from the wells, and after 10 min, 200 μL of PBS was added to suspend the cells.

    Article Title: Mechanical and Biochemical Effects of Progesterone on Engineered Cervical Tissue
    Article Snippet: After washing and overnight incubation in developing buffer (0.01 M Tris base, 0.03 M Tris-HCl, 0.2 M NaCl, 6.6 mM CaCl2 , 0.02% Tween 20), gels were stained with 0.5% Coomassie Brilliant Blue R250 for 30 min. .. Ethylenediaminetetraacetic acid (15 mM) was used for positive control of MMP inhibition and recombinant MMP2 (MilliporeSigma, Burlington, MA) was used as a positive control.

    Article Title: Identification of Peptides Derived from the C-terminal Domain of Fibulin-7 Active for Endothelial Cell Adhesion and Tube Formation Disruption
    Article Snippet: .. For the inhibition of cell attachment with heparin (Sigma-Aldrich, USA), EDTA (Sigma-Aldrich, USA) and integrin β1-blocking antibody (EMD Millipore, USA), HUVECs were first incubated for 20 min at 37°C in the presence of either 10 μg/ml heparin, 5mM EDTA, or 10 μg/ml integrin β1-blocking antibody (clone 6S6, Millipore). ..

    Article Title: The NLRP6 inflammasome recognizes lipoteichoic acid and regulates Gram-positive pathogen infection
    Article Snippet: .. To prepare bacterial extracts, 109 cfu of bacteria were suspended in 1 ml of PBS, incubated with 100 µg/ml lysozyme (Fisher Scientific), 5 mM EDTA and protease inhibitor cocktail (Sigma-Aldrich) for 30 min at room temperature, sonicated, and centrifuged at 21,000×g for 1 min. .. The extracts were treated with DNase I (10 µg/ml), RNase A (10 µg/ml), PDE (1 unit/reaction), or Proteinase K (100 µg/ml) in the presence of 14 mM MgCl2 at 37 ºC for 3 h, then heated at 100 ºC for 10 min. 1 µg of bacterial ligand or 1.25 µl of bacterial extract was suspended in 10 µl of Opti-MEM.

    Article Title: A Divalent Ion Is Crucial in the Structure and Dominant-Negative Function of ID Proteins, a Class of Helix-Loop-Helix Transcription Regulators
    Article Snippet: EGTA, EDTA and 18C6 knockdowns of ID2 and ID3 in EMSAs Stock solutions of the chemicals were prepared (0.5 M for EGTA [Sigma-Aldrich] and EDTA [1st Base], 5 M for 18C6 [Sigma-Aldrich]). .. EMSAs were performed as described above, with the addition of 1 ul of stock solution to the incubation mix of ID and E proteins.

    Activity Assay:

    Article Title: Viability Quantitative PCR Utilizing Propidium Monoazide, Spheroplast Formation, and Campylobacter coli as a Bacterial Model
    Article Snippet: In brief, 0.5 ml of a hyperosmotic buffer (S1) containing PBS, sucrose, EDTA, and lysozyme (all reagents from Sigma-Aldrich, Steinheim, Germany) was added to each C. coli sample at a ratio of 1:1 in a microcentrifuge tube (2.0 ml; Kisker Biotech GmbH & Co., Steinfurt, Germany). .. C. coli cell suspensions were incubated with the S1 buffer at 37°C in order to attain optimum lysozyme activity according to the manufacturer’s instructions.

    Cell Culture:

    Article Title: Expression and oncogenic properties of membranous Notch1 in oral leukoplakia and oral squamous cell carcinoma
    Article Snippet: Cell line and reagents The WSU-HN4 cell line previously described ( ) was cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco-BRL) at 37°C in a humidified 5% CO2 atmosphere. .. For the EDTA (Sigma-Aldrich; Merck KGaA, Taufkirchen, Bayern, Germany) activation assay, WSU-HN4 cells were washed twice in phosphate-buffered saline (PBS) and incubated in PBS, 2.5 mM EDTA or 2.5 mM CaCl2 (Sigma-Aldrich; Merck KGaA) for 15 min at 37°C.

    Article Title: Influence of PEGylation of Vitamin-K-Loaded Mixed Micelles on the Uptake by and Transport through Caco-2 Cells
    Article Snippet: Uptake of Mixed Micelles by Caco-2 Cells As Studied by Fluorescence Activated Cell Sorting Analysis (FACS) Caco-2 cells were seeded in a 24-well plate at a density of 1 × 105 cells per well and cultured for 3 weeks as described in . .. Subsequently, 90 μL of solution of 0.02% EDTA and 0.05% trypsin solutions (Sigma) were added to detach the cells from the wells, and after 10 min, 200 μL of PBS was added to suspend the cells.

    Article Title: The NLRP6 inflammasome recognizes lipoteichoic acid and regulates Gram-positive pathogen infection
    Article Snippet: Gentamicin (10 µg/ml) was added to the cultures 1 h after infection when the cells were continuously cultured for more than 2 h. For transfection, cells were primed with poly(I:C) (1 µg/ml) for 4 h and culture medium was replaced with Opti-MEM (Gibco). .. To prepare bacterial extracts, 109 cfu of bacteria were suspended in 1 ml of PBS, incubated with 100 µg/ml lysozyme (Fisher Scientific), 5 mM EDTA and protease inhibitor cocktail (Sigma-Aldrich) for 30 min at room temperature, sonicated, and centrifuged at 21,000×g for 1 min.

    Bradford Assay:

    Article Title: Mechanical and Biochemical Effects of Progesterone on Engineered Cervical Tissue
    Article Snippet: The supernatant was harvested and the protein content quantified using the Bradford Assay. .. Ethylenediaminetetraacetic acid (15 mM) was used for positive control of MMP inhibition and recombinant MMP2 (MilliporeSigma, Burlington, MA) was used as a positive control.

    Modification:

    Article Title: Expression and oncogenic properties of membranous Notch1 in oral leukoplakia and oral squamous cell carcinoma
    Article Snippet: Cell line and reagents The WSU-HN4 cell line previously described ( ) was cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco-BRL) at 37°C in a humidified 5% CO2 atmosphere. .. For the EDTA (Sigma-Aldrich; Merck KGaA, Taufkirchen, Bayern, Germany) activation assay, WSU-HN4 cells were washed twice in phosphate-buffered saline (PBS) and incubated in PBS, 2.5 mM EDTA or 2.5 mM CaCl2 (Sigma-Aldrich; Merck KGaA) for 15 min at 37°C.

    Western Blot:

    Article Title: Expression and oncogenic properties of membranous Notch1 in oral leukoplakia and oral squamous cell carcinoma
    Article Snippet: For the EDTA (Sigma-Aldrich; Merck KGaA, Taufkirchen, Bayern, Germany) activation assay, WSU-HN4 cells were washed twice in phosphate-buffered saline (PBS) and incubated in PBS, 2.5 mM EDTA or 2.5 mM CaCl2 (Sigma-Aldrich; Merck KGaA) for 15 min at 37°C. .. The cells were then recovered in DMEM for 4 h and subjected to western blot analysis or immunofluorescence staining.

    Transfection:

    Article Title: The NLRP6 inflammasome recognizes lipoteichoic acid and regulates Gram-positive pathogen infection
    Article Snippet: Gentamicin (10 µg/ml) was added to the cultures 1 h after infection when the cells were continuously cultured for more than 2 h. For transfection, cells were primed with poly(I:C) (1 µg/ml) for 4 h and culture medium was replaced with Opti-MEM (Gibco). .. To prepare bacterial extracts, 109 cfu of bacteria were suspended in 1 ml of PBS, incubated with 100 µg/ml lysozyme (Fisher Scientific), 5 mM EDTA and protease inhibitor cocktail (Sigma-Aldrich) for 30 min at room temperature, sonicated, and centrifuged at 21,000×g for 1 min.

    Activation Assay:

    Article Title: Expression and oncogenic properties of membranous Notch1 in oral leukoplakia and oral squamous cell carcinoma
    Article Snippet: .. For the EDTA (Sigma-Aldrich; Merck KGaA, Taufkirchen, Bayern, Germany) activation assay, WSU-HN4 cells were washed twice in phosphate-buffered saline (PBS) and incubated in PBS, 2.5 mM EDTA or 2.5 mM CaCl2 (Sigma-Aldrich; Merck KGaA) for 15 min at 37°C. .. The cells were then recovered in DMEM for 4 h and subjected to western blot analysis or immunofluorescence staining.

    Protease Inhibitor:

    Article Title: The NLRP6 inflammasome recognizes lipoteichoic acid and regulates Gram-positive pathogen infection
    Article Snippet: .. To prepare bacterial extracts, 109 cfu of bacteria were suspended in 1 ml of PBS, incubated with 100 µg/ml lysozyme (Fisher Scientific), 5 mM EDTA and protease inhibitor cocktail (Sigma-Aldrich) for 30 min at room temperature, sonicated, and centrifuged at 21,000×g for 1 min. .. The extracts were treated with DNase I (10 µg/ml), RNase A (10 µg/ml), PDE (1 unit/reaction), or Proteinase K (100 µg/ml) in the presence of 14 mM MgCl2 at 37 ºC for 3 h, then heated at 100 ºC for 10 min. 1 µg of bacterial ligand or 1.25 µl of bacterial extract was suspended in 10 µl of Opti-MEM.

    Infection:

    Article Title: The NLRP6 inflammasome recognizes lipoteichoic acid and regulates Gram-positive pathogen infection
    Article Snippet: Gentamicin (10 µg/ml) was added to the cultures 1 h after infection when the cells were continuously cultured for more than 2 h. For transfection, cells were primed with poly(I:C) (1 µg/ml) for 4 h and culture medium was replaced with Opti-MEM (Gibco). .. To prepare bacterial extracts, 109 cfu of bacteria were suspended in 1 ml of PBS, incubated with 100 µg/ml lysozyme (Fisher Scientific), 5 mM EDTA and protease inhibitor cocktail (Sigma-Aldrich) for 30 min at room temperature, sonicated, and centrifuged at 21,000×g for 1 min.

    Inhibition:

    Article Title: Mechanical and Biochemical Effects of Progesterone on Engineered Cervical Tissue
    Article Snippet: .. Ethylenediaminetetraacetic acid (15 mM) was used for positive control of MMP inhibition and recombinant MMP2 (MilliporeSigma, Burlington, MA) was used as a positive control. ..

    Article Title: Identification of Peptides Derived from the C-terminal Domain of Fibulin-7 Active for Endothelial Cell Adhesion and Tube Formation Disruption
    Article Snippet: .. For the inhibition of cell attachment with heparin (Sigma-Aldrich, USA), EDTA (Sigma-Aldrich, USA) and integrin β1-blocking antibody (EMD Millipore, USA), HUVECs were first incubated for 20 min at 37°C in the presence of either 10 μg/ml heparin, 5mM EDTA, or 10 μg/ml integrin β1-blocking antibody (clone 6S6, Millipore). ..

    Sonication:

    Article Title: The NLRP6 inflammasome recognizes lipoteichoic acid and regulates Gram-positive pathogen infection
    Article Snippet: .. To prepare bacterial extracts, 109 cfu of bacteria were suspended in 1 ml of PBS, incubated with 100 µg/ml lysozyme (Fisher Scientific), 5 mM EDTA and protease inhibitor cocktail (Sigma-Aldrich) for 30 min at room temperature, sonicated, and centrifuged at 21,000×g for 1 min. .. The extracts were treated with DNase I (10 µg/ml), RNase A (10 µg/ml), PDE (1 unit/reaction), or Proteinase K (100 µg/ml) in the presence of 14 mM MgCl2 at 37 ºC for 3 h, then heated at 100 ºC for 10 min. 1 µg of bacterial ligand or 1.25 µl of bacterial extract was suspended in 10 µl of Opti-MEM.

    Injection:

    Article Title: TNF Signaling Impacts Glucagon-Like Peptide-1 Expression and Secretion
    Article Snippet: When performing IPGTT and OGTT, sera were harvested at 0, 15, and 30 minutes after intraperitoneal injection or oral gavage of glucose, and insulin levels in these sera were determined using Ultra Sensitive Mouse Insulin ELISA Kit (Crystal Chemical) per manufacturer’s instructions. .. Plasma were obtained in the presence of EDTA (10% of blood), DPP4 inhibitor (10 μl/ml blood, Millipore), and protease and phosphatase inhibitor mini table (0.00987 g/mouse, Thermo Fisher).

    Recombinant:

    Article Title: Mechanical and Biochemical Effects of Progesterone on Engineered Cervical Tissue
    Article Snippet: .. Ethylenediaminetetraacetic acid (15 mM) was used for positive control of MMP inhibition and recombinant MMP2 (MilliporeSigma, Burlington, MA) was used as a positive control. ..

    Article Title: Identification of Peptides Derived from the C-terminal Domain of Fibulin-7 Active for Endothelial Cell Adhesion and Tube Formation Disruption
    Article Snippet: Paragraph title: Cell adhesion assay using either Fbln7-C recombinant protein or Fbln7-synthetic peptides-coated plastic plates ... For the inhibition of cell attachment with heparin (Sigma-Aldrich, USA), EDTA (Sigma-Aldrich, USA) and integrin β1-blocking antibody (EMD Millipore, USA), HUVECs were first incubated for 20 min at 37°C in the presence of either 10 μg/ml heparin, 5mM EDTA, or 10 μg/ml integrin β1-blocking antibody (clone 6S6, Millipore).

    Immunofluorescence:

    Article Title: Expression and oncogenic properties of membranous Notch1 in oral leukoplakia and oral squamous cell carcinoma
    Article Snippet: For the EDTA (Sigma-Aldrich; Merck KGaA, Taufkirchen, Bayern, Germany) activation assay, WSU-HN4 cells were washed twice in phosphate-buffered saline (PBS) and incubated in PBS, 2.5 mM EDTA or 2.5 mM CaCl2 (Sigma-Aldrich; Merck KGaA) for 15 min at 37°C. .. The cells were then recovered in DMEM for 4 h and subjected to western blot analysis or immunofluorescence staining.

    Molecular Weight:

    Article Title: Liquid Cladding Mediated Optical Fiber Sensors for Copper Ion Detection
    Article Snippet: .. Chitosan (low molecular weight), EDTA (99%), (3-aminopropyl)triethoxysilane (APTES, 98%), isopropanol (C3 H8 O, 99.7%), acetic acid (C2 H4 O2 , 99.7%), 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS, 98%), and copper (II) nitrate trihydrate (Cu(NO3 )2 .3H2 O, 99%) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). .. Deionized water was produced by Biosesang Co. (Seongnam-Si, Korea).

    Article Title: Liquid Cladding Mediated Optical Fiber Sensors for Copper Ion Detection
    Article Snippet: .. Materials and Reagents Chitosan (low molecular weight), EDTA (99%), (3-aminopropyl)triethoxysilane (APTES, 98%), isopropanol (C3 H8 O, 99.7%), acetic acid (C2 H4 O2 , 99.7%), 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS, 98%), and copper (II) nitrate trihydrate (Cu(NO3 )2 .3H2 O, 99%) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). .. Deionized water was produced by Biosesang Co. (Seongnam-Si, Korea).

    FACS:

    Article Title: Influence of PEGylation of Vitamin-K-Loaded Mixed Micelles on the Uptake by and Transport through Caco-2 Cells
    Article Snippet: Paragraph title: Uptake of Mixed Micelles by Caco-2 Cells As Studied by Fluorescence Activated Cell Sorting Analysis (FACS) ... Subsequently, 90 μL of solution of 0.02% EDTA and 0.05% trypsin solutions (Sigma) were added to detach the cells from the wells, and after 10 min, 200 μL of PBS was added to suspend the cells.

    Fluorescence:

    Article Title: Influence of PEGylation of Vitamin-K-Loaded Mixed Micelles on the Uptake by and Transport through Caco-2 Cells
    Article Snippet: Paragraph title: Uptake of Mixed Micelles by Caco-2 Cells As Studied by Fluorescence Activated Cell Sorting Analysis (FACS) ... Subsequently, 90 μL of solution of 0.02% EDTA and 0.05% trypsin solutions (Sigma) were added to detach the cells from the wells, and after 10 min, 200 μL of PBS was added to suspend the cells.

    Labeling:

    Article Title: Influence of PEGylation of Vitamin-K-Loaded Mixed Micelles on the Uptake by and Transport through Caco-2 Cells
    Article Snippet: Fluorescein conjugated DSPE-PEG labeled mixed micelles were used for fluorescence activated cell sorting analysis (FACS) (FACSCalibur; BD Biosciences) because the FACS instrument has a specific FITC channel. .. Subsequently, 90 μL of solution of 0.02% EDTA and 0.05% trypsin solutions (Sigma) were added to detach the cells from the wells, and after 10 min, 200 μL of PBS was added to suspend the cells.

    Mouse Assay:

    Article Title: TNF Signaling Impacts Glucagon-Like Peptide-1 Expression and Secretion
    Article Snippet: Mice were then euthanized for harvesting plasma at 0, 15, and 30 minutes after oral gavage of glucose. .. Plasma were obtained in the presence of EDTA (10% of blood), DPP4 inhibitor (10 μl/ml blood, Millipore), and protease and phosphatase inhibitor mini table (0.00987 g/mouse, Thermo Fisher).

    Cell Adhesion Assay:

    Article Title: Identification of Peptides Derived from the C-terminal Domain of Fibulin-7 Active for Endothelial Cell Adhesion and Tube Formation Disruption
    Article Snippet: Paragraph title: Cell adhesion assay using either Fbln7-C recombinant protein or Fbln7-synthetic peptides-coated plastic plates ... For the inhibition of cell attachment with heparin (Sigma-Aldrich, USA), EDTA (Sigma-Aldrich, USA) and integrin β1-blocking antibody (EMD Millipore, USA), HUVECs were first incubated for 20 min at 37°C in the presence of either 10 μg/ml heparin, 5mM EDTA, or 10 μg/ml integrin β1-blocking antibody (clone 6S6, Millipore).

    Lysis:

    Article Title: Mechanical and Biochemical Effects of Progesterone on Engineered Cervical Tissue
    Article Snippet: The scaffold was then centrifuged with 500 μL of cold zymography lysis buffer (1.25 mL 1 M Tris-HCl solution, 2.5 mL 2 M NaCl solution, 5 mL 10% IGEPAL, and 41.25 mL deionized water) mixed with protease inhibitors (1 μL 5 mg/mL aprotinin, 0.5 μL 2 mg/mL leupeptin, and 1 μL 2 M benzamidine) at 16,000 g for 10 min. .. Ethylenediaminetetraacetic acid (15 mM) was used for positive control of MMP inhibition and recombinant MMP2 (MilliporeSigma, Burlington, MA) was used as a positive control.

    Software:

    Article Title: Mechanical and Biochemical Effects of Progesterone on Engineered Cervical Tissue
    Article Snippet: After de-staining (Coomassie R-250 de-staining solution, methanol: acetic acid: water, 50:10:40), gels were imaged and quantified using Image J software. .. Ethylenediaminetetraacetic acid (15 mM) was used for positive control of MMP inhibition and recombinant MMP2 (MilliporeSigma, Burlington, MA) was used as a positive control.

    Electrophoresis:

    Article Title: Mechanical and Biochemical Effects of Progesterone on Engineered Cervical Tissue
    Article Snippet: Electrophoresis was performed with 5 μg of protein per lane in 8.0% sodium dodecyl sulfate polyacrylamide gels and 0.1% gelatin at 125 V for 90 min. .. Ethylenediaminetetraacetic acid (15 mM) was used for positive control of MMP inhibition and recombinant MMP2 (MilliporeSigma, Burlington, MA) was used as a positive control.

    Cell Attachment Assay:

    Article Title: Identification of Peptides Derived from the C-terminal Domain of Fibulin-7 Active for Endothelial Cell Adhesion and Tube Formation Disruption
    Article Snippet: .. For the inhibition of cell attachment with heparin (Sigma-Aldrich, USA), EDTA (Sigma-Aldrich, USA) and integrin β1-blocking antibody (EMD Millipore, USA), HUVECs were first incubated for 20 min at 37°C in the presence of either 10 μg/ml heparin, 5mM EDTA, or 10 μg/ml integrin β1-blocking antibody (clone 6S6, Millipore). ..

    Produced:

    Article Title: Liquid Cladding Mediated Optical Fiber Sensors for Copper Ion Detection
    Article Snippet: Chitosan (low molecular weight), EDTA (99%), (3-aminopropyl)triethoxysilane (APTES, 98%), isopropanol (C3 H8 O, 99.7%), acetic acid (C2 H4 O2 , 99.7%), 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS, 98%), and copper (II) nitrate trihydrate (Cu(NO3 )2 .3H2 O, 99%) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). .. Deionized water was produced by Biosesang Co. (Seongnam-Si, Korea).

    Article Title: Liquid Cladding Mediated Optical Fiber Sensors for Copper Ion Detection
    Article Snippet: Materials and Reagents Chitosan (low molecular weight), EDTA (99%), (3-aminopropyl)triethoxysilane (APTES, 98%), isopropanol (C3 H8 O, 99.7%), acetic acid (C2 H4 O2 , 99.7%), 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS, 98%), and copper (II) nitrate trihydrate (Cu(NO3 )2 .3H2 O, 99%) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). .. Deionized water was produced by Biosesang Co. (Seongnam-Si, Korea).

    Concentration Assay:

    Article Title: Viability Quantitative PCR Utilizing Propidium Monoazide, Spheroplast Formation, and Campylobacter coli as a Bacterial Model
    Article Snippet: In brief, 0.5 ml of a hyperosmotic buffer (S1) containing PBS, sucrose, EDTA, and lysozyme (all reagents from Sigma-Aldrich, Steinheim, Germany) was added to each C. coli sample at a ratio of 1:1 in a microcentrifuge tube (2.0 ml; Kisker Biotech GmbH & Co., Steinfurt, Germany). .. The final concentration was 10 mM for PBS (pH 7.0), 250 mM for sucrose, and 0.5 mg ml−1 for lysozyme, whereas various final concentrations of EDTA (12.5, 25, 50, and 100 mM) were investigated.

    Staining:

    Article Title: Expression and oncogenic properties of membranous Notch1 in oral leukoplakia and oral squamous cell carcinoma
    Article Snippet: For the EDTA (Sigma-Aldrich; Merck KGaA, Taufkirchen, Bayern, Germany) activation assay, WSU-HN4 cells were washed twice in phosphate-buffered saline (PBS) and incubated in PBS, 2.5 mM EDTA or 2.5 mM CaCl2 (Sigma-Aldrich; Merck KGaA) for 15 min at 37°C. .. The cells were then recovered in DMEM for 4 h and subjected to western blot analysis or immunofluorescence staining.

    Article Title: Mechanical and Biochemical Effects of Progesterone on Engineered Cervical Tissue
    Article Snippet: After washing and overnight incubation in developing buffer (0.01 M Tris base, 0.03 M Tris-HCl, 0.2 M NaCl, 6.6 mM CaCl2 , 0.02% Tween 20), gels were stained with 0.5% Coomassie Brilliant Blue R250 for 30 min. .. Ethylenediaminetetraacetic acid (15 mM) was used for positive control of MMP inhibition and recombinant MMP2 (MilliporeSigma, Burlington, MA) was used as a positive control.

    Article Title: Identification of Peptides Derived from the C-terminal Domain of Fibulin-7 Active for Endothelial Cell Adhesion and Tube Formation Disruption
    Article Snippet: Attached cells were fixed and stained for 10 min with 0.2% crystal violet in 20% methanol. .. For the inhibition of cell attachment with heparin (Sigma-Aldrich, USA), EDTA (Sigma-Aldrich, USA) and integrin β1-blocking antibody (EMD Millipore, USA), HUVECs were first incubated for 20 min at 37°C in the presence of either 10 μg/ml heparin, 5mM EDTA, or 10 μg/ml integrin β1-blocking antibody (clone 6S6, Millipore).

    other:

    Article Title: An ABC transport system that maintains lipid asymmetry in the Gram-negative outer membrane
    Article Snippet: SDS and EDTA, purchased from Sigma-Aldrich, were prepared as filter sterilized stock solutions of 10% SDS and 50 mM EDTA (pH 7.5).

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  • 96
    Millipore pbs edta
    Representative absorbance spectra of AMB-PEG 2 and unconjugated AMB formulations prepared in buffers with varying hydrophobicity. 20 mM AMB-PEG 2 and unconjugated AMB formulations were prepared in DMSO and resuspended in 20% and 48% ACN buffers containing 4.3% acetic acid, as well as <t>PBS-EDTA,</t> to a final concentration of 2 mM AMB. As buffer hydrophobicity increases with higher ACN concentrations, the A 348 /A 409 ratio decreases, implying that AMB-PEG is increasingly in its monomeric form. As AMB-PEG 1 and 2 have similar UV-visible absorption profiles, with identical peak height ratios in all buffers tested, data for AMB-PEG 1 is not shown. AMB-PEG formulations that have been subjected to buffer exchange to PBS-EDTA through a 10 kDa centrifugal filter have the same UV-visible absorption spectra as the initial formulation of AMB-PEG in PBS-EDTA, which contains 10% DMSO.
    Pbs Edta, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbs edta/product/Millipore
    Average 96 stars, based on 23 article reviews
    Price from $9.99 to $1999.99
    pbs edta - by Bioz Stars, 2020-04
    96/100 stars
      Buy from Supplier

    94
    Millipore calpain reaction buffer
    Schematic diagram showing the relationship of <t>calpain/NF-κB/inflammation/NVU</t> damage after CCI in mice. Traumatic brain injury induces calcium overload, which, in turn, upregulates calpain. Calpain may downregulate IκB and activate NF-κB. NF-κB induces activation of TNF-α, iNOS, ICAM-1, and MMP-9. These inflammatory substances induce degradation of basal lamina and tight junction proteins, resulting in NVU disruption, leading to brain edema. MDL28170 could reverse those changes. NF-κB: Nuclear factor-κB; NVU: Neurovascular unit; CCI: Controlled cortical impact; IκB: Inhibitory-κB; TNF-α: Tumor necrosis factor-α; iNOS: Inducible nitric oxide synthase; ICAM-1: Intracellular adhesion molecule-1; MMP-9: Matrix metalloproteinase-9.
    Calpain Reaction Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Millipore m edta
    Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) induce Ca 2+ mobilization and phospholipase D (PLD) activation in type II alveolar epithelial cells. (a, c) A549 cells were labelled with 3 μ m Fluo-3/AM, treated or not with <t>EDTA,</t> <t>EGTA</t>
    M Edta, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m edta/product/Millipore
    Average 98 stars, based on 2 article reviews
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    Millipore immunoprecipitation buffer
    Binding of Wt1 protein to the 5′-flanking region of the Adamts16 gene. ChIP was performed to detect Wt1 protein bound to the 5′-flanking region of the Adamts16 gene in its native chromosomal configuration. The drawing ( A ) delineates the three predicted Wt1 binding sites ( Wt1-A , Wt1-B , and Wt1-C ) in the promoter and 5′-UTR of the Adamts16 gene and allocates the PCR primers used for DNA amplification. Specific antibodies against Wt1 and histone proteins were chosen for <t>immunoprecipitation</t> of M15 whole cell lysates. Amplicons encompassing the 5′-flanking region of the Adamts16 gene were enriched ∼2.5-fold with the use of anti-Wt1 antibody compared with normal rabbit IgG (NRb-IgG). No differences in actin DNA were observed between anti-Wt1 antibody and normal rabbit IgG ( B ). The gene encoding anti-Müllerian hormone receptor 2 ( Amhr2 ), served as a positive control ( B ). Binding of Wt1(−KTS) protein to the Adamts16 promoter was confirmed in stimulated UB27 cells ( C ). Wt1(+KTS) protein failed to interact with the promoter of the Adamts16 gene in UD28 cells ( D ). Data shown are means ± S.E. ( error bars ). Statistical significances are indicated by asterisks (*, p
    Immunoprecipitation Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 187 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Representative absorbance spectra of AMB-PEG 2 and unconjugated AMB formulations prepared in buffers with varying hydrophobicity. 20 mM AMB-PEG 2 and unconjugated AMB formulations were prepared in DMSO and resuspended in 20% and 48% ACN buffers containing 4.3% acetic acid, as well as PBS-EDTA, to a final concentration of 2 mM AMB. As buffer hydrophobicity increases with higher ACN concentrations, the A 348 /A 409 ratio decreases, implying that AMB-PEG is increasingly in its monomeric form. As AMB-PEG 1 and 2 have similar UV-visible absorption profiles, with identical peak height ratios in all buffers tested, data for AMB-PEG 1 is not shown. AMB-PEG formulations that have been subjected to buffer exchange to PBS-EDTA through a 10 kDa centrifugal filter have the same UV-visible absorption spectra as the initial formulation of AMB-PEG in PBS-EDTA, which contains 10% DMSO.

    Journal: PLoS ONE

    Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy

    doi: 10.1371/journal.pone.0152112

    Figure Lengend Snippet: Representative absorbance spectra of AMB-PEG 2 and unconjugated AMB formulations prepared in buffers with varying hydrophobicity. 20 mM AMB-PEG 2 and unconjugated AMB formulations were prepared in DMSO and resuspended in 20% and 48% ACN buffers containing 4.3% acetic acid, as well as PBS-EDTA, to a final concentration of 2 mM AMB. As buffer hydrophobicity increases with higher ACN concentrations, the A 348 /A 409 ratio decreases, implying that AMB-PEG is increasingly in its monomeric form. As AMB-PEG 1 and 2 have similar UV-visible absorption profiles, with identical peak height ratios in all buffers tested, data for AMB-PEG 1 is not shown. AMB-PEG formulations that have been subjected to buffer exchange to PBS-EDTA through a 10 kDa centrifugal filter have the same UV-visible absorption spectra as the initial formulation of AMB-PEG in PBS-EDTA, which contains 10% DMSO.

    Article Snippet: AMB-PEG Particle Size Determination Using DLS 2 mM solutions of AMB-PEG were prepared in PBS-EDTA, and diafiltration carried out with PBS-EDTA using 10 kDa centrifugal filters (Millipore).

    Techniques: Concentration Assay, Buffer Exchange

    (A) Reverse phase chromatogram of AMB-PEG and unconjugated AMB, with eluted peaks detected at 406 nm. 50 μL of AMB-PEG 1 and 2, and 10 μL of unconjugated AMB dispersed in PBS-EDTA and 48% ACN respectively were injected into a C18 reverse phase column and eluted isocratically in a 48% ACN buffer at a flow rate of 0.5 ml/min for 40 minutes. Peaks were detected at 406 nm. The AMB-PEG conjugate has a shorter retention time, implying that it is more hydrophilic. From the AMB-PEG samples, AMB-PEG conjugate and free AMB (based on the retention time of unconjugated AMB) fractions were collected for further analysis via size exclusion chromatography. (B) Size exclusion chromatogram of AMB-PEG 2 and unconjugated AMB, as well as the relevant fractions collected from RPC, in a 20% ACN mobile phase. 20 mM of AMB-PEG 2 formulations and unconjugated AMB in DMSO were prepared and resuspended at 2 mM in a 20% ACN buffer. 50 μL of these samples (unconjugated AMB diluted tenfold prior to analysis) and the peak fractions collected previously from RPC were passed through a size exclusion column and eluted peaks were detected at 406 nm. Unconjugated AMB was eluted later compared to the AMB-PEG conjugate, implying that it has a smaller hydrodynamic volume compared to AMB-PEG in 20% ACN. The previously collected AMB-PEG conjugate and free AMB peak fractions had similar retention times as the AMB-PEG 2 formulation and unconjugated AMB samples respectively, thus verifying their respective peak identities. AMB-PEG 1 and 2 have identical elution profiles. (C) Size exclusion chromatogram of AMB-PEG and unconjugated AMB dispersed in PBS-EDTA. 3 μL of 2 mM AMB-PEG formulations that have been retained by 10 kDa centrifugal filters (Millipore) and 50 μL of the supernatant obtained from unconjugated AMB were analysed using a Superdex 75 size exclusion column and eluted peaks detected at 406 nm. In a PBS-EDTA mobile phase, unconjugated AMB has a shorter retention time of 20 minutes compared to AMB-PEG at 40 minutes, implying that AMB-PEG has a smaller hydrodynamic volume under these experimental conditions.

    Journal: PLoS ONE

    Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy

    doi: 10.1371/journal.pone.0152112

    Figure Lengend Snippet: (A) Reverse phase chromatogram of AMB-PEG and unconjugated AMB, with eluted peaks detected at 406 nm. 50 μL of AMB-PEG 1 and 2, and 10 μL of unconjugated AMB dispersed in PBS-EDTA and 48% ACN respectively were injected into a C18 reverse phase column and eluted isocratically in a 48% ACN buffer at a flow rate of 0.5 ml/min for 40 minutes. Peaks were detected at 406 nm. The AMB-PEG conjugate has a shorter retention time, implying that it is more hydrophilic. From the AMB-PEG samples, AMB-PEG conjugate and free AMB (based on the retention time of unconjugated AMB) fractions were collected for further analysis via size exclusion chromatography. (B) Size exclusion chromatogram of AMB-PEG 2 and unconjugated AMB, as well as the relevant fractions collected from RPC, in a 20% ACN mobile phase. 20 mM of AMB-PEG 2 formulations and unconjugated AMB in DMSO were prepared and resuspended at 2 mM in a 20% ACN buffer. 50 μL of these samples (unconjugated AMB diluted tenfold prior to analysis) and the peak fractions collected previously from RPC were passed through a size exclusion column and eluted peaks were detected at 406 nm. Unconjugated AMB was eluted later compared to the AMB-PEG conjugate, implying that it has a smaller hydrodynamic volume compared to AMB-PEG in 20% ACN. The previously collected AMB-PEG conjugate and free AMB peak fractions had similar retention times as the AMB-PEG 2 formulation and unconjugated AMB samples respectively, thus verifying their respective peak identities. AMB-PEG 1 and 2 have identical elution profiles. (C) Size exclusion chromatogram of AMB-PEG and unconjugated AMB dispersed in PBS-EDTA. 3 μL of 2 mM AMB-PEG formulations that have been retained by 10 kDa centrifugal filters (Millipore) and 50 μL of the supernatant obtained from unconjugated AMB were analysed using a Superdex 75 size exclusion column and eluted peaks detected at 406 nm. In a PBS-EDTA mobile phase, unconjugated AMB has a shorter retention time of 20 minutes compared to AMB-PEG at 40 minutes, implying that AMB-PEG has a smaller hydrodynamic volume under these experimental conditions.

    Article Snippet: AMB-PEG Particle Size Determination Using DLS 2 mM solutions of AMB-PEG were prepared in PBS-EDTA, and diafiltration carried out with PBS-EDTA using 10 kDa centrifugal filters (Millipore).

    Techniques: Injection, Flow Cytometry, Size-exclusion Chromatography

    (A) AMB-PEG reaction scheme. The NHS ester on MS(PEG) 4 reacts with the primary amine (–NH2) group on AMB at a 1:1 molar ratio, generating conjugated AMB-PEG via amide bond formation. (B) Solubility of AMB-PEG at varying AMB:PEG molar ratios. AMB-PEG mixtures and unreacted AMB were prepared in DMSO, incubated for 2 hours and then (i) dispersed to a final concentration of 2 mM in PBS-EDTA, containing 10% DMSO by volume. (ii) The mixtures were then centrifuged to facilitate the observation of insoluble precipitates. Higher molar ratios yielded a suspension of yellow particles that precipitated upon centrifugation, similar to what was observed with unconjugated AMB.

    Journal: PLoS ONE

    Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy

    doi: 10.1371/journal.pone.0152112

    Figure Lengend Snippet: (A) AMB-PEG reaction scheme. The NHS ester on MS(PEG) 4 reacts with the primary amine (–NH2) group on AMB at a 1:1 molar ratio, generating conjugated AMB-PEG via amide bond formation. (B) Solubility of AMB-PEG at varying AMB:PEG molar ratios. AMB-PEG mixtures and unreacted AMB were prepared in DMSO, incubated for 2 hours and then (i) dispersed to a final concentration of 2 mM in PBS-EDTA, containing 10% DMSO by volume. (ii) The mixtures were then centrifuged to facilitate the observation of insoluble precipitates. Higher molar ratios yielded a suspension of yellow particles that precipitated upon centrifugation, similar to what was observed with unconjugated AMB.

    Article Snippet: AMB-PEG Particle Size Determination Using DLS 2 mM solutions of AMB-PEG were prepared in PBS-EDTA, and diafiltration carried out with PBS-EDTA using 10 kDa centrifugal filters (Millipore).

    Techniques: Mass Spectrometry, Solubility, Incubation, Concentration Assay, Centrifugation

    Aqueous solubility of AMB-PEG 1 and 2. Increasing amounts of AMB-PEG formulations were added to PBS-EDTA, and post-centrifugation, the concentration of soluble AMB-PEG in the supernatants quantified based on their absorbance at 365 nm. Error bars denote the standard deviation between 3 independent formulations. Dotted line represents the theoretical condition where AMB formulation is completely soluble (i.e. concentration of total AMB = concentration of soluble AMB).

    Journal: PLoS ONE

    Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy

    doi: 10.1371/journal.pone.0152112

    Figure Lengend Snippet: Aqueous solubility of AMB-PEG 1 and 2. Increasing amounts of AMB-PEG formulations were added to PBS-EDTA, and post-centrifugation, the concentration of soluble AMB-PEG in the supernatants quantified based on their absorbance at 365 nm. Error bars denote the standard deviation between 3 independent formulations. Dotted line represents the theoretical condition where AMB formulation is completely soluble (i.e. concentration of total AMB = concentration of soluble AMB).

    Article Snippet: AMB-PEG Particle Size Determination Using DLS 2 mM solutions of AMB-PEG were prepared in PBS-EDTA, and diafiltration carried out with PBS-EDTA using 10 kDa centrifugal filters (Millipore).

    Techniques: Solubility, Centrifugation, Concentration Assay, Standard Deviation

    Schematic diagram showing the relationship of calpain/NF-κB/inflammation/NVU damage after CCI in mice. Traumatic brain injury induces calcium overload, which, in turn, upregulates calpain. Calpain may downregulate IκB and activate NF-κB. NF-κB induces activation of TNF-α, iNOS, ICAM-1, and MMP-9. These inflammatory substances induce degradation of basal lamina and tight junction proteins, resulting in NVU disruption, leading to brain edema. MDL28170 could reverse those changes. NF-κB: Nuclear factor-κB; NVU: Neurovascular unit; CCI: Controlled cortical impact; IκB: Inhibitory-κB; TNF-α: Tumor necrosis factor-α; iNOS: Inducible nitric oxide synthase; ICAM-1: Intracellular adhesion molecule-1; MMP-9: Matrix metalloproteinase-9.

    Journal: Chinese Medical Journal

    Article Title: Protective Effects of Calpain Inhibition on Neurovascular Unit Injury through Downregulating Nuclear Factor-κB-related Inflammation during Traumatic Brain Injury in Mice

    doi: 10.4103/0366-6999.198001

    Figure Lengend Snippet: Schematic diagram showing the relationship of calpain/NF-κB/inflammation/NVU damage after CCI in mice. Traumatic brain injury induces calcium overload, which, in turn, upregulates calpain. Calpain may downregulate IκB and activate NF-κB. NF-κB induces activation of TNF-α, iNOS, ICAM-1, and MMP-9. These inflammatory substances induce degradation of basal lamina and tight junction proteins, resulting in NVU disruption, leading to brain edema. MDL28170 could reverse those changes. NF-κB: Nuclear factor-κB; NVU: Neurovascular unit; CCI: Controlled cortical impact; IκB: Inhibitory-κB; TNF-α: Tumor necrosis factor-α; iNOS: Inducible nitric oxide synthase; ICAM-1: Intracellular adhesion molecule-1; MMP-9: Matrix metalloproteinase-9.

    Article Snippet: [ ] In brief, cytosolic and mitochondrial proteins (30 μg) were incubated with calpain reaction buffer (20 mmol/L HEPES, 1 mmol/L EDTA, 50 mmol/L NaCl, and 0.1% (v/v) 2-mercaptoethanol, containing 10 μmol/L calpain I fluorescent substrate [Calbiochem Co., La Jolla, CA, USA], pH 7.6).

    Techniques: Mouse Assay, Activation Assay

    MDL28170 treatment suppresses the calpain activity in the cytosolic and mitochondrial fractions and upregulates the expression of calpastatin in the cytosolic fractions. (a and b) The bar graphs reflect the calpain activity in the cytosolic fractions and mitochondrial fractions at 6 h and 24 h. (c) Representative Western blots of calpastatin and β-actin from each group; (d) the results were quantified and are shown as the mean ± SD. * P

    Journal: Chinese Medical Journal

    Article Title: Protective Effects of Calpain Inhibition on Neurovascular Unit Injury through Downregulating Nuclear Factor-κB-related Inflammation during Traumatic Brain Injury in Mice

    doi: 10.4103/0366-6999.198001

    Figure Lengend Snippet: MDL28170 treatment suppresses the calpain activity in the cytosolic and mitochondrial fractions and upregulates the expression of calpastatin in the cytosolic fractions. (a and b) The bar graphs reflect the calpain activity in the cytosolic fractions and mitochondrial fractions at 6 h and 24 h. (c) Representative Western blots of calpastatin and β-actin from each group; (d) the results were quantified and are shown as the mean ± SD. * P

    Article Snippet: [ ] In brief, cytosolic and mitochondrial proteins (30 μg) were incubated with calpain reaction buffer (20 mmol/L HEPES, 1 mmol/L EDTA, 50 mmol/L NaCl, and 0.1% (v/v) 2-mercaptoethanol, containing 10 μmol/L calpain I fluorescent substrate [Calbiochem Co., La Jolla, CA, USA], pH 7.6).

    Techniques: Activity Assay, Expressing, Western Blot

    Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) induce Ca 2+ mobilization and phospholipase D (PLD) activation in type II alveolar epithelial cells. (a, c) A549 cells were labelled with 3 μ m Fluo-3/AM, treated or not with EDTA, EGTA

    Journal: Immunology

    Article Title: Natural lysophospholipids reduce Mycobacterium tuberculosis-induced cytotoxicity and induce anti-mycobacterial activity by a phagolysosome maturation-dependent mechanism in A549 type II alveolar epithelial cells

    doi: 10.1111/j.1365-2567.2009.03145.x

    Figure Lengend Snippet: Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) induce Ca 2+ mobilization and phospholipase D (PLD) activation in type II alveolar epithelial cells. (a, c) A549 cells were labelled with 3 μ m Fluo-3/AM, treated or not with EDTA, EGTA

    Article Snippet: In several experiments, 2 m m EDTA or 3 m m EGTA (Calbiochem) were added 15 min before the addition of LPA or S1P, whereas 20 μ m 1,2-bis(2-aminophenoxy)ethane-N,N,N1,N1-tetraacetic acid tetraicis (acetoxymethyl ester) (BAPTA-AM) (Sigma-Aldrich, Milan, Italy) was added 30 min before stimulation with LPA or S1P.

    Techniques: Activation Assay

    Binding of Wt1 protein to the 5′-flanking region of the Adamts16 gene. ChIP was performed to detect Wt1 protein bound to the 5′-flanking region of the Adamts16 gene in its native chromosomal configuration. The drawing ( A ) delineates the three predicted Wt1 binding sites ( Wt1-A , Wt1-B , and Wt1-C ) in the promoter and 5′-UTR of the Adamts16 gene and allocates the PCR primers used for DNA amplification. Specific antibodies against Wt1 and histone proteins were chosen for immunoprecipitation of M15 whole cell lysates. Amplicons encompassing the 5′-flanking region of the Adamts16 gene were enriched ∼2.5-fold with the use of anti-Wt1 antibody compared with normal rabbit IgG (NRb-IgG). No differences in actin DNA were observed between anti-Wt1 antibody and normal rabbit IgG ( B ). The gene encoding anti-Müllerian hormone receptor 2 ( Amhr2 ), served as a positive control ( B ). Binding of Wt1(−KTS) protein to the Adamts16 promoter was confirmed in stimulated UB27 cells ( C ). Wt1(+KTS) protein failed to interact with the promoter of the Adamts16 gene in UD28 cells ( D ). Data shown are means ± S.E. ( error bars ). Statistical significances are indicated by asterisks (*, p

    Journal: The Journal of Biological Chemistry

    Article Title: Transcriptional Regulation by the Wilms Tumor Protein, Wt1, Suggests a Role of the Metalloproteinase Adamts16 in Murine Genitourinary Development *

    doi: 10.1074/jbc.M113.464644

    Figure Lengend Snippet: Binding of Wt1 protein to the 5′-flanking region of the Adamts16 gene. ChIP was performed to detect Wt1 protein bound to the 5′-flanking region of the Adamts16 gene in its native chromosomal configuration. The drawing ( A ) delineates the three predicted Wt1 binding sites ( Wt1-A , Wt1-B , and Wt1-C ) in the promoter and 5′-UTR of the Adamts16 gene and allocates the PCR primers used for DNA amplification. Specific antibodies against Wt1 and histone proteins were chosen for immunoprecipitation of M15 whole cell lysates. Amplicons encompassing the 5′-flanking region of the Adamts16 gene were enriched ∼2.5-fold with the use of anti-Wt1 antibody compared with normal rabbit IgG (NRb-IgG). No differences in actin DNA were observed between anti-Wt1 antibody and normal rabbit IgG ( B ). The gene encoding anti-Müllerian hormone receptor 2 ( Amhr2 ), served as a positive control ( B ). Binding of Wt1(−KTS) protein to the Adamts16 promoter was confirmed in stimulated UB27 cells ( C ). Wt1(+KTS) protein failed to interact with the promoter of the Adamts16 gene in UD28 cells ( D ). Data shown are means ± S.E. ( error bars ). Statistical significances are indicated by asterisks (*, p

    Article Snippet: The supernatants were diluted in immunoprecipitation buffer (0.01% SDS, 1.1% Triton X-100, 1.2 m m EDTA, 16.7 m m Tris-HCl, pH 8.1) and precleared for 1 h at 4 °C with DNA-blocked protein G-agarose (Millipore).

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Immunoprecipitation, Positive Control