Structured Review

Millipore edta
Effects of <t>EDTA,</t> heparin, and <t>integrin</t> β1 blocking antibody on the attachment of HUVECs on peptide-coated plates (A) Effect of EDTA and heparin on HUVECs attachment to the peptide-coated plates. 96-well plates were coated with peptides and Fbln7-C as indicated. Either 5 mM EDTA or 10 μg/ml heparin was added to the cell suspension. After a 10 min incubation at 37°C, cells were added to the wells. After 1 hour, the cells were fixed and stained with Crystal Violet and absorbance at 570 nm was measured. Each value represents the mean of six replicates ± SD. p=0.01–0.05; **p=0.001–0.01; ***p=0.0001–0.001. (B) Effects of anti-integrin β1 blocking antibody on HUVECs attachment to the peptides. Cells were preincubated for 10 min at 37°C with 10 μg/ml of the integrin β1-blocking antibody and then added to the wells. Quantification was assessed as described in (A). Each value represents the mean of six replicates ± SD. *p=0.01–0.05; **p=0.001–0.01. Fbln7-C recombinant protein and AG73 and EF-1 peptides were used as controls.
Edta, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Identification of Peptides Derived from the C-terminal Domain of Fibulin-7 Active for Endothelial Cell Adhesion and Tube Formation Disruption"

Article Title: Identification of Peptides Derived from the C-terminal Domain of Fibulin-7 Active for Endothelial Cell Adhesion and Tube Formation Disruption

Journal: Biopolymers

doi: 10.1002/bip.22754

Effects of EDTA, heparin, and integrin β1 blocking antibody on the attachment of HUVECs on peptide-coated plates (A) Effect of EDTA and heparin on HUVECs attachment to the peptide-coated plates. 96-well plates were coated with peptides and Fbln7-C as indicated. Either 5 mM EDTA or 10 μg/ml heparin was added to the cell suspension. After a 10 min incubation at 37°C, cells were added to the wells. After 1 hour, the cells were fixed and stained with Crystal Violet and absorbance at 570 nm was measured. Each value represents the mean of six replicates ± SD. p=0.01–0.05; **p=0.001–0.01; ***p=0.0001–0.001. (B) Effects of anti-integrin β1 blocking antibody on HUVECs attachment to the peptides. Cells were preincubated for 10 min at 37°C with 10 μg/ml of the integrin β1-blocking antibody and then added to the wells. Quantification was assessed as described in (A). Each value represents the mean of six replicates ± SD. *p=0.01–0.05; **p=0.001–0.01. Fbln7-C recombinant protein and AG73 and EF-1 peptides were used as controls.
Figure Legend Snippet: Effects of EDTA, heparin, and integrin β1 blocking antibody on the attachment of HUVECs on peptide-coated plates (A) Effect of EDTA and heparin on HUVECs attachment to the peptide-coated plates. 96-well plates were coated with peptides and Fbln7-C as indicated. Either 5 mM EDTA or 10 μg/ml heparin was added to the cell suspension. After a 10 min incubation at 37°C, cells were added to the wells. After 1 hour, the cells were fixed and stained with Crystal Violet and absorbance at 570 nm was measured. Each value represents the mean of six replicates ± SD. p=0.01–0.05; **p=0.001–0.01; ***p=0.0001–0.001. (B) Effects of anti-integrin β1 blocking antibody on HUVECs attachment to the peptides. Cells were preincubated for 10 min at 37°C with 10 μg/ml of the integrin β1-blocking antibody and then added to the wells. Quantification was assessed as described in (A). Each value represents the mean of six replicates ± SD. *p=0.01–0.05; **p=0.001–0.01. Fbln7-C recombinant protein and AG73 and EF-1 peptides were used as controls.

Techniques Used: Blocking Assay, Incubation, Staining, Recombinant

Effects of EDTA, heparin, and integrin β1 blocking antibody on the attachment of HUVECs on peptide-coated plates (A) Effect of EDTA and heparin on HUVECs attachment to the peptide-coated plates. 96-well plates were coated with peptides and Fbln7-C as indicated. Either 5 mM EDTA or 10 μg/ml heparin was added to the cell suspension. After a 10 min incubation at 37°C, cells were added to the wells. After 1 hour, the cells were fixed and stained with Crystal Violet and absorbance at 570 nm was measured. Each value represents the mean of six replicates ± SD. p=0.01–0.05; **p=0.001–0.01; ***p=0.0001–0.001. (B) Effects of anti-integrin β1 blocking antibody on HUVECs attachment to the peptides. Cells were preincubated for 10 min at 37°C with 10 μg/ml of the integrin β1-blocking antibody and then added to the wells. Quantification was assessed as described in (A). Each value represents the mean of six replicates ± SD. *p=0.01–0.05; **p=0.001–0.01. Fbln7-C recombinant protein and AG73 and EF-1 peptides were used as controls.
Figure Legend Snippet: Effects of EDTA, heparin, and integrin β1 blocking antibody on the attachment of HUVECs on peptide-coated plates (A) Effect of EDTA and heparin on HUVECs attachment to the peptide-coated plates. 96-well plates were coated with peptides and Fbln7-C as indicated. Either 5 mM EDTA or 10 μg/ml heparin was added to the cell suspension. After a 10 min incubation at 37°C, cells were added to the wells. After 1 hour, the cells were fixed and stained with Crystal Violet and absorbance at 570 nm was measured. Each value represents the mean of six replicates ± SD. p=0.01–0.05; **p=0.001–0.01; ***p=0.0001–0.001. (B) Effects of anti-integrin β1 blocking antibody on HUVECs attachment to the peptides. Cells were preincubated for 10 min at 37°C with 10 μg/ml of the integrin β1-blocking antibody and then added to the wells. Quantification was assessed as described in (A). Each value represents the mean of six replicates ± SD. *p=0.01–0.05; **p=0.001–0.01. Fbln7-C recombinant protein and AG73 and EF-1 peptides were used as controls.

Techniques Used: Blocking Assay, Incubation, Staining, Recombinant

2) Product Images from "A Divalent Ion Is Crucial in the Structure and Dominant-Negative Function of ID Proteins, a Class of Helix-Loop-Helix Transcription Regulators"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0048591

Identification of the positive ion in electrophoretic mobility shift (EMSA) assays. (A) Top: Wild type ID2 titrated against 0.5 uM E47. Bottom: Wild type ID2 titrated against 0.5 uM E47 in the presence of 250 uM 18C6. (B) Top: Wild type ID2 titrated against 0.5 uM E47 in the presence of 25 uM EDTA. Bottom: Wild type ID2 titrated against 0.5 uM E47 in the presence of 25 uM EGTA. (C) A repeat of (B) with ID3 instead of ID2. (D) Normalized quantification (to E47 Control for each lane; see Methods for details) of the ID2-E47 EMSAs in (A). (E) The y-axis represents calcium levels (uM) in ID2 and ID2-E47 complexes (IE), tested using the AbcamColormetric Calcium Detection Kit (See Methods), with 400 uM protein sample concentrations.
Figure Legend Snippet: Identification of the positive ion in electrophoretic mobility shift (EMSA) assays. (A) Top: Wild type ID2 titrated against 0.5 uM E47. Bottom: Wild type ID2 titrated against 0.5 uM E47 in the presence of 250 uM 18C6. (B) Top: Wild type ID2 titrated against 0.5 uM E47 in the presence of 25 uM EDTA. Bottom: Wild type ID2 titrated against 0.5 uM E47 in the presence of 25 uM EGTA. (C) A repeat of (B) with ID3 instead of ID2. (D) Normalized quantification (to E47 Control for each lane; see Methods for details) of the ID2-E47 EMSAs in (A). (E) The y-axis represents calcium levels (uM) in ID2 and ID2-E47 complexes (IE), tested using the AbcamColormetric Calcium Detection Kit (See Methods), with 400 uM protein sample concentrations.

Techniques Used: Electrophoretic Mobility Shift Assay

3) Product Images from "An ABC transport system that maintains lipid asymmetry in the Gram-negative outer membrane"

Article Title: An ABC transport system that maintains lipid asymmetry in the Gram-negative outer membrane

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0903229106

Suppressors of Δ mlaC SDS-EDTA S . ( A ) Each suppressor class is labeled 1–4. The pldA translation start is indicated as +1. The predicted −10 hexamer and the ribosomal binding site ( rbs ), the inferred −35
Figure Legend Snippet: Suppressors of Δ mlaC SDS-EDTA S . ( A ) Each suppressor class is labeled 1–4. The pldA translation start is indicated as +1. The predicted −10 hexamer and the ribosomal binding site ( rbs ), the inferred −35

Techniques Used: Labeling, Binding Assay

The mla genes and the SDS-EDTA S profiles of various strains. ( A ) The relative chromosomal positions of the mlaFEDCB operon and the mlaA gene are given in minutes, and the original gene names are indicated in parentheses. ( B ) CFU/mL on agar media containing
Figure Legend Snippet: The mla genes and the SDS-EDTA S profiles of various strains. ( A ) The relative chromosomal positions of the mlaFEDCB operon and the mlaA gene are given in minutes, and the original gene names are indicated in parentheses. ( B ) CFU/mL on agar media containing

Techniques Used:

4) Product Images from "Liquid Cladding Mediated Optical Fiber Sensors for Copper Ion Detection"

Article Title: Liquid Cladding Mediated Optical Fiber Sensors for Copper Ion Detection

Journal: Micromachines

doi: 10.3390/mi9090471

Scanning electron microscopy (SEM) images of ( a ) optical fiber core; ( b ) fiber core treated with APTES; ( c ) fiber core/APTES functionalized with chitosan conjugated EDTA (the inset image is a part of fiber cross-section that contains parts of immobilized layers and the fiber core).
Figure Legend Snippet: Scanning electron microscopy (SEM) images of ( a ) optical fiber core; ( b ) fiber core treated with APTES; ( c ) fiber core/APTES functionalized with chitosan conjugated EDTA (the inset image is a part of fiber cross-section that contains parts of immobilized layers and the fiber core).

Techniques Used: Electron Microscopy

The fiber sensor device output power vs. Cu 2+ concentrations (0 to 2 mM) with APTES/chitosan-EDTA functionalization on the surface.
Figure Legend Snippet: The fiber sensor device output power vs. Cu 2+ concentrations (0 to 2 mM) with APTES/chitosan-EDTA functionalization on the surface.

Techniques Used:

Atomic force microscope (AFM) image of ( a ) silica surface; ( b ) APTES-treated silica surface; ( c ) surface functionalized with chitosan conjugated EDTA.
Figure Legend Snippet: Atomic force microscope (AFM) image of ( a ) silica surface; ( b ) APTES-treated silica surface; ( c ) surface functionalized with chitosan conjugated EDTA.

Techniques Used: Microscopy

5) Product Images from "Liquid Cladding Mediated Optical Fiber Sensors for Copper Ion Detection"

Article Title: Liquid Cladding Mediated Optical Fiber Sensors for Copper Ion Detection

Journal: Micromachines

doi: 10.3390/mi9090471

Scanning electron microscopy (SEM) images of ( a ) optical fiber core; ( b ) fiber core treated with APTES; ( c ) fiber core/APTES functionalized with chitosan conjugated EDTA (the inset image is a part of fiber cross-section that contains parts of immobilized layers and the fiber core).
Figure Legend Snippet: Scanning electron microscopy (SEM) images of ( a ) optical fiber core; ( b ) fiber core treated with APTES; ( c ) fiber core/APTES functionalized with chitosan conjugated EDTA (the inset image is a part of fiber cross-section that contains parts of immobilized layers and the fiber core).

Techniques Used: Electron Microscopy

The fiber sensor device output power vs. Cu 2+ concentrations (0 to 2 mM) with APTES/chitosan-EDTA functionalization on the surface.
Figure Legend Snippet: The fiber sensor device output power vs. Cu 2+ concentrations (0 to 2 mM) with APTES/chitosan-EDTA functionalization on the surface.

Techniques Used:

Atomic force microscope (AFM) image of ( a ) silica surface; ( b ) APTES-treated silica surface; ( c ) surface functionalized with chitosan conjugated EDTA.
Figure Legend Snippet: Atomic force microscope (AFM) image of ( a ) silica surface; ( b ) APTES-treated silica surface; ( c ) surface functionalized with chitosan conjugated EDTA.

Techniques Used: Microscopy

6) Product Images from "Expression and oncogenic properties of membranous Notch1 in oral leukoplakia and oral squamous cell carcinoma"

Article Title: Expression and oncogenic properties of membranous Notch1 in oral leukoplakia and oral squamous cell carcinoma

Journal: Oncology Reports

doi: 10.3892/or.2018.6335

Validation of Notch1 antibody for detection of membranous and nuclear Notch1 in HN4 cells. (A) Immunofluorescent staining was observed after HN4 cells were treated with PBS (a-c) or 2.5 mM EDTA (d-f) for 10 min and immunostained with the anti-Notch1 antibody. The cells treated with PBS demonstrated strong membranous staining of Notch1 (a-c). An obvious nuclear enrichment of Notch1 staining was observed in the cells treated with EDTA (d-f). Scale bars, 10 µm. (B) Western blot analysis (left) and the quantification (right) of protein extracted from HN4 cells with different treatments revealed transmembranous (TM) Notch1 (S2 cleaved and S3 uncleaved, solid dot) and activated NICD (S3 cleaved, hollow circle). In PBS-treated HN4 cells, the Notch1 protein was mostly in the transmembranous form. The EDTA treatment induced NICD S3-cleaved, as a smaller size band was detected by the Notch1 antibody, and the cleaved state was further confirmed by the Notch1 Val1744-specific antibody. The treatment with CaCl 2 neutralized the function of EDTA and reversed the S3-cleaved status induced by EDTA. The PBS-treated cells were set as the control group. The quantification analysis was calculated from three independent experiments.
Figure Legend Snippet: Validation of Notch1 antibody for detection of membranous and nuclear Notch1 in HN4 cells. (A) Immunofluorescent staining was observed after HN4 cells were treated with PBS (a-c) or 2.5 mM EDTA (d-f) for 10 min and immunostained with the anti-Notch1 antibody. The cells treated with PBS demonstrated strong membranous staining of Notch1 (a-c). An obvious nuclear enrichment of Notch1 staining was observed in the cells treated with EDTA (d-f). Scale bars, 10 µm. (B) Western blot analysis (left) and the quantification (right) of protein extracted from HN4 cells with different treatments revealed transmembranous (TM) Notch1 (S2 cleaved and S3 uncleaved, solid dot) and activated NICD (S3 cleaved, hollow circle). In PBS-treated HN4 cells, the Notch1 protein was mostly in the transmembranous form. The EDTA treatment induced NICD S3-cleaved, as a smaller size band was detected by the Notch1 antibody, and the cleaved state was further confirmed by the Notch1 Val1744-specific antibody. The treatment with CaCl 2 neutralized the function of EDTA and reversed the S3-cleaved status induced by EDTA. The PBS-treated cells were set as the control group. The quantification analysis was calculated from three independent experiments.

Techniques Used: Staining, Western Blot

7) Product Images from "A Solid-State Hard Microfluidic-Nanopore Biosensor with Multilayer Fluidics and On-Chip Bioassay/Purification Chamber"

Article Title: A Solid-State Hard Microfluidic-Nanopore Biosensor with Multilayer Fluidics and On-Chip Bioassay/Purification Chamber

Journal: Advanced functional materials

doi: 10.1002/adfm.201804182

a) An open pore current trace of a 7 nm diameter nanopore (25 nm thickness) in a conventional chamber. The open pore current is 6.85 ± 0.18 nA. b) An open pore current trace of the same pore, but assembled into the microfluidic chamber. The open pore current is 6.96 ± 0.19 nA. Both traces were measured using a 100 KHz bandwidth. c) Comparison of the power spectral density of the current in the conventional system and microfluidic system. The electrolyte solution is 1 M KCl 50 mM phosphate 5 mM EDTA. +500 mV bias toward trans chamber. Signals were filtered using the Axon 200B built-in Bessel filter at 100 KHz.
Figure Legend Snippet: a) An open pore current trace of a 7 nm diameter nanopore (25 nm thickness) in a conventional chamber. The open pore current is 6.85 ± 0.18 nA. b) An open pore current trace of the same pore, but assembled into the microfluidic chamber. The open pore current is 6.96 ± 0.19 nA. Both traces were measured using a 100 KHz bandwidth. c) Comparison of the power spectral density of the current in the conventional system and microfluidic system. The electrolyte solution is 1 M KCl 50 mM phosphate 5 mM EDTA. +500 mV bias toward trans chamber. Signals were filtered using the Axon 200B built-in Bessel filter at 100 KHz.

Techniques Used:

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: 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.

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.

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).

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.

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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    Millipore edta
    Treatment of <t>ARDS</t> tracheal aspirates with MMP-8 and MMP-9 inhibitors. A. Tracheal aspirates from ARDS subjects (n = 4) with high MMP-8 activity were examined in the presence of different MMP inhibitors. MMP-8 was reduced by 40–50% relative to basal activity when incubated with 5 mM of <t>EDTA</t> ( † p = 0.04 ) and 30 ng/ml of a specific MMP-8 inhibitor ( ‡ p = 0.04 ) versus basal activity and vehicle controls. Incubation with 100 mcg/ml of doxycycline resulted in a 10% decrease in MMP-8 activity compared to basal levels (* p = 0.05 ). B. Tracheal aspirates from ARDS subjects (n = 4) with high MMP-9 activity were examined in the presence of different MMP inhibitors. MMP-9 activity decreased by 70–100% relative to basal activity in the presence of EDTA (5 mM; † p = 0.008 ) and a MMP-9 specific inhibitor (50 ng/ml; ‡ p = 0.008 ). Inhibition with doxycycline (100 mcg/ml) decreased MMP-9 activity by approximately 40% of basal activity (* p = 0.04 ).
    Edta, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/edta/product/Millipore
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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
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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
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    Average 98 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
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    Image Search Results


    Treatment of ARDS tracheal aspirates with MMP-8 and MMP-9 inhibitors. A. Tracheal aspirates from ARDS subjects (n = 4) with high MMP-8 activity were examined in the presence of different MMP inhibitors. MMP-8 was reduced by 40–50% relative to basal activity when incubated with 5 mM of EDTA ( † p = 0.04 ) and 30 ng/ml of a specific MMP-8 inhibitor ( ‡ p = 0.04 ) versus basal activity and vehicle controls. Incubation with 100 mcg/ml of doxycycline resulted in a 10% decrease in MMP-8 activity compared to basal levels (* p = 0.05 ). B. Tracheal aspirates from ARDS subjects (n = 4) with high MMP-9 activity were examined in the presence of different MMP inhibitors. MMP-9 activity decreased by 70–100% relative to basal activity in the presence of EDTA (5 mM; † p = 0.008 ) and a MMP-9 specific inhibitor (50 ng/ml; ‡ p = 0.008 ). Inhibition with doxycycline (100 mcg/ml) decreased MMP-9 activity by approximately 40% of basal activity (* p = 0.04 ).

    Journal: PLoS ONE

    Article Title: Early Elevation of Matrix Metalloproteinase-8 and -9 in Pediatric ARDS Is Associated with an Increased Risk of Prolonged Mechanical Ventilation

    doi: 10.1371/journal.pone.0022596

    Figure Lengend Snippet: Treatment of ARDS tracheal aspirates with MMP-8 and MMP-9 inhibitors. A. Tracheal aspirates from ARDS subjects (n = 4) with high MMP-8 activity were examined in the presence of different MMP inhibitors. MMP-8 was reduced by 40–50% relative to basal activity when incubated with 5 mM of EDTA ( † p = 0.04 ) and 30 ng/ml of a specific MMP-8 inhibitor ( ‡ p = 0.04 ) versus basal activity and vehicle controls. Incubation with 100 mcg/ml of doxycycline resulted in a 10% decrease in MMP-8 activity compared to basal levels (* p = 0.05 ). B. Tracheal aspirates from ARDS subjects (n = 4) with high MMP-9 activity were examined in the presence of different MMP inhibitors. MMP-9 activity decreased by 70–100% relative to basal activity in the presence of EDTA (5 mM; † p = 0.008 ) and a MMP-9 specific inhibitor (50 ng/ml; ‡ p = 0.008 ). Inhibition with doxycycline (100 mcg/ml) decreased MMP-9 activity by approximately 40% of basal activity (* p = 0.04 ).

    Article Snippet: Treatment of ARDS lower airway samples with MMP-8 and -9 inhibitors Tracheal aspirates from ARDS subjects with high MMP-8 and -9 activity were incubated with EDTA (5 mM), MMP-8 specific inhibitor (Calbiochem #44237 at 30 ng/ml), MMP-9 specific inhibitor (Calbiochem #444278 at 50 ng/ml) and doxycyline (100 mcg/ml) or vehicle (DMSO, 1∶1000) for 2 hours followed by MMP-8 and -9 activity measurement (R & D Systems).

    Techniques: Activity Assay, Incubation, Inhibition

    TEM images of particles formed by various DNA samples upon condensation with the Tat-NLS peptide. ( A ) Condensates formed by the nicked-DNA duplexes of oligonucleotides N1 and N2. ( B ) Condensates formed by the gapped-DNA duplexes of oligonucleotides G1 and G2. ( C ) Condensates formed by the nicked-gapped-DNA duplex of oligonucleotides N1 and G2. ( D ) Condensates formed by 3kbDNA . ( E ) Condensates formed by 21mer duplex. For all samples, DNA was 15 μM in base pair, and was condensed by mixing with the Tat-NLS peptide at a charge ratio of 1:2 (DNA phosphate:cationic charged group of the peptide) in 5 mM Bis-Tris, 50 μM EDTA (pH 7.0). Scale bar is 100 nm.

    Journal: Nucleic Acids Research

    Article Title: Condensation of oligonucleotides assembled into nicked and gapped duplexes: potential structures for oligonucleotide delivery

    doi: 10.1093/nar/gki156

    Figure Lengend Snippet: TEM images of particles formed by various DNA samples upon condensation with the Tat-NLS peptide. ( A ) Condensates formed by the nicked-DNA duplexes of oligonucleotides N1 and N2. ( B ) Condensates formed by the gapped-DNA duplexes of oligonucleotides G1 and G2. ( C ) Condensates formed by the nicked-gapped-DNA duplex of oligonucleotides N1 and G2. ( D ) Condensates formed by 3kbDNA . ( E ) Condensates formed by 21mer duplex. For all samples, DNA was 15 μM in base pair, and was condensed by mixing with the Tat-NLS peptide at a charge ratio of 1:2 (DNA phosphate:cationic charged group of the peptide) in 5 mM Bis-Tris, 50 μM EDTA (pH 7.0). Scale bar is 100 nm.

    Article Snippet: The linearized plasmid DNA was rinsed five times with 5 mM Bis-Tris and 50 μM EDTA (pH 7.0) using a Microcon YM-30 spin column (Millipore, Bedford, MA) to remove excess salt introduced during restriction digestion.

    Techniques: Transmission Electron Microscopy

    Condensation of 3kbDNA , nicked-DNA and a 21mer duplex by hexammine cobalt chloride, as monitored by light scattering. For each data point shown, DNA concentration was 7.5 μM in base pair (5 mM Bis-Tris, 50 μM EDTA, pH 7.0). Light-scattering intensities shown are averages from measurements taken over a 5 min period. Inset : 3kbDNA and nicked-DNA scattering intensities, as a function of hexammine cobalt chloride concentration, normalized to maximum observed intensity. Note : nicked-DNA condensation occurs at a lower hexammine cobalt chloride concentration than 3kbDNA .

    Journal: Nucleic Acids Research

    Article Title: Condensation of oligonucleotides assembled into nicked and gapped duplexes: potential structures for oligonucleotide delivery

    doi: 10.1093/nar/gki156

    Figure Lengend Snippet: Condensation of 3kbDNA , nicked-DNA and a 21mer duplex by hexammine cobalt chloride, as monitored by light scattering. For each data point shown, DNA concentration was 7.5 μM in base pair (5 mM Bis-Tris, 50 μM EDTA, pH 7.0). Light-scattering intensities shown are averages from measurements taken over a 5 min period. Inset : 3kbDNA and nicked-DNA scattering intensities, as a function of hexammine cobalt chloride concentration, normalized to maximum observed intensity. Note : nicked-DNA condensation occurs at a lower hexammine cobalt chloride concentration than 3kbDNA .

    Article Snippet: The linearized plasmid DNA was rinsed five times with 5 mM Bis-Tris and 50 μM EDTA (pH 7.0) using a Microcon YM-30 spin column (Millipore, Bedford, MA) to remove excess salt introduced during restriction digestion.

    Techniques: Concentration Assay

    TEM images of particles formed by various DNA samples upon condensation with hexammine cobalt chloride. ( A ) Condensates formed by the nicked-DNA duplexes of oligonucleotides N1 and N2. ( B ) Condensates formed by the gapped-DNA duplexes of oligonucleotides G1 and G2. ( C ) Condensates formed by the nicked-gapped-DNA duplex of oligonucleotides N1 and G2. ( D ) Condensates formed by 3kbDNA . For all samples, DNA was 15 μM in base pair, and condensed by mixing with an equal volume of 200 μM hexammine cobalt chloride in 5 mM Bis-Tris, 50 μM EDTA (pH 7.0). Scale bar is 100 nm.

    Journal: Nucleic Acids Research

    Article Title: Condensation of oligonucleotides assembled into nicked and gapped duplexes: potential structures for oligonucleotide delivery

    doi: 10.1093/nar/gki156

    Figure Lengend Snippet: TEM images of particles formed by various DNA samples upon condensation with hexammine cobalt chloride. ( A ) Condensates formed by the nicked-DNA duplexes of oligonucleotides N1 and N2. ( B ) Condensates formed by the gapped-DNA duplexes of oligonucleotides G1 and G2. ( C ) Condensates formed by the nicked-gapped-DNA duplex of oligonucleotides N1 and G2. ( D ) Condensates formed by 3kbDNA . For all samples, DNA was 15 μM in base pair, and condensed by mixing with an equal volume of 200 μM hexammine cobalt chloride in 5 mM Bis-Tris, 50 μM EDTA (pH 7.0). Scale bar is 100 nm.

    Article Snippet: The linearized plasmid DNA was rinsed five times with 5 mM Bis-Tris and 50 μM EDTA (pH 7.0) using a Microcon YM-30 spin column (Millipore, Bedford, MA) to remove excess salt introduced during restriction digestion.

    Techniques: Transmission Electron Microscopy

    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

    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