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Applichem dnaase i
Dnaase I, supplied by Applichem, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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dnaase i - by Bioz Stars, 2020-04
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Related Articles

Clone Assay:

Article Title: NO• Binds Human Cystathionine β-Synthase Quickly and Tightly *
Article Snippet: Human CBS cDNA was cloned into a pET28b vector with restriction sites for NdeI and XhoI, yielding an N-terminally His-tagged protein, as described in Ref. . .. Following a 4-h incubation at 22 °C and 120 rpm, cells were harvested by centrifugation and the pellet resuspended in buffer A (50 m m potassium phosphate, 300 m m KCl, pH 7.0, 10% glycerol; 10 ml of buffer per liter of culture) containing 1 mg/ml lysozyme (AppliChem), 1 m m phenylmethylsulfonyl fluoride (Merck), and a few grains of deoxyribonuclease I (AppliChem).

Centrifugation:

Article Title: Evolutionary and Structural Analyses of Mammalian Haloacid Dehalogenase-type Phosphatases AUM and Chronophin Provide Insight into the Basis of Their Different Substrate Specificities *
Article Snippet: .. Cells were lysed in the presence of 150 units/ml DNase I (Applichem) using a cell disruptor (Constant Systems), and cell debris was removed by centrifugation (10,000 × g , 30 min, 4 °C). .. For His6 -AUM purification, cleared supernatants were loaded on a nickel-nitrilotriacetic acid-agarose column (HisTrap HP, GE Healthcare) operated on an ÄKTA liquid chromatography system (GE Healthcare) in TNM, and His6 -tagged proteins were eluted using a linear 10–400 m m imidazole gradient in TNM.

Article Title: S-Adenosyl-l-methionine Modulates CO and NO• Binding to the Human H2S-generating Enzyme Cystathionine β-Synthase *
Article Snippet: .. After a 16-h incubation at 25 °C and 120 rpm, cells were harvested by centrifugation and resuspended in 50 m m potassium phosphate, 300 m m KCl, 10% glycerol, pH 7.0 (buffer A, 10 ml was used to resuspend cells from 1 liter of culture), containing 1 m m phenylmethylsulfonyl fluoride (Merck), 1 mg/ml lysozyme (Applichem), and deoxyribonuclease I (Applichem). .. The cleared supernatant was supplemented with 10 m m imidazole and loaded at 2.5 ml/min onto a HisTrap FF crude 5-ml column (GE Healthcare) previously equilibrated with buffer A containing 10 m m imidazole (buffer B).

Article Title: NO• Binds Human Cystathionine β-Synthase Quickly and Tightly *
Article Snippet: .. Following a 4-h incubation at 22 °C and 120 rpm, cells were harvested by centrifugation and the pellet resuspended in buffer A (50 m m potassium phosphate, 300 m m KCl, pH 7.0, 10% glycerol; 10 ml of buffer per liter of culture) containing 1 mg/ml lysozyme (AppliChem), 1 m m phenylmethylsulfonyl fluoride (Merck), and a few grains of deoxyribonuclease I (AppliChem). .. After a 30-min incubation on ice, cells were disrupted by sonication, centrifuged at 8200 × g (5 min, 4 °C), and imidazole (10 m m ) was added to the cleared supernatant.

Amplification:

Article Title: The Salmonella Spi1 Virulence Regulatory Protein HilD Directly Activates Transcription of the Flagellar Master Operon flhDC
Article Snippet: The flhDC DNA fragment comprising the putative HilD binding site was PCR amplified from Salmonella Typhimurium LT2 genomic DNA using 5′ digoxigenin (DIG)-labeled primers (5′-GATCATGCTGACACGTACGGATTCTTATGTAAAGAATCGTGGC-3′ and 5′-DIG-GTCAACACCAAATTCTTTTTTG-3′). .. DNase I (AppliChem) was added to the binding reaction mixtures containing 100 ng DIG-labeled DNA fragments and increasing amounts of HilD after 20 min at RT.

Cytometry:

Article Title: IgE promotes type 2 innate lymphoid cells in murine food allergy
Article Snippet: Leukocytes were isolated from the jejunal lamina propria for analysis by flow cytometry and cell culture according to previously described procedures [ , ]. .. Remaining tissue was chopped finely prior to digestion for 30–45min with 1.6mg/ml collagenase IV (Worthington Biochemicals), 0.3mg/ml hyaluronidase (Worthington Biochemicals) and 100μg/ml DNAse I (Applichem) in complete RPMI supplemented with 20% FCS, 2mM CaCl2 and 2mM MgCl2 (Sigma Aldrich).

Construct:

Article Title: Evolutionary and Structural Analyses of Mammalian Haloacid Dehalogenase-type Phosphatases AUM and Chronophin Provide Insight into the Basis of Their Different Substrate Specificities *
Article Snippet: pETM11 constructs encoding for His6 -tagged AUMWT and AUM variants were transformed into Escherichia coli BL21 (DE3) and expressed for 20 h at 28 °C after induction with 0.5 m m isopropyl β- d -1-thiogalactopyranoside supplemented with 20 μg/ml chloramphenicol, 50 μg/ml kanamycin, and 1 ng/ml tetracycline. .. Cells were lysed in the presence of 150 units/ml DNase I (Applichem) using a cell disruptor (Constant Systems), and cell debris was removed by centrifugation (10,000 × g , 30 min, 4 °C).

Article Title: S-Adenosyl-l-methionine Modulates CO and NO• Binding to the Human H2S-generating Enzyme Cystathionine β-Synthase *
Article Snippet: The resulting pET28b-ΔhCBS construct was used to transform Escherichia coli BL21(DE3) Rosetta cells. .. After a 16-h incubation at 25 °C and 120 rpm, cells were harvested by centrifugation and resuspended in 50 m m potassium phosphate, 300 m m KCl, 10% glycerol, pH 7.0 (buffer A, 10 ml was used to resuspend cells from 1 liter of culture), containing 1 m m phenylmethylsulfonyl fluoride (Merck), 1 mg/ml lysozyme (Applichem), and deoxyribonuclease I (Applichem).

Real-time Polymerase Chain Reaction:

Article Title: Correct Capsid Assembly Mediated by a Conserved YXXLGL Motif in Prototype Foamy Virus Gag Is Essential for Infectivity and Reverse Transcription of the Viral Genome ▿
Article Snippet: .. Viral supernatants for real-time PCR analysis were generated as described above, with the modification that during the sodium butyrate induction step and the subsequent viral supernatant production step, DNase I ( > 3,000 U/mg; Applichem) was added to the medium to a final concentration of 500 μg/ml. .. Forty-eight hours posttransfection, the cell-free viral supernatant was harvested by sterile filtration (pore size, 0.45 μm) and centrifuged at 4°C and 25,000 rpm for 2 h in an SW40 or SW28 rotor through a 20% sucrose cushion.

Incubation:

Article Title: S-Adenosyl-l-methionine Modulates CO and NO• Binding to the Human H2S-generating Enzyme Cystathionine β-Synthase *
Article Snippet: .. After a 16-h incubation at 25 °C and 120 rpm, cells were harvested by centrifugation and resuspended in 50 m m potassium phosphate, 300 m m KCl, 10% glycerol, pH 7.0 (buffer A, 10 ml was used to resuspend cells from 1 liter of culture), containing 1 m m phenylmethylsulfonyl fluoride (Merck), 1 mg/ml lysozyme (Applichem), and deoxyribonuclease I (Applichem). .. The cleared supernatant was supplemented with 10 m m imidazole and loaded at 2.5 ml/min onto a HisTrap FF crude 5-ml column (GE Healthcare) previously equilibrated with buffer A containing 10 m m imidazole (buffer B).

Article Title: IgE promotes type 2 innate lymphoid cells in murine food allergy
Article Snippet: The intestinal epithelium was removed from 1cm intestinal segments with a 30min incubation at 37°C in a rotary incubator in PBS containing 2% FCS, 10mM EDTA, and 1.5mM dithioerythritol. .. Remaining tissue was chopped finely prior to digestion for 30–45min with 1.6mg/ml collagenase IV (Worthington Biochemicals), 0.3mg/ml hyaluronidase (Worthington Biochemicals) and 100μg/ml DNAse I (Applichem) in complete RPMI supplemented with 20% FCS, 2mM CaCl2 and 2mM MgCl2 (Sigma Aldrich).

Article Title: Extracellular nucleases and extracellular DNA play important roles in Vibrio cholerae biofilm formation
Article Snippet: At the respective time points the wells were rinsed four times with spent LB (obtained from a 24 or 72 h old biofilm culture of the respective strain) before DNase I (AppliChem), λExonuclease (New England Biolabs) or in combination were added to a final concentration of 133 Kunitz units per ml. .. The biofilm was incubated with the respective solution for 3 or 7 h at RT respectively.

Article Title: The Salmonella Spi1 Virulence Regulatory Protein HilD Directly Activates Transcription of the Flagellar Master Operon flhDC
Article Snippet: .. An flhDC promoter fragment comprising nucleotides −668 to −388 upstream from the flhDC coding region was incubated with increasing concentrations of purified HilD protein and, after partial digestion with DNase I, the resulting fragments were subjected to denaturing gel electrophoresis ( ). ..

Article Title: Differentiated Type II Pneumocytes Can Be Reprogrammed by Ectopic Sox2 Expression
Article Snippet: .. Lungs were isolated, incubated for 45 minutes in 1 ml Dispase at room temperature and transferred to a culture dish containing 5 ml DMEM/F12 medium (Gibco) supplemented with 0.04 mg/ml DNase I (AppliChem), 3.6 mg/ml D-(+)-Glucose (AppliChem) and 1% Penicillin/Streptomycin (P/S). ..

Article Title: NO• Binds Human Cystathionine β-Synthase Quickly and Tightly *
Article Snippet: .. Following a 4-h incubation at 22 °C and 120 rpm, cells were harvested by centrifugation and the pellet resuspended in buffer A (50 m m potassium phosphate, 300 m m KCl, pH 7.0, 10% glycerol; 10 ml of buffer per liter of culture) containing 1 mg/ml lysozyme (AppliChem), 1 m m phenylmethylsulfonyl fluoride (Merck), and a few grains of deoxyribonuclease I (AppliChem). .. After a 30-min incubation on ice, cells were disrupted by sonication, centrifuged at 8200 × g (5 min, 4 °C), and imidazole (10 m m ) was added to the cleared supernatant.

Activity Assay:

Article Title: Evolutionary and Structural Analyses of Mammalian Haloacid Dehalogenase-type Phosphatases AUM and Chronophin Provide Insight into the Basis of Their Different Substrate Specificities *
Article Snippet: Cells were lysed in the presence of 150 units/ml DNase I (Applichem) using a cell disruptor (Constant Systems), and cell debris was removed by centrifugation (10,000 × g , 30 min, 4 °C). .. Peak fractions were tested for phosphatase activity using p NPP as a substrate (see below).

Cell Culture:

Article Title: IgE promotes type 2 innate lymphoid cells in murine food allergy
Article Snippet: Leukocytes were isolated from the jejunal lamina propria for analysis by flow cytometry and cell culture according to previously described procedures [ , ]. .. Remaining tissue was chopped finely prior to digestion for 30–45min with 1.6mg/ml collagenase IV (Worthington Biochemicals), 0.3mg/ml hyaluronidase (Worthington Biochemicals) and 100μg/ml DNAse I (Applichem) in complete RPMI supplemented with 20% FCS, 2mM CaCl2 and 2mM MgCl2 (Sigma Aldrich).

Article Title: Differentiated Type II Pneumocytes Can Be Reprogrammed by Ectopic Sox2 Expression
Article Snippet: Lungs were isolated, incubated for 45 minutes in 1 ml Dispase at room temperature and transferred to a culture dish containing 5 ml DMEM/F12 medium (Gibco) supplemented with 0.04 mg/ml DNase I (AppliChem), 3.6 mg/ml D-(+)-Glucose (AppliChem) and 1% Penicillin/Streptomycin (P/S). .. AVTII cells were cultured in DMEM/F10 containing 10% FCS and 1% P/S in tissue culture cover slip immersed in 12 well plates (Corning, NY) previously coated with collagen (Inamed); cultures were maintained in a 5% CO2 /air incubator.

Expressing:

Article Title: Evolutionary and Structural Analyses of Mammalian Haloacid Dehalogenase-type Phosphatases AUM and Chronophin Provide Insight into the Basis of Their Different Substrate Specificities *
Article Snippet: Paragraph title: Recombinant Protein Expression and Purification ... Cells were lysed in the presence of 150 units/ml DNase I (Applichem) using a cell disruptor (Constant Systems), and cell debris was removed by centrifugation (10,000 × g , 30 min, 4 °C).

Article Title: S-Adenosyl-l-methionine Modulates CO and NO• Binding to the Human H2S-generating Enzyme Cystathionine β-Synthase *
Article Snippet: Paragraph title: Expression and Purification of CBS ... After a 16-h incubation at 25 °C and 120 rpm, cells were harvested by centrifugation and resuspended in 50 m m potassium phosphate, 300 m m KCl, 10% glycerol, pH 7.0 (buffer A, 10 ml was used to resuspend cells from 1 liter of culture), containing 1 m m phenylmethylsulfonyl fluoride (Merck), 1 mg/ml lysozyme (Applichem), and deoxyribonuclease I (Applichem).

Article Title: NO• Binds Human Cystathionine β-Synthase Quickly and Tightly *
Article Snippet: Paragraph title: Protein Expression and Purification ... Following a 4-h incubation at 22 °C and 120 rpm, cells were harvested by centrifugation and the pellet resuspended in buffer A (50 m m potassium phosphate, 300 m m KCl, pH 7.0, 10% glycerol; 10 ml of buffer per liter of culture) containing 1 mg/ml lysozyme (AppliChem), 1 m m phenylmethylsulfonyl fluoride (Merck), and a few grains of deoxyribonuclease I (AppliChem).

Modification:

Article Title: Correct Capsid Assembly Mediated by a Conserved YXXLGL Motif in Prototype Foamy Virus Gag Is Essential for Infectivity and Reverse Transcription of the Viral Genome ▿
Article Snippet: .. Viral supernatants for real-time PCR analysis were generated as described above, with the modification that during the sodium butyrate induction step and the subsequent viral supernatant production step, DNase I ( > 3,000 U/mg; Applichem) was added to the medium to a final concentration of 500 μg/ml. .. Forty-eight hours posttransfection, the cell-free viral supernatant was harvested by sterile filtration (pore size, 0.45 μm) and centrifuged at 4°C and 25,000 rpm for 2 h in an SW40 or SW28 rotor through a 20% sucrose cushion.

Western Blot:

Article Title: Correct Capsid Assembly Mediated by a Conserved YXXLGL Motif in Prototype Foamy Virus Gag Is Essential for Infectivity and Reverse Transcription of the Viral Genome ▿
Article Snippet: Viral supernatants for real-time PCR analysis were generated as described above, with the modification that during the sodium butyrate induction step and the subsequent viral supernatant production step, DNase I ( > 3,000 U/mg; Applichem) was added to the medium to a final concentration of 500 μg/ml. .. Seventy-five microliters of the sample was added to an equal volume of 2× sodium dodecyl sulfate protein sample buffer, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and analyzed by Western blotting as described above.

Transformation Assay:

Article Title: Evolutionary and Structural Analyses of Mammalian Haloacid Dehalogenase-type Phosphatases AUM and Chronophin Provide Insight into the Basis of Their Different Substrate Specificities *
Article Snippet: pETM11 constructs encoding for His6 -tagged AUMWT and AUM variants were transformed into Escherichia coli BL21 (DE3) and expressed for 20 h at 28 °C after induction with 0.5 m m isopropyl β- d -1-thiogalactopyranoside supplemented with 20 μg/ml chloramphenicol, 50 μg/ml kanamycin, and 1 ng/ml tetracycline. .. Cells were lysed in the presence of 150 units/ml DNase I (Applichem) using a cell disruptor (Constant Systems), and cell debris was removed by centrifugation (10,000 × g , 30 min, 4 °C).

Over Expression:

Article Title: NO• Binds Human Cystathionine β-Synthase Quickly and Tightly *
Article Snippet: Protein overexpression was induced by the addition of 0.5 m m isopropyl-β- d -thiogalactopyranoside (Nzytech). δ-Aminolevulinic acid (Sigma) was added to the culture medium to a final concentration of 75 mg/liter. .. Following a 4-h incubation at 22 °C and 120 rpm, cells were harvested by centrifugation and the pellet resuspended in buffer A (50 m m potassium phosphate, 300 m m KCl, pH 7.0, 10% glycerol; 10 ml of buffer per liter of culture) containing 1 mg/ml lysozyme (AppliChem), 1 m m phenylmethylsulfonyl fluoride (Merck), and a few grains of deoxyribonuclease I (AppliChem).

Flow Cytometry:

Article Title: IgE promotes type 2 innate lymphoid cells in murine food allergy
Article Snippet: Leukocytes were isolated from the jejunal lamina propria for analysis by flow cytometry and cell culture according to previously described procedures [ , ]. .. Remaining tissue was chopped finely prior to digestion for 30–45min with 1.6mg/ml collagenase IV (Worthington Biochemicals), 0.3mg/ml hyaluronidase (Worthington Biochemicals) and 100μg/ml DNAse I (Applichem) in complete RPMI supplemented with 20% FCS, 2mM CaCl2 and 2mM MgCl2 (Sigma Aldrich).

Protease Inhibitor:

Article Title: Evolutionary and Structural Analyses of Mammalian Haloacid Dehalogenase-type Phosphatases AUM and Chronophin Provide Insight into the Basis of Their Different Substrate Specificities *
Article Snippet: Cells were harvested at 8000 × g for 10 min and resuspended in TNM (50 m m triethanolamine (TEA), 200 m m NaCl, 5 m m MgCl2 ; pH 7.5) supplemented with 10 m m imidazole and protease inhibitors (EDTA-free protease inhibitor tablets; Roche Applied Science). .. Cells were lysed in the presence of 150 units/ml DNase I (Applichem) using a cell disruptor (Constant Systems), and cell debris was removed by centrifugation (10,000 × g , 30 min, 4 °C).

Solubility:

Article Title: Evolutionary and Structural Analyses of Mammalian Haloacid Dehalogenase-type Phosphatases AUM and Chronophin Provide Insight into the Basis of Their Different Substrate Specificities *
Article Snippet: To increase solubility, AUM was co-expressed with the chaperones groES-groEL-tig from the pG-Tf2 plasmid (Takara) according to the manufacturer's instructions. .. Cells were lysed in the presence of 150 units/ml DNase I (Applichem) using a cell disruptor (Constant Systems), and cell debris was removed by centrifugation (10,000 × g , 30 min, 4 °C).

Generated:

Article Title: Correct Capsid Assembly Mediated by a Conserved YXXLGL Motif in Prototype Foamy Virus Gag Is Essential for Infectivity and Reverse Transcription of the Viral Genome ▿
Article Snippet: .. Viral supernatants for real-time PCR analysis were generated as described above, with the modification that during the sodium butyrate induction step and the subsequent viral supernatant production step, DNase I ( > 3,000 U/mg; Applichem) was added to the medium to a final concentration of 500 μg/ml. .. Forty-eight hours posttransfection, the cell-free viral supernatant was harvested by sterile filtration (pore size, 0.45 μm) and centrifuged at 4°C and 25,000 rpm for 2 h in an SW40 or SW28 rotor through a 20% sucrose cushion.

Article Title: S-Adenosyl-l-methionine Modulates CO and NO• Binding to the Human H2S-generating Enzyme Cystathionine β-Synthase *
Article Snippet: The 1227G > A mutant (cDNA numbering) carrying a premature stop codon at position 409 was generated from the full-length CBS-expressing pET28b-based vector, using the XL QuikChange kit (Agilent) and the primers 5′-GAAGAAGCCCTGGTG A TGGCACCTCCGTG (forward) and 5′-CACGGAGGTGCCA T CACCAGGGCTTCTTC (reverse). .. After a 16-h incubation at 25 °C and 120 rpm, cells were harvested by centrifugation and resuspended in 50 m m potassium phosphate, 300 m m KCl, 10% glycerol, pH 7.0 (buffer A, 10 ml was used to resuspend cells from 1 liter of culture), containing 1 m m phenylmethylsulfonyl fluoride (Merck), 1 mg/ml lysozyme (Applichem), and deoxyribonuclease I (Applichem).

Polymerase Chain Reaction:

Article Title: The Salmonella Spi1 Virulence Regulatory Protein HilD Directly Activates Transcription of the Flagellar Master Operon flhDC
Article Snippet: The flhDC DNA fragment comprising the putative HilD binding site was PCR amplified from Salmonella Typhimurium LT2 genomic DNA using 5′ digoxigenin (DIG)-labeled primers (5′-GATCATGCTGACACGTACGGATTCTTATGTAAAGAATCGTGGC-3′ and 5′-DIG-GTCAACACCAAATTCTTTTTTG-3′). .. DNase I (AppliChem) was added to the binding reaction mixtures containing 100 ng DIG-labeled DNA fragments and increasing amounts of HilD after 20 min at RT.

Sonication:

Article Title: NO• Binds Human Cystathionine β-Synthase Quickly and Tightly *
Article Snippet: Following a 4-h incubation at 22 °C and 120 rpm, cells were harvested by centrifugation and the pellet resuspended in buffer A (50 m m potassium phosphate, 300 m m KCl, pH 7.0, 10% glycerol; 10 ml of buffer per liter of culture) containing 1 mg/ml lysozyme (AppliChem), 1 m m phenylmethylsulfonyl fluoride (Merck), and a few grains of deoxyribonuclease I (AppliChem). .. After a 30-min incubation on ice, cells were disrupted by sonication, centrifuged at 8200 × g (5 min, 4 °C), and imidazole (10 m m ) was added to the cleared supernatant.

Injection:

Article Title: Vaccination-induced skin-resident memory CD8+ T cells mediate strong protection against cutaneous melanoma
Article Snippet: Alternatively, mice were intraperitoneally injected with seven 25 µg doses of FTY720 (Sigma-Aldrich, ref SML0700) during consecutive days before CD8+ T cell analysis in blood. .. Skin pieces were then disaggregated using microscope slides with ground edges (Sail Brand, ref 7105), and single-cell suspension was obtained using a 70 μm cell strainer (BD Falcon®, ref 352350) followed by a second digestion with 5 mL of supplemented RPMI 1640 medium containing 25 µg/mL of DNAse I (AppliChem, ref A3778,0010) during 5 min on ice, to obtain cell suspensions.

Binding Assay:

Article Title: The Salmonella Spi1 Virulence Regulatory Protein HilD Directly Activates Transcription of the Flagellar Master Operon flhDC
Article Snippet: .. DNase I (AppliChem) was added to the binding reaction mixtures containing 100 ng DIG-labeled DNA fragments and increasing amounts of HilD after 20 min at RT. ..

FACS:

Article Title: Post-Aire Maturation of Thymic Medullary Epithelial Cells Involves Selective Expression of Keratinocyte-Specific Autoantigens
Article Snippet: Paragraph title: FACS sorting and analysis of sorted cells ... GentleMACS C-Tubes (Miltenyi Biotec) were used for mechanical disruption followed by enzymatic digestion in 0.5 mg/ml dispase/collagenase (Roche) and 5 μg/ml DNase I (AppliChem) in PBS at 37°C for 3 × 30 min with gentle agitation.

Article Title: The appropriate frequency and function of decidual Tim-3+CTLA-4+CD8+ T cells are important in maintaining normal pregnancy
Article Snippet: DICs and DSCs were obtained from the normal or RSA decidual tissue digesting in RPMI 1640 (HyClone, USA) supplemented with collagenase type IV (1.0 mg/ml, CLS-1, Worthington Biomedical, USA) and DNase I (150 U/ml, Applichem, Germany) as described previously . .. CD8+ T cells were isolated by magnetic affinity cell sorting using CD8 microbeads (MiltenyiBiotec, Germany).

Nucleic Acid Electrophoresis:

Article Title: Correct Capsid Assembly Mediated by a Conserved YXXLGL Motif in Prototype Foamy Virus Gag Is Essential for Infectivity and Reverse Transcription of the Viral Genome ▿
Article Snippet: Viral supernatants for real-time PCR analysis were generated as described above, with the modification that during the sodium butyrate induction step and the subsequent viral supernatant production step, DNase I ( > 3,000 U/mg; Applichem) was added to the medium to a final concentration of 500 μg/ml. .. Seventy-five microliters of the sample was added to an equal volume of 2× sodium dodecyl sulfate protein sample buffer, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and analyzed by Western blotting as described above.

Article Title: The Salmonella Spi1 Virulence Regulatory Protein HilD Directly Activates Transcription of the Flagellar Master Operon flhDC
Article Snippet: .. An flhDC promoter fragment comprising nucleotides −668 to −388 upstream from the flhDC coding region was incubated with increasing concentrations of purified HilD protein and, after partial digestion with DNase I, the resulting fragments were subjected to denaturing gel electrophoresis ( ). ..

Mutagenesis:

Article Title: S-Adenosyl-l-methionine Modulates CO and NO• Binding to the Human H2S-generating Enzyme Cystathionine β-Synthase *
Article Snippet: The 1227G > A mutant (cDNA numbering) carrying a premature stop codon at position 409 was generated from the full-length CBS-expressing pET28b-based vector, using the XL QuikChange kit (Agilent) and the primers 5′-GAAGAAGCCCTGGTG A TGGCACCTCCGTG (forward) and 5′-CACGGAGGTGCCA T CACCAGGGCTTCTTC (reverse). .. After a 16-h incubation at 25 °C and 120 rpm, cells were harvested by centrifugation and resuspended in 50 m m potassium phosphate, 300 m m KCl, 10% glycerol, pH 7.0 (buffer A, 10 ml was used to resuspend cells from 1 liter of culture), containing 1 m m phenylmethylsulfonyl fluoride (Merck), 1 mg/ml lysozyme (Applichem), and deoxyribonuclease I (Applichem).

Isolation:

Article Title: IgE promotes type 2 innate lymphoid cells in murine food allergy
Article Snippet: Paragraph title: Isolation of intestinal leukocytes ... Remaining tissue was chopped finely prior to digestion for 30–45min with 1.6mg/ml collagenase IV (Worthington Biochemicals), 0.3mg/ml hyaluronidase (Worthington Biochemicals) and 100μg/ml DNAse I (Applichem) in complete RPMI supplemented with 20% FCS, 2mM CaCl2 and 2mM MgCl2 (Sigma Aldrich).

Article Title: Differentiated Type II Pneumocytes Can Be Reprogrammed by Ectopic Sox2 Expression
Article Snippet: .. Lungs were isolated, incubated for 45 minutes in 1 ml Dispase at room temperature and transferred to a culture dish containing 5 ml DMEM/F12 medium (Gibco) supplemented with 0.04 mg/ml DNase I (AppliChem), 3.6 mg/ml D-(+)-Glucose (AppliChem) and 1% Penicillin/Streptomycin (P/S). ..

Article Title: The appropriate frequency and function of decidual Tim-3+CTLA-4+CD8+ T cells are important in maintaining normal pregnancy
Article Snippet: Trophoblasts were isolated by trypsin-DNase I (Applichem, Germany) digestion and discontinuous Percoll gradient centrifugation from the placenta tissues as described previously . .. DICs and DSCs were obtained from the normal or RSA decidual tissue digesting in RPMI 1640 (HyClone, USA) supplemented with collagenase type IV (1.0 mg/ml, CLS-1, Worthington Biomedical, USA) and DNase I (150 U/ml, Applichem, Germany) as described previously .

Microscopy:

Article Title: Vaccination-induced skin-resident memory CD8+ T cells mediate strong protection against cutaneous melanoma
Article Snippet: .. Skin pieces were then disaggregated using microscope slides with ground edges (Sail Brand, ref 7105), and single-cell suspension was obtained using a 70 μm cell strainer (BD Falcon®, ref 352350) followed by a second digestion with 5 mL of supplemented RPMI 1640 medium containing 25 µg/mL of DNAse I (AppliChem, ref A3778,0010) during 5 min on ice, to obtain cell suspensions. .. For lung preparations, lungs were excised, cut in small fragments and digested in 1 mL of non-supplemented RPMI 1640 medium (ThermoFisher Scientific, ref 61870-036) containing 5 mg/mL of collagenase type IV (Gibco, ref 17104019) and 5 µg/mL of DNAse I (AppliChem, ref A3778,0010) for 60 min at 37°C, with shaking at 230 RPM.

Purification:

Article Title: Evolutionary and Structural Analyses of Mammalian Haloacid Dehalogenase-type Phosphatases AUM and Chronophin Provide Insight into the Basis of Their Different Substrate Specificities *
Article Snippet: Paragraph title: Recombinant Protein Expression and Purification ... Cells were lysed in the presence of 150 units/ml DNase I (Applichem) using a cell disruptor (Constant Systems), and cell debris was removed by centrifugation (10,000 × g , 30 min, 4 °C).

Article Title: S-Adenosyl-l-methionine Modulates CO and NO• Binding to the Human H2S-generating Enzyme Cystathionine β-Synthase *
Article Snippet: Paragraph title: Expression and Purification of CBS ... After a 16-h incubation at 25 °C and 120 rpm, cells were harvested by centrifugation and resuspended in 50 m m potassium phosphate, 300 m m KCl, 10% glycerol, pH 7.0 (buffer A, 10 ml was used to resuspend cells from 1 liter of culture), containing 1 m m phenylmethylsulfonyl fluoride (Merck), 1 mg/ml lysozyme (Applichem), and deoxyribonuclease I (Applichem).

Article Title: The Salmonella Spi1 Virulence Regulatory Protein HilD Directly Activates Transcription of the Flagellar Master Operon flhDC
Article Snippet: .. An flhDC promoter fragment comprising nucleotides −668 to −388 upstream from the flhDC coding region was incubated with increasing concentrations of purified HilD protein and, after partial digestion with DNase I, the resulting fragments were subjected to denaturing gel electrophoresis ( ). ..

Article Title: NO• Binds Human Cystathionine β-Synthase Quickly and Tightly *
Article Snippet: Paragraph title: Protein Expression and Purification ... Following a 4-h incubation at 22 °C and 120 rpm, cells were harvested by centrifugation and the pellet resuspended in buffer A (50 m m potassium phosphate, 300 m m KCl, pH 7.0, 10% glycerol; 10 ml of buffer per liter of culture) containing 1 mg/ml lysozyme (AppliChem), 1 m m phenylmethylsulfonyl fluoride (Merck), and a few grains of deoxyribonuclease I (AppliChem).

Protein Purification:

Article Title: S-Adenosyl-l-methionine Modulates CO and NO• Binding to the Human H2S-generating Enzyme Cystathionine β-Synthase *
Article Snippet: After a 16-h incubation at 25 °C and 120 rpm, cells were harvested by centrifugation and resuspended in 50 m m potassium phosphate, 300 m m KCl, 10% glycerol, pH 7.0 (buffer A, 10 ml was used to resuspend cells from 1 liter of culture), containing 1 m m phenylmethylsulfonyl fluoride (Merck), 1 mg/ml lysozyme (Applichem), and deoxyribonuclease I (Applichem). .. Protein purification was carried out in an ÅKTA Prime fast performance liquid chromatography system (GE Healthcare).

Sequencing:

Article Title: The Salmonella Spi1 Virulence Regulatory Protein HilD Directly Activates Transcription of the Flagellar Master Operon flhDC
Article Snippet: DNase I (AppliChem) was added to the binding reaction mixtures containing 100 ng DIG-labeled DNA fragments and increasing amounts of HilD after 20 min at RT. .. Sequencing reactions were carried out using the USB Thermo Sequenase cycle sequencing kit (Affimetrix).

Affinity Chromatography:

Article Title: NO• Binds Human Cystathionine β-Synthase Quickly and Tightly *
Article Snippet: Following a 4-h incubation at 22 °C and 120 rpm, cells were harvested by centrifugation and the pellet resuspended in buffer A (50 m m potassium phosphate, 300 m m KCl, pH 7.0, 10% glycerol; 10 ml of buffer per liter of culture) containing 1 mg/ml lysozyme (AppliChem), 1 m m phenylmethylsulfonyl fluoride (Merck), and a few grains of deoxyribonuclease I (AppliChem). .. Purification of the His-tagged protein was performed by affinity chromatography, using a HisTrap FF crude 1-ml column (GE Healthcare) previously equilibrated with buffer A containing 10 m m imidazole (buffer B).

Recombinant:

Article Title: Evolutionary and Structural Analyses of Mammalian Haloacid Dehalogenase-type Phosphatases AUM and Chronophin Provide Insight into the Basis of Their Different Substrate Specificities *
Article Snippet: Paragraph title: Recombinant Protein Expression and Purification ... Cells were lysed in the presence of 150 units/ml DNase I (Applichem) using a cell disruptor (Constant Systems), and cell debris was removed by centrifugation (10,000 × g , 30 min, 4 °C).

Lysis:

Article Title: Vaccination-induced skin-resident memory CD8+ T cells mediate strong protection against cutaneous melanoma
Article Snippet: Skin pieces were then disaggregated using microscope slides with ground edges (Sail Brand, ref 7105), and single-cell suspension was obtained using a 70 μm cell strainer (BD Falcon®, ref 352350) followed by a second digestion with 5 mL of supplemented RPMI 1640 medium containing 25 µg/mL of DNAse I (AppliChem, ref A3778,0010) during 5 min on ice, to obtain cell suspensions. .. Blood was obtained by tail bleeding in 1.5 mL tubes containing heparin (Sanderson Laboratories), and red blood cells were lysed with RBC lysis buffer (Biolegend, ref 420301).

Mouse Assay:

Article Title: Post-Aire Maturation of Thymic Medullary Epithelial Cells Involves Selective Expression of Keratinocyte-Specific Autoantigens
Article Snippet: FACS sorting and analysis of sorted cells Fifteen thymi from 6- to 8-week-old heterozygous mice were dissected and collected into ice-cold PBS. .. GentleMACS C-Tubes (Miltenyi Biotec) were used for mechanical disruption followed by enzymatic digestion in 0.5 mg/ml dispase/collagenase (Roche) and 5 μg/ml DNase I (AppliChem) in PBS at 37°C for 3 × 30 min with gentle agitation.

Article Title: Differentiated Type II Pneumocytes Can Be Reprogrammed by Ectopic Sox2 Expression
Article Snippet: Isolation and culturing of alveolar type II cells (AVTII) Alveolar epithelial cells were isolated from the lungs of Sox2SPC-rtTA mice by Dispase (BD, Pharmingen) digestion as described previously with few modifications . .. Lungs were isolated, incubated for 45 minutes in 1 ml Dispase at room temperature and transferred to a culture dish containing 5 ml DMEM/F12 medium (Gibco) supplemented with 0.04 mg/ml DNase I (AppliChem), 3.6 mg/ml D-(+)-Glucose (AppliChem) and 1% Penicillin/Streptomycin (P/S).

Article Title: Vaccination-induced skin-resident memory CD8+ T cells mediate strong protection against cutaneous melanoma
Article Snippet: Preparation of tissue cell suspensions : Spleens from vaccinated mice were mechanically disaggregated using microscope slides with ground edges (Sail Brand, ref 7105), and single-cell suspension was obtained using a 70 μm cell strainer (BD Falcon, ref 352350). .. Skin pieces were then disaggregated using microscope slides with ground edges (Sail Brand, ref 7105), and single-cell suspension was obtained using a 70 μm cell strainer (BD Falcon®, ref 352350) followed by a second digestion with 5 mL of supplemented RPMI 1640 medium containing 25 µg/mL of DNAse I (AppliChem, ref A3778,0010) during 5 min on ice, to obtain cell suspensions.

Liquid Chromatography:

Article Title: Evolutionary and Structural Analyses of Mammalian Haloacid Dehalogenase-type Phosphatases AUM and Chronophin Provide Insight into the Basis of Their Different Substrate Specificities *
Article Snippet: Cells were lysed in the presence of 150 units/ml DNase I (Applichem) using a cell disruptor (Constant Systems), and cell debris was removed by centrifugation (10,000 × g , 30 min, 4 °C). .. For His6 -AUM purification, cleared supernatants were loaded on a nickel-nitrilotriacetic acid-agarose column (HisTrap HP, GE Healthcare) operated on an ÄKTA liquid chromatography system (GE Healthcare) in TNM, and His6 -tagged proteins were eluted using a linear 10–400 m m imidazole gradient in TNM.

Article Title: S-Adenosyl-l-methionine Modulates CO and NO• Binding to the Human H2S-generating Enzyme Cystathionine β-Synthase *
Article Snippet: After a 16-h incubation at 25 °C and 120 rpm, cells were harvested by centrifugation and resuspended in 50 m m potassium phosphate, 300 m m KCl, 10% glycerol, pH 7.0 (buffer A, 10 ml was used to resuspend cells from 1 liter of culture), containing 1 m m phenylmethylsulfonyl fluoride (Merck), 1 mg/ml lysozyme (Applichem), and deoxyribonuclease I (Applichem). .. Protein purification was carried out in an ÅKTA Prime fast performance liquid chromatography system (GE Healthcare).

Plasmid Preparation:

Article Title: Evolutionary and Structural Analyses of Mammalian Haloacid Dehalogenase-type Phosphatases AUM and Chronophin Provide Insight into the Basis of Their Different Substrate Specificities *
Article Snippet: To increase solubility, AUM was co-expressed with the chaperones groES-groEL-tig from the pG-Tf2 plasmid (Takara) according to the manufacturer's instructions. .. Cells were lysed in the presence of 150 units/ml DNase I (Applichem) using a cell disruptor (Constant Systems), and cell debris was removed by centrifugation (10,000 × g , 30 min, 4 °C).

Article Title: S-Adenosyl-l-methionine Modulates CO and NO• Binding to the Human H2S-generating Enzyme Cystathionine β-Synthase *
Article Snippet: The 1227G > A mutant (cDNA numbering) carrying a premature stop codon at position 409 was generated from the full-length CBS-expressing pET28b-based vector, using the XL QuikChange kit (Agilent) and the primers 5′-GAAGAAGCCCTGGTG A TGGCACCTCCGTG (forward) and 5′-CACGGAGGTGCCA T CACCAGGGCTTCTTC (reverse). .. After a 16-h incubation at 25 °C and 120 rpm, cells were harvested by centrifugation and resuspended in 50 m m potassium phosphate, 300 m m KCl, 10% glycerol, pH 7.0 (buffer A, 10 ml was used to resuspend cells from 1 liter of culture), containing 1 m m phenylmethylsulfonyl fluoride (Merck), 1 mg/ml lysozyme (Applichem), and deoxyribonuclease I (Applichem).

Article Title: NO• Binds Human Cystathionine β-Synthase Quickly and Tightly *
Article Snippet: The resulting vector was used to transform Escherichia coli BL21(DE3) Rosetta cells. .. Following a 4-h incubation at 22 °C and 120 rpm, cells were harvested by centrifugation and the pellet resuspended in buffer A (50 m m potassium phosphate, 300 m m KCl, pH 7.0, 10% glycerol; 10 ml of buffer per liter of culture) containing 1 mg/ml lysozyme (AppliChem), 1 m m phenylmethylsulfonyl fluoride (Merck), and a few grains of deoxyribonuclease I (AppliChem).

Selection:

Article Title: Post-Aire Maturation of Thymic Medullary Epithelial Cells Involves Selective Expression of Keratinocyte-Specific Autoantigens
Article Snippet: GentleMACS C-Tubes (Miltenyi Biotec) were used for mechanical disruption followed by enzymatic digestion in 0.5 mg/ml dispase/collagenase (Roche) and 5 μg/ml DNase I (AppliChem) in PBS at 37°C for 3 × 30 min with gentle agitation. .. In this population, a negative selection was performed with CD45 microbeads and AutoMACS (Miltenyi Biotec) to obtain the CD45- cells.

Sterile Filtration:

Article Title: Correct Capsid Assembly Mediated by a Conserved YXXLGL Motif in Prototype Foamy Virus Gag Is Essential for Infectivity and Reverse Transcription of the Viral Genome ▿
Article Snippet: Viral supernatants for real-time PCR analysis were generated as described above, with the modification that during the sodium butyrate induction step and the subsequent viral supernatant production step, DNase I ( > 3,000 U/mg; Applichem) was added to the medium to a final concentration of 500 μg/ml. .. Forty-eight hours posttransfection, the cell-free viral supernatant was harvested by sterile filtration (pore size, 0.45 μm) and centrifuged at 4°C and 25,000 rpm for 2 h in an SW40 or SW28 rotor through a 20% sucrose cushion.

Concentration Assay:

Article Title: Correct Capsid Assembly Mediated by a Conserved YXXLGL Motif in Prototype Foamy Virus Gag Is Essential for Infectivity and Reverse Transcription of the Viral Genome ▿
Article Snippet: .. Viral supernatants for real-time PCR analysis were generated as described above, with the modification that during the sodium butyrate induction step and the subsequent viral supernatant production step, DNase I ( > 3,000 U/mg; Applichem) was added to the medium to a final concentration of 500 μg/ml. .. Forty-eight hours posttransfection, the cell-free viral supernatant was harvested by sterile filtration (pore size, 0.45 μm) and centrifuged at 4°C and 25,000 rpm for 2 h in an SW40 or SW28 rotor through a 20% sucrose cushion.

Article Title: Extracellular nucleases and extracellular DNA play important roles in Vibrio cholerae biofilm formation
Article Snippet: .. At the respective time points the wells were rinsed four times with spent LB (obtained from a 24 or 72 h old biofilm culture of the respective strain) before DNase I (AppliChem), λExonuclease (New England Biolabs) or in combination were added to a final concentration of 133 Kunitz units per ml. ..

Article Title: NO• Binds Human Cystathionine β-Synthase Quickly and Tightly *
Article Snippet: Protein overexpression was induced by the addition of 0.5 m m isopropyl-β- d -thiogalactopyranoside (Nzytech). δ-Aminolevulinic acid (Sigma) was added to the culture medium to a final concentration of 75 mg/liter. .. Following a 4-h incubation at 22 °C and 120 rpm, cells were harvested by centrifugation and the pellet resuspended in buffer A (50 m m potassium phosphate, 300 m m KCl, pH 7.0, 10% glycerol; 10 ml of buffer per liter of culture) containing 1 mg/ml lysozyme (AppliChem), 1 m m phenylmethylsulfonyl fluoride (Merck), and a few grains of deoxyribonuclease I (AppliChem).

Staining:

Article Title: Post-Aire Maturation of Thymic Medullary Epithelial Cells Involves Selective Expression of Keratinocyte-Specific Autoantigens
Article Snippet: GentleMACS C-Tubes (Miltenyi Biotec) were used for mechanical disruption followed by enzymatic digestion in 0.5 mg/ml dispase/collagenase (Roche) and 5 μg/ml DNase I (AppliChem) in PBS at 37°C for 3 × 30 min with gentle agitation. .. To get the populations of diverse mTECs, the CD45− thymic stromal cells were stained with LacZ (X-gal, ImaGene Red C12RG β-galactosidase substrate, Invitrogen), anti-EpCAM monoclonal Ab, and anti-MHCII monoclonal Ab (Miltenyi Biotec), followed by FACS sorting with a FACS Vantage (BD).

Article Title: Extracellular nucleases and extracellular DNA play important roles in Vibrio cholerae biofilm formation
Article Snippet: At the respective time points the wells were rinsed four times with spent LB (obtained from a 24 or 72 h old biofilm culture of the respective strain) before DNase I (AppliChem), λExonuclease (New England Biolabs) or in combination were added to a final concentration of 133 Kunitz units per ml. .. Afterwards the wells were rinsed four times with distilled water and the remaining biofilm was stained with crystal violet and quantified as described above.

Gradient Centrifugation:

Article Title: The appropriate frequency and function of decidual Tim-3+CTLA-4+CD8+ T cells are important in maintaining normal pregnancy
Article Snippet: Trophoblasts were isolated by trypsin-DNase I (Applichem, Germany) digestion and discontinuous Percoll gradient centrifugation from the placenta tissues as described previously . .. DICs and DSCs were obtained from the normal or RSA decidual tissue digesting in RPMI 1640 (HyClone, USA) supplemented with collagenase type IV (1.0 mg/ml, CLS-1, Worthington Biomedical, USA) and DNase I (150 U/ml, Applichem, Germany) as described previously .

Cell Isolation:

Article Title: The appropriate frequency and function of decidual Tim-3+CTLA-4+CD8+ T cells are important in maintaining normal pregnancy
Article Snippet: Paragraph title: Human cell isolation ... DICs and DSCs were obtained from the normal or RSA decidual tissue digesting in RPMI 1640 (HyClone, USA) supplemented with collagenase type IV (1.0 mg/ml, CLS-1, Worthington Biomedical, USA) and DNase I (150 U/ml, Applichem, Germany) as described previously .

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    PanReac AppliChem dnase i
    d (−) lactic acid induced the release of cell-free DNA from neutrophils that activated bovine umbilical vein endothelial cell (BUVEC) sheets by ICAM display. (A) Supernatants collected from neutrophils treated with 5 mM d (−) lactic acid plus or minus <t>DNase</t> I were perfused onto BUVEC for 10 min. Next, fresh, untreated neutrophils were perfused on endothelial monolayers, n = 5, ** P
    Dnase I, supplied by PanReac AppliChem, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase i/product/PanReac AppliChem
    Average 94 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    dnase i - by Bioz Stars, 2020-04
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      Buy from Supplier

    95
    Applichem dnase i
    <t>DNase</t> I footprinting demonstrates HilD binding to the flhDC P5 promoter region. (A) DNase I footprinting of an flhDC P5 promoter DNA fragment. A DNA fragment covering a region of nucleotides from position −668 to −388 upstream from the flhD start codon was DIG labeled on the noncoding strand and incubated alone (lane P) and with increasing amounts of purified HilD protein (lane 1, 4.23 pmol; lane 2, 8.45 pmol; lane 3, 12.68 pmol; lane 4, 16.9 pmol; lane 5, 21.13 pmol; lane 6, 42.25 pmol) and digested with DNase I before being loaded on a sequencing gel. The vertical line indicates the region protected from DNase I digestion. Lanes C, T, A, and G show the specific nucleotides of the noncoding strand. Exposed nucleotides are highlighted by dots. (B) Partial nucleotide sequence of the P5 promoter of flhDC that is relevant for HilD binding. A horizontal line marks the protected region, and the four most sensitive nucleotides are highlighted by dots. The transcriptional start site (marked as +1) and the −10 element of the P5 flhDC promoter are indicated. (C) Comparison of HilD binding sites in the flhDC , rtsA , hilC , hilD , and hilA ). The proposed consensus is displayed at the bottom; uppercase letters indicate predominant nucleotides ( > 80% conserved), and lowercase letters indicate conserved nucleotides ( > 60% conserved).
    Dnase I, supplied by Applichem, used in various techniques. Bioz Stars score: 95/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase i/product/Applichem
    Average 95 stars, based on 30 article reviews
    Price from $9.99 to $1999.99
    dnase i - by Bioz Stars, 2020-04
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      Buy from Supplier

    Image Search Results


    d (−) lactic acid induced the release of cell-free DNA from neutrophils that activated bovine umbilical vein endothelial cell (BUVEC) sheets by ICAM display. (A) Supernatants collected from neutrophils treated with 5 mM d (−) lactic acid plus or minus DNase I were perfused onto BUVEC for 10 min. Next, fresh, untreated neutrophils were perfused on endothelial monolayers, n = 5, ** P

    Journal: Frontiers in Immunology

    Article Title: d(−) Lactic Acid-Induced Adhesion of Bovine Neutrophils onto Endothelial Cells Is Dependent on Neutrophils Extracellular Traps Formation and CD11b Expression

    doi: 10.3389/fimmu.2017.00975

    Figure Lengend Snippet: d (−) lactic acid induced the release of cell-free DNA from neutrophils that activated bovine umbilical vein endothelial cell (BUVEC) sheets by ICAM display. (A) Supernatants collected from neutrophils treated with 5 mM d (−) lactic acid plus or minus DNase I were perfused onto BUVEC for 10 min. Next, fresh, untreated neutrophils were perfused on endothelial monolayers, n = 5, ** P

    Article Snippet: Briefly, BUVECs were treated with 5 mM of d (−) lactic acid, vehicle (pH buffer), or with supernatants from neutrophils exposed to 5 mM of d (−) lactic acid, DNase I, or 0.01% vehicle (ethanol) for 30 min at 37°C.

    Techniques:

    PAD4 activation regulates d (−) lactic acid-induced neutrophil extracellular trap (NET) formation and subsequent adhesion. (A) Immunofluorescence of bovine neutrophils pre-treated with monocarboxylate transporter 1 inhibitor (Ar-c155858), PAD4 inhibitor (Cl-amidine), or vehicle (0.01% DMSO) for 60 min, followed by stimulation with either 5 mM d (−) lactic acid or vehicle concomitant with DNase I (90 U) or controls for 30 min. Neutrophils were probed with anti-H 4 citrullinated 3/alexa 405, CD11b/alexa 635, and PicoGreen as a probe for DNA; representative images from three independent experiments, scale bar: 50 µm. (B) Fold of control [(number of NETs/number of neutrophils) × 100] from neutrophils treated with 1 µM Ar-c155858, 200 µM Cl-amidine or vehicle for 1 h and stimulated with 5 mM of d (−) lactic acid or vehicle (control) together or not with DNAse I (90 U) for 30 min, n = 3. *** P

    Journal: Frontiers in Immunology

    Article Title: d(−) Lactic Acid-Induced Adhesion of Bovine Neutrophils onto Endothelial Cells Is Dependent on Neutrophils Extracellular Traps Formation and CD11b Expression

    doi: 10.3389/fimmu.2017.00975

    Figure Lengend Snippet: PAD4 activation regulates d (−) lactic acid-induced neutrophil extracellular trap (NET) formation and subsequent adhesion. (A) Immunofluorescence of bovine neutrophils pre-treated with monocarboxylate transporter 1 inhibitor (Ar-c155858), PAD4 inhibitor (Cl-amidine), or vehicle (0.01% DMSO) for 60 min, followed by stimulation with either 5 mM d (−) lactic acid or vehicle concomitant with DNase I (90 U) or controls for 30 min. Neutrophils were probed with anti-H 4 citrullinated 3/alexa 405, CD11b/alexa 635, and PicoGreen as a probe for DNA; representative images from three independent experiments, scale bar: 50 µm. (B) Fold of control [(number of NETs/number of neutrophils) × 100] from neutrophils treated with 1 µM Ar-c155858, 200 µM Cl-amidine or vehicle for 1 h and stimulated with 5 mM of d (−) lactic acid or vehicle (control) together or not with DNAse I (90 U) for 30 min, n = 3. *** P

    Article Snippet: Briefly, BUVECs were treated with 5 mM of d (−) lactic acid, vehicle (pH buffer), or with supernatants from neutrophils exposed to 5 mM of d (−) lactic acid, DNase I, or 0.01% vehicle (ethanol) for 30 min at 37°C.

    Techniques: Activation Assay, Immunofluorescence

    DNase I footprinting demonstrates HilD binding to the flhDC P5 promoter region. (A) DNase I footprinting of an flhDC P5 promoter DNA fragment. A DNA fragment covering a region of nucleotides from position −668 to −388 upstream from the flhD start codon was DIG labeled on the noncoding strand and incubated alone (lane P) and with increasing amounts of purified HilD protein (lane 1, 4.23 pmol; lane 2, 8.45 pmol; lane 3, 12.68 pmol; lane 4, 16.9 pmol; lane 5, 21.13 pmol; lane 6, 42.25 pmol) and digested with DNase I before being loaded on a sequencing gel. The vertical line indicates the region protected from DNase I digestion. Lanes C, T, A, and G show the specific nucleotides of the noncoding strand. Exposed nucleotides are highlighted by dots. (B) Partial nucleotide sequence of the P5 promoter of flhDC that is relevant for HilD binding. A horizontal line marks the protected region, and the four most sensitive nucleotides are highlighted by dots. The transcriptional start site (marked as +1) and the −10 element of the P5 flhDC promoter are indicated. (C) Comparison of HilD binding sites in the flhDC , rtsA , hilC , hilD , and hilA ). The proposed consensus is displayed at the bottom; uppercase letters indicate predominant nucleotides ( > 80% conserved), and lowercase letters indicate conserved nucleotides ( > 60% conserved).

    Journal: Journal of Bacteriology

    Article Title: The Salmonella Spi1 Virulence Regulatory Protein HilD Directly Activates Transcription of the Flagellar Master Operon flhDC

    doi: 10.1128/JB.01438-13

    Figure Lengend Snippet: DNase I footprinting demonstrates HilD binding to the flhDC P5 promoter region. (A) DNase I footprinting of an flhDC P5 promoter DNA fragment. A DNA fragment covering a region of nucleotides from position −668 to −388 upstream from the flhD start codon was DIG labeled on the noncoding strand and incubated alone (lane P) and with increasing amounts of purified HilD protein (lane 1, 4.23 pmol; lane 2, 8.45 pmol; lane 3, 12.68 pmol; lane 4, 16.9 pmol; lane 5, 21.13 pmol; lane 6, 42.25 pmol) and digested with DNase I before being loaded on a sequencing gel. The vertical line indicates the region protected from DNase I digestion. Lanes C, T, A, and G show the specific nucleotides of the noncoding strand. Exposed nucleotides are highlighted by dots. (B) Partial nucleotide sequence of the P5 promoter of flhDC that is relevant for HilD binding. A horizontal line marks the protected region, and the four most sensitive nucleotides are highlighted by dots. The transcriptional start site (marked as +1) and the −10 element of the P5 flhDC promoter are indicated. (C) Comparison of HilD binding sites in the flhDC , rtsA , hilC , hilD , and hilA ). The proposed consensus is displayed at the bottom; uppercase letters indicate predominant nucleotides ( > 80% conserved), and lowercase letters indicate conserved nucleotides ( > 60% conserved).

    Article Snippet: An flhDC promoter fragment comprising nucleotides −668 to −388 upstream from the flhDC coding region was incubated with increasing concentrations of purified HilD protein and, after partial digestion with DNase I, the resulting fragments were subjected to denaturing gel electrophoresis ( ).

    Techniques: Footprinting, Binding Assay, Labeling, Incubation, Purification, Sequencing