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Fisher Scientific dnase i
Dirofilaria immitis -induced dose- and viability- and diphenyleneiodonium (DPI)-independent neutrophil extracellular trap (NET) formation. (A) D. immitis microfilariae (MF; 100) were cocultured with canine polymorphonuclear neutrophils (PMN) for 180 min. For NADPH oxidase inhibition, DPI pre-treatment was used. To resolve NET formation, <t>DNase</t> I was added to coculture. (B) Canine PMN were cocultured with vital or heat-inactivated microfilariae (MF-HI). (C) Vital and heat-inactivated D. immitis L3 were exposed to canine PMN. Sytox Orange-derived fluorescence intensities were analyzed by spectrofluorometric analysis at an excitation wavelength of 547 nm and emission wavelength 570 nm using an automated plate monochrome reader. As negative control, PMN in plain medium were used. PMN stimulated with zymosan served as positive control.
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1) Product Images from "Dirofilaria immitis Microfilariae and Third-Stage Larvae Induce Canine NETosis Resulting in Different Types of Neutrophil Extracellular Traps"

Article Title: Dirofilaria immitis Microfilariae and Third-Stage Larvae Induce Canine NETosis Resulting in Different Types of Neutrophil Extracellular Traps

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00968

Dirofilaria immitis -induced dose- and viability- and diphenyleneiodonium (DPI)-independent neutrophil extracellular trap (NET) formation. (A) D. immitis microfilariae (MF; 100) were cocultured with canine polymorphonuclear neutrophils (PMN) for 180 min. For NADPH oxidase inhibition, DPI pre-treatment was used. To resolve NET formation, DNase I was added to coculture. (B) Canine PMN were cocultured with vital or heat-inactivated microfilariae (MF-HI). (C) Vital and heat-inactivated D. immitis L3 were exposed to canine PMN. Sytox Orange-derived fluorescence intensities were analyzed by spectrofluorometric analysis at an excitation wavelength of 547 nm and emission wavelength 570 nm using an automated plate monochrome reader. As negative control, PMN in plain medium were used. PMN stimulated with zymosan served as positive control.
Figure Legend Snippet: Dirofilaria immitis -induced dose- and viability- and diphenyleneiodonium (DPI)-independent neutrophil extracellular trap (NET) formation. (A) D. immitis microfilariae (MF; 100) were cocultured with canine polymorphonuclear neutrophils (PMN) for 180 min. For NADPH oxidase inhibition, DPI pre-treatment was used. To resolve NET formation, DNase I was added to coculture. (B) Canine PMN were cocultured with vital or heat-inactivated microfilariae (MF-HI). (C) Vital and heat-inactivated D. immitis L3 were exposed to canine PMN. Sytox Orange-derived fluorescence intensities were analyzed by spectrofluorometric analysis at an excitation wavelength of 547 nm and emission wavelength 570 nm using an automated plate monochrome reader. As negative control, PMN in plain medium were used. PMN stimulated with zymosan served as positive control.

Techniques Used: Inhibition, Hi-C, Derivative Assay, Fluorescence, Negative Control, Positive Control

Dirofilaria immitis- induced parasite entrapment. Canine polymorphonuclear neutrophils (PMN) were exposed to vital D. immitis microfilariae (A) or L3 (B,C) for 60 and 180 min. (D) In parallel settings, the same number of PMN was incubated either with heat-inactivated (HI) microfilariae (HI MF) or pretreated with diphenyleneiodonium (DPI) before exposure to vital microfilariae. Furthermore, DNase I was added at the moment of exposure to vital microfilariae. Larvae were considered as entrapped when PMN and/or PMN-derived neutrophil extracellular traps (NETs) were in contact with larvae. The data are expressed as percentage of entrapped larvae relative to the total amount of larvae per condition. The formation of “clasp”-like NET structures sticking mainly to the anterior part of the larvae is displayed in white arrow: image [ (C) , 2].
Figure Legend Snippet: Dirofilaria immitis- induced parasite entrapment. Canine polymorphonuclear neutrophils (PMN) were exposed to vital D. immitis microfilariae (A) or L3 (B,C) for 60 and 180 min. (D) In parallel settings, the same number of PMN was incubated either with heat-inactivated (HI) microfilariae (HI MF) or pretreated with diphenyleneiodonium (DPI) before exposure to vital microfilariae. Furthermore, DNase I was added at the moment of exposure to vital microfilariae. Larvae were considered as entrapped when PMN and/or PMN-derived neutrophil extracellular traps (NETs) were in contact with larvae. The data are expressed as percentage of entrapped larvae relative to the total amount of larvae per condition. The formation of “clasp”-like NET structures sticking mainly to the anterior part of the larvae is displayed in white arrow: image [ (C) , 2].

Techniques Used: Incubation, Derivative Assay

2) Product Images from "The BAF (BRG1/BRM-Associated Factor) Chromatin-Remodeling Complex Exhibits Ethanol Sensitivity in Fetal Neural Progenitor Cells and Regulates Transcription at the Mir-9-2 Encoding Gene Locus"

Article Title: The BAF (BRG1/BRM-Associated Factor) Chromatin-Remodeling Complex Exhibits Ethanol Sensitivity in Fetal Neural Progenitor Cells and Regulates Transcription at the Mir-9-2 Encoding Gene Locus

Journal: Alcohol (Fayetteville, N.Y.)

doi: 10.1016/j.alcohol.2017.01.003

ChIP analysis indicates that ethanol exposure increases BRG1 binding to DNAse I-resistant/CpG island containing region 3 and the pre-miR-9-2 coding exon Bar graph shows the effect of ethanol on BRG1-association with regions 1 to 5 and the pre-miR-9-2 exon-coding region of the primary (pri)-miR-9-2 coding locus. Primers for a gene desert on chromosome 6 (not predicted to bind any transcription factors) shows the specificity of the immuno-precipitation. Vertical axis shows the fold change of the specific associated DNA region to BRG1 in ethanol treated group relative to control group. Error bars indicate standard error of the mean.
Figure Legend Snippet: ChIP analysis indicates that ethanol exposure increases BRG1 binding to DNAse I-resistant/CpG island containing region 3 and the pre-miR-9-2 coding exon Bar graph shows the effect of ethanol on BRG1-association with regions 1 to 5 and the pre-miR-9-2 exon-coding region of the primary (pri)-miR-9-2 coding locus. Primers for a gene desert on chromosome 6 (not predicted to bind any transcription factors) shows the specificity of the immuno-precipitation. Vertical axis shows the fold change of the specific associated DNA region to BRG1 in ethanol treated group relative to control group. Error bars indicate standard error of the mean.

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation

BRG1-containing complexes associate with both DNAse I-hypersensitive and insensitive sites on the miR-9-2 gene locus (a) Positional representation of POU5F1/Oct4, c-myc and REST transcription regulatory factor binding sites along the human pre-miR-9-2 gene locus identified with the UCSC genome browser ENCODE analysis hub track. Colored circles indicate locations for primer pairs for pri-miR-9-2 regions 1,2,3,4 and 5 and the pre-miR-9-2 coding region. (b) qPCR results of BRG1-ChIP from neurospheres. Primers were used to amplify 6 distinct positions along the pri-miR-9-2 gene locus and a genetically sparse region on mouse chromosome 6. Vertical axis shows fold change relative to an IgG pulldown control. Error bars indicate standard error of the mean. (c) qPCR of neurosphere DNA following digestion with 20, 60 and 120U of DNAse I at the 6 positions along the pri-miR-9-2 gene locus. The vertical axis shows fold amplification relative to DNAse I-untreated neurosphere DNA.
Figure Legend Snippet: BRG1-containing complexes associate with both DNAse I-hypersensitive and insensitive sites on the miR-9-2 gene locus (a) Positional representation of POU5F1/Oct4, c-myc and REST transcription regulatory factor binding sites along the human pre-miR-9-2 gene locus identified with the UCSC genome browser ENCODE analysis hub track. Colored circles indicate locations for primer pairs for pri-miR-9-2 regions 1,2,3,4 and 5 and the pre-miR-9-2 coding region. (b) qPCR results of BRG1-ChIP from neurospheres. Primers were used to amplify 6 distinct positions along the pri-miR-9-2 gene locus and a genetically sparse region on mouse chromosome 6. Vertical axis shows fold change relative to an IgG pulldown control. Error bars indicate standard error of the mean. (c) qPCR of neurosphere DNA following digestion with 20, 60 and 120U of DNAse I at the 6 positions along the pri-miR-9-2 gene locus. The vertical axis shows fold amplification relative to DNAse I-untreated neurosphere DNA.

Techniques Used: Binding Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Amplification

3) Product Images from "Dirofilaria immitis Microfilariae and Third-Stage Larvae Induce Canine NETosis Resulting in Different Types of Neutrophil Extracellular Traps"

Article Title: Dirofilaria immitis Microfilariae and Third-Stage Larvae Induce Canine NETosis Resulting in Different Types of Neutrophil Extracellular Traps

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00968

Dirofilaria immitis -induced dose- and viability- and diphenyleneiodonium (DPI)-independent neutrophil extracellular trap (NET) formation. (A) D. immitis microfilariae (MF; 100) were cocultured with canine polymorphonuclear neutrophils (PMN) for 180 min. For NADPH oxidase inhibition, DPI pre-treatment was used. To resolve NET formation, DNase I was added to coculture. (B) Canine PMN were cocultured with vital or heat-inactivated microfilariae (MF-HI). (C) Vital and heat-inactivated D. immitis L3 were exposed to canine PMN. Sytox Orange-derived fluorescence intensities were analyzed by spectrofluorometric analysis at an excitation wavelength of 547 nm and emission wavelength 570 nm using an automated plate monochrome reader. As negative control, PMN in plain medium were used. PMN stimulated with zymosan served as positive control.
Figure Legend Snippet: Dirofilaria immitis -induced dose- and viability- and diphenyleneiodonium (DPI)-independent neutrophil extracellular trap (NET) formation. (A) D. immitis microfilariae (MF; 100) were cocultured with canine polymorphonuclear neutrophils (PMN) for 180 min. For NADPH oxidase inhibition, DPI pre-treatment was used. To resolve NET formation, DNase I was added to coculture. (B) Canine PMN were cocultured with vital or heat-inactivated microfilariae (MF-HI). (C) Vital and heat-inactivated D. immitis L3 were exposed to canine PMN. Sytox Orange-derived fluorescence intensities were analyzed by spectrofluorometric analysis at an excitation wavelength of 547 nm and emission wavelength 570 nm using an automated plate monochrome reader. As negative control, PMN in plain medium were used. PMN stimulated with zymosan served as positive control.

Techniques Used: Inhibition, Hi-C, Derivative Assay, Fluorescence, Negative Control, Positive Control

Dirofilaria immitis- induced parasite entrapment. Canine polymorphonuclear neutrophils (PMN) were exposed to vital D. immitis microfilariae (A) or L3 (B,C) for 60 and 180 min. (D) In parallel settings, the same number of PMN was incubated either with heat-inactivated (HI) microfilariae (HI MF) or pretreated with diphenyleneiodonium (DPI) before exposure to vital microfilariae. Furthermore, DNase I was added at the moment of exposure to vital microfilariae. Larvae were considered as entrapped when PMN and/or PMN-derived neutrophil extracellular traps (NETs) were in contact with larvae. The data are expressed as percentage of entrapped larvae relative to the total amount of larvae per condition. The formation of “clasp”-like NET structures sticking mainly to the anterior part of the larvae is displayed in white arrow: image [ (C) , 2].
Figure Legend Snippet: Dirofilaria immitis- induced parasite entrapment. Canine polymorphonuclear neutrophils (PMN) were exposed to vital D. immitis microfilariae (A) or L3 (B,C) for 60 and 180 min. (D) In parallel settings, the same number of PMN was incubated either with heat-inactivated (HI) microfilariae (HI MF) or pretreated with diphenyleneiodonium (DPI) before exposure to vital microfilariae. Furthermore, DNase I was added at the moment of exposure to vital microfilariae. Larvae were considered as entrapped when PMN and/or PMN-derived neutrophil extracellular traps (NETs) were in contact with larvae. The data are expressed as percentage of entrapped larvae relative to the total amount of larvae per condition. The formation of “clasp”-like NET structures sticking mainly to the anterior part of the larvae is displayed in white arrow: image [ (C) , 2].

Techniques Used: Incubation, Derivative Assay

Related Articles

Clone Assay:

Article Title: Comprehensive Experimental and Computational Analysis of Binding Energy Hot Spots at the NF-?B Essential Modulator (NEMO)/IKK? Protein-Protein Interface
Article Snippet: Dithiothreitol (DTT), B-PER bacterial protein extraction reagent, lysozyme, DNase I, Gelcode Blue Safe Protein Stain, phenylmethanesulfonylfluoride (PMSF) and HisPur Ni-NTA resin were purchased from Fisher Scientific, and Roche Complete EDTA-free protease inhibitors were obtained from Roche (Penzberg, Germany). .. All restriction enzymes (NdeI, NheI, XhoI, NcoI and DpnI), deoxyribonucleotide triphosphates (dNTPs), Factor Xa, Quick Ligation Kit (Quick Ligase buffer and Quick Ligase), and high efficiency E. coli competent cloning cells (NEB 5-alpha) were purchased from New England BioLabs (Ipswich, MA) and were used according to the manufacturer’s instructions.

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Centrifugation:

Article Title: The Third Transmembrane Domain of EscR Is Critical for Function of the Enteropathogenic Escherichia coli Type III Secretion System
Article Snippet: Ten micrograms of RNase A and DNase I per milliliter was added, and the samples were sonicated (Fisher Scientific; 3 times for 15 s each). .. Intact bacteria were removed by centrifugation (at 2,300 × g for 15 min), and the cleared supernatants containing cytoplasmic and membrane proteins were transferred to new tubes.

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Article Title: EscE and EscG Are Cochaperones for the Type III Needle Protein EscF of Enteropathogenic Escherichia coli
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Amplification:

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Single-cell Isolation:

Article Title: Control of intestinal stem cell function and proliferation by mitochondrial pyruvate metabolism
Article Snippet: .. Single cell isolation was carried out as described previously by resuspending crypts in TrypLE Express (Invitrogen #1260413) with DNase I (Gold Biotechnology #D-300–1) for 10 minutes at 37 degree C. Dissociated cells were washed in culture media without growth factors, centrifuged at 700g , resuspended and passed through a 35-um strainer (Fisher Scientific #08–771-23) and analyzed by FACS. .. Neagtive staining was carried out with DAPI and single live cells were collected, pelleted, and snap frozen for analysis or resuspended for culture.

Incubation:

Article Title: The Third Transmembrane Domain of EscR Is Critical for Function of the Enteropathogenic Escherichia coli Type III Secretion System
Article Snippet: The cultures were harvested, washed in PBS, resuspended in 1 ml of buffer A (50 mM Tris [pH 7.5], 20% [wt/vol] sucrose, protease inhibitor cocktail [Roche Applied Science], and lysozyme [100 µg/ml]), and incubated for 30 min at room temperature to generate spheroplasts. .. Ten micrograms of RNase A and DNase I per milliliter was added, and the samples were sonicated (Fisher Scientific; 3 times for 15 s each).

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Article Title: Direct Assessment of Viral Diversity in Soils by Random PCR Amplification of Polymorphic DNA
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Article Title: Dirofilaria immitis Microfilariae and Third-Stage Larvae Induce Canine NETosis Resulting in Different Types of Neutrophil Extracellular Traps
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Article Title: EscE and EscG Are Cochaperones for the Type III Needle Protein EscF of Enteropathogenic Escherichia coli
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Article Title: The Role of the Small Export Apparatus Protein, SctS, in the Activity of the Type III Secretion System
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Article Title: The BAF (BRG1/BRM-Associated Factor) Chromatin-Remodeling Complex Exhibits Ethanol Sensitivity in Fetal Neural Progenitor Cells and Regulates Transcription at the Mir-9-2 Encoding Gene Locus
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Expressing:

Article Title: Comprehensive Experimental and Computational Analysis of Binding Energy Hot Spots at the NF-?B Essential Modulator (NEMO)/IKK? Protein-Protein Interface
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Article Title: The Nla28S/Nla28 Two-Component Signal Transduction System Regulates Sporulation in Myxococcus xanthus
Article Snippet: Paragraph title: Protein expression and purification. ... Cells were harvested, resuspended in lysis buffer (100 mM sodium phosphate [pH 8.0], 300 mM NaCl, 10% glycerol, 1 mg/ml of lysozyme, 5 U/ml of DNase I, 1 μg/ml of pepstatin, 1 μg/ml of leupeptin), and lysed by three 30-s sonication bursts using a model 100 Sonic Dismembrator (Fisher Scientific) set at an intensity of 4.

Cell Fractionation:

Article Title: The Ruler Protein EscP of the Enteropathogenic Escherichia coli Type III Secretion System Is Involved in Calcium Sensing and Secretion Hierarchy Regulation by Interacting with the Gatekeeper Protein SepL
Article Snippet: Paragraph title: Bacterial cell fractionation. ... Ten micrograms of RNase A and DNase I per milliliter was added, and the samples were sonicated (Fisher Scientific, 3 × 15 s).

Ligation:

Article Title: Comprehensive Experimental and Computational Analysis of Binding Energy Hot Spots at the NF-?B Essential Modulator (NEMO)/IKK? Protein-Protein Interface
Article Snippet: Dithiothreitol (DTT), B-PER bacterial protein extraction reagent, lysozyme, DNase I, Gelcode Blue Safe Protein Stain, phenylmethanesulfonylfluoride (PMSF) and HisPur Ni-NTA resin were purchased from Fisher Scientific, and Roche Complete EDTA-free protease inhibitors were obtained from Roche (Penzberg, Germany). .. All restriction enzymes (NdeI, NheI, XhoI, NcoI and DpnI), deoxyribonucleotide triphosphates (dNTPs), Factor Xa, Quick Ligation Kit (Quick Ligase buffer and Quick Ligase), and high efficiency E. coli competent cloning cells (NEB 5-alpha) were purchased from New England BioLabs (Ipswich, MA) and were used according to the manufacturer’s instructions.

Article Title: The BAF (BRG1/BRM-Associated Factor) Chromatin-Remodeling Complex Exhibits Ethanol Sensitivity in Fetal Neural Progenitor Cells and Regulates Transcription at the Mir-9-2 Encoding Gene Locus
Article Snippet: As described in , nuclear pellets were treated with either 0, 20, 60 or 120U of DNAse I (Ambion/Thermo Fisher Scientific) for one hour on ice. .. Digestion into 200–500bp fragments was confirmed by size fractionation on an agarose DNA gel, and fragmented DNA was then blunt ended using T4 polymerase, followed by ligation to a double stranded linker DNA.

Protease Inhibitor:

Article Title: The Third Transmembrane Domain of EscR Is Critical for Function of the Enteropathogenic Escherichia coli Type III Secretion System
Article Snippet: The cultures were harvested, washed in PBS, resuspended in 1 ml of buffer A (50 mM Tris [pH 7.5], 20% [wt/vol] sucrose, protease inhibitor cocktail [Roche Applied Science], and lysozyme [100 µg/ml]), and incubated for 30 min at room temperature to generate spheroplasts. .. Ten micrograms of RNase A and DNase I per milliliter was added, and the samples were sonicated (Fisher Scientific; 3 times for 15 s each).

Article Title: The Ruler Protein EscP of the Enteropathogenic Escherichia coli Type III Secretion System Is Involved in Calcium Sensing and Secretion Hierarchy Regulation by Interacting with the Gatekeeper Protein SepL
Article Snippet: The cultures were harvested, washed in PBS, and resuspended in 1 ml of buffer A (50 mM Tris [pH 7.5], 20% [wt/vol] sucrose, protease inhibitor cocktail [Roche Applied Science], and lysozyme [100 µg/ml]) and incubated for 30 min at room temperature to generate spheroplasts. .. Ten micrograms of RNase A and DNase I per milliliter was added, and the samples were sonicated (Fisher Scientific, 3 × 15 s).

Article Title: EscE and EscG Are Cochaperones for the Type III Needle Protein EscF of Enteropathogenic Escherichia coli
Article Snippet: The culture was harvested, washed in phosphate-buffered saline (PBS), resuspended in 1 ml of buffer A (50 mM Tris [pH 7.5], 20% [wt/vol] sucrose, protease inhibitor cocktail [Roche Applied Science], 10 mM EDTA, and lysozyme [10 μg/ml]), and incubated for 30 min at room temperature to generate spheroplasts. .. RNase A and DNase I (each at 10 μg/ml) were added, and the samples were sonicated (3 times, for 15 s each time; Fisher Scientific).

Article Title: The Role of the Small Export Apparatus Protein, SctS, in the Activity of the Type III Secretion System
Article Snippet: Cells were harvested, washed in PBS, and resuspended in 0.25 mL buffer A [50 mM Tris (pH 7.5), 20% (w/v) sucrose, 5 mM EDTA, protease inhibitor cocktail (Roche Applied Science), and lysozyme (100 μg/mL)] and incubated for 15 min, at room temperature, while rotating, to generate spheroplasts. .. RNase A and DNase I (10 μg/mL) were added and the samples were sonicated (Fisher Scientific, 3 × 15 s).

Cell Culture:

Article Title: Novel Role Of Surfactant Protein A In Bacterial Sinusitis
Article Snippet: Briefly, middle meatus tissues were dissociated in minimum essential medium (MEM) media containing pronase and DNase I for 48h at 4°C; cells were spun down and plated in primaria 10-cm plates (Fisher Scientific, Waltham, MA) for 4 h 37°C in 5% CO2 to remove fibroblasts. .. Non-adherent cells were plated into 10-cm that have been coated with collagen and cultured in BEGM media (Lonza, New Jersey, NJ) until they reached 80% confluence.

Sequencing:

Article Title: Molecular tools enabling pennycress (Thlaspi arvense) as a model plant and oilseed cash cover crop
Article Snippet: For T7 endonuclease I analysis, 10 μL of each 20 μL PCR reaction was denatured by heating at 95 °C for 5 min in a thermocycler (Fisher) and annealed using gradual cooling: −2 °C per second decrease from 95 to 85 °C then −0.1 °C per second decrease from 85 to 25 °C, followed by T7 endonuclease I (Fisher Scientific cat. #50‐995‐224 to New England Biolabs cat. #M0302L, Ipswich, MA) digestion for 30 min. .. The digested product was electrophoresed in a 1% agarose gel to identify samples that partially digested, indicating an SpCas9‐induced edit in TaFAE1 , which were confirmed by Sanger sequence analyses.

Sonication:

Article Title: The Third Transmembrane Domain of EscR Is Critical for Function of the Enteropathogenic Escherichia coli Type III Secretion System
Article Snippet: .. Ten micrograms of RNase A and DNase I per milliliter was added, and the samples were sonicated (Fisher Scientific; 3 times for 15 s each). .. Intact bacteria were removed by centrifugation (at 2,300 × g for 15 min), and the cleared supernatants containing cytoplasmic and membrane proteins were transferred to new tubes.

Article Title: The Nla28S/Nla28 Two-Component Signal Transduction System Regulates Sporulation in Myxococcus xanthus
Article Snippet: .. Cells were harvested, resuspended in lysis buffer (100 mM sodium phosphate [pH 8.0], 300 mM NaCl, 10% glycerol, 1 mg/ml of lysozyme, 5 U/ml of DNase I, 1 μg/ml of pepstatin, 1 μg/ml of leupeptin), and lysed by three 30-s sonication bursts using a model 100 Sonic Dismembrator (Fisher Scientific) set at an intensity of 4. .. GST and the GST fusion proteins were purified using 5-ml GSTrapFF columns (GE Healthcare) on an AKTA purifier UPC 10 fast-performance liquid chromatography (FPLC) system (GE).

Article Title: The Ruler Protein EscP of the Enteropathogenic Escherichia coli Type III Secretion System Is Involved in Calcium Sensing and Secretion Hierarchy Regulation by Interacting with the Gatekeeper Protein SepL
Article Snippet: .. Ten micrograms of RNase A and DNase I per milliliter was added, and the samples were sonicated (Fisher Scientific, 3 × 15 s). .. Intact bacteria were removed by centrifugation at 2,300 × g for 15 min, and the cleared supernatants containing cytoplasmic and membrane proteins were transferred to new tubes.

Article Title: EscE and EscG Are Cochaperones for the Type III Needle Protein EscF of Enteropathogenic Escherichia coli
Article Snippet: .. RNase A and DNase I (each at 10 μg/ml) were added, and the samples were sonicated (3 times, for 15 s each time; Fisher Scientific). .. Intact bacteria were removed by centrifugation (at 2,300 × g for 15 min), and the cleared supernatant containing cytoplasmic and membrane proteins was transferred to new tubes.

Article Title: The Role of the Small Export Apparatus Protein, SctS, in the Activity of the Type III Secretion System
Article Snippet: .. RNase A and DNase I (10 μg/mL) were added and the samples were sonicated (Fisher Scientific, 3 × 15 s). .. Intact bacteria were removed by centrifugation (2,300 × g for 15 min), and the cleared supernatants containing cytoplasmic and membrane proteins were transferred to new tubes.

Staining:

Article Title: Control of intestinal stem cell function and proliferation by mitochondrial pyruvate metabolism
Article Snippet: Single cell isolation was carried out as described previously by resuspending crypts in TrypLE Express (Invitrogen #1260413) with DNase I (Gold Biotechnology #D-300–1) for 10 minutes at 37 degree C. Dissociated cells were washed in culture media without growth factors, centrifuged at 700g , resuspended and passed through a 35-um strainer (Fisher Scientific #08–771-23) and analyzed by FACS. .. Neagtive staining was carried out with DAPI and single live cells were collected, pelleted, and snap frozen for analysis or resuspended for culture.

Article Title: Comprehensive Experimental and Computational Analysis of Binding Energy Hot Spots at the NF-?B Essential Modulator (NEMO)/IKK? Protein-Protein Interface
Article Snippet: .. Dithiothreitol (DTT), B-PER bacterial protein extraction reagent, lysozyme, DNase I, Gelcode Blue Safe Protein Stain, phenylmethanesulfonylfluoride (PMSF) and HisPur Ni-NTA resin were purchased from Fisher Scientific, and Roche Complete EDTA-free protease inhibitors were obtained from Roche (Penzberg, Germany). .. Pfu Turbo DNA polymerase and Pfu reaction buffer, used for all polymerase chain reaction (PCR) experiments, were purchased from Strat-agene/Agilent Technolog ies (Santa Clara, CA) along with E. coli BL21(DE3) competent expression cells.

DNA Extraction:

Article Title: Genome analysis of the staphylococcal temperate phage DW2 and functional studies on the endolysin and tail hydrolase
Article Snippet: .. Phage DNA preparation In order to remove contaminating host RNA and DNA from a filter sterilized lysate of phage DW2, prior to phage DNA extraction, DNase I was added to a final concentration of 3.3 U/µl (Fisher Scientific, 11873795) and RNase to a final concentration of 6.7 U/ml (Roche, 10109142001). .. The sample was then incubated at 37 °C for 10 min. To lyse the protein capsid 150 µl of lysis buffer (0.4 M EDTA, 1% SDS and 0.05 M TRIS-HCl pH 8) and 10 µl of 10 mg/ml proteinase K (Roche, 3115879001) was added to 750 µl aliquots of the phage lysate sample and they were incubated at 65 °C for 30 min. Proteins were removed by two chloroform: isoamylalcohol: phenol (24:1:25) and a chloroform: isoamylalcohol steps.

Mutagenesis:

Article Title: Comprehensive Experimental and Computational Analysis of Binding Energy Hot Spots at the NF-?B Essential Modulator (NEMO)/IKK? Protein-Protein Interface
Article Snippet: Dithiothreitol (DTT), B-PER bacterial protein extraction reagent, lysozyme, DNase I, Gelcode Blue Safe Protein Stain, phenylmethanesulfonylfluoride (PMSF) and HisPur Ni-NTA resin were purchased from Fisher Scientific, and Roche Complete EDTA-free protease inhibitors were obtained from Roche (Penzberg, Germany). .. All oligonucleotide primers used for site-directed mutagenesis of IKKβ were obtained from Integrated DNA Technologies (Coralville, IA).

Article Title: The Ruler Protein EscP of the Enteropathogenic Escherichia coli Type III Secretion System Is Involved in Calcium Sensing and Secretion Hierarchy Regulation by Interacting with the Gatekeeper Protein SepL
Article Snippet: Briefly, EPEC (WT and Δlee mutant) strains from an overnight culture were subcultured 1:50 in 50 ml of DMEM for 6 h at 37°C in a CO2 tissue culture incubator. .. Ten micrograms of RNase A and DNase I per milliliter was added, and the samples were sonicated (Fisher Scientific, 3 × 15 s).

Article Title: Molecular tools enabling pennycress (Thlaspi arvense) as a model plant and oilseed cash cover crop
Article Snippet: Template for PCR reactions was a 50:50 mix of each putative mutant prep and wild type to ensure that even in the case of a homozygous mutation, a DNA mismatch would be PCR amplified and detected. .. For T7 endonuclease I analysis, 10 μL of each 20 μL PCR reaction was denatured by heating at 95 °C for 5 min in a thermocycler (Fisher) and annealed using gradual cooling: −2 °C per second decrease from 95 to 85 °C then −0.1 °C per second decrease from 85 to 25 °C, followed by T7 endonuclease I (Fisher Scientific cat. #50‐995‐224 to New England Biolabs cat. #M0302L, Ipswich, MA) digestion for 30 min.

Microscopy:

Article Title: Dirofilaria immitis Microfilariae and Third-Stage Larvae Induce Canine NETosis Resulting in Different Types of Neutrophil Extracellular Traps
Article Snippet: Treatments of PMN with 90U DNase I (Fisher Scientific) at the moment of parasite exposure were used to dissolve NETs. .. Following incubation, all samples were fixed (4% paraformaldehyde), and PMN-entrapped larvae were scored microscopically by using an inverted DMIRB® phase-contrast microscope (Leica).

Purification:

Article Title: The Nla28S/Nla28 Two-Component Signal Transduction System Regulates Sporulation in Myxococcus xanthus
Article Snippet: Paragraph title: Protein expression and purification. ... Cells were harvested, resuspended in lysis buffer (100 mM sodium phosphate [pH 8.0], 300 mM NaCl, 10% glycerol, 1 mg/ml of lysozyme, 5 U/ml of DNase I, 1 μg/ml of pepstatin, 1 μg/ml of leupeptin), and lysed by three 30-s sonication bursts using a model 100 Sonic Dismembrator (Fisher Scientific) set at an intensity of 4.

Article Title: Protein features for assembly of the RNA editing helicase 2 subcomplex (REH2C) in Trypanosome holo-editosomes
Article Snippet: Paragraph title: Cell extracts and purification of protein and RNA from IgG pulldowns ... The pellet was resuspended into STM buffer (0.25M Sucrose, 20 mM Tris-HCl pH 8.0, 2 mM MgCl2 ) supplemented with DNase I, RNase-free (EN0523; Fisher Scientific).

Article Title: The BAF (BRG1/BRM-Associated Factor) Chromatin-Remodeling Complex Exhibits Ethanol Sensitivity in Fetal Neural Progenitor Cells and Regulates Transcription at the Mir-9-2 Encoding Gene Locus
Article Snippet: As described in , nuclear pellets were treated with either 0, 20, 60 or 120U of DNAse I (Ambion/Thermo Fisher Scientific) for one hour on ice. .. The nuclei were then lysed open using NER buffer, and fragmented DNA was purified using isoamyl alcohol-chloroform.

Polymerase Chain Reaction:

Article Title: Comprehensive Experimental and Computational Analysis of Binding Energy Hot Spots at the NF-?B Essential Modulator (NEMO)/IKK? Protein-Protein Interface
Article Snippet: Dithiothreitol (DTT), B-PER bacterial protein extraction reagent, lysozyme, DNase I, Gelcode Blue Safe Protein Stain, phenylmethanesulfonylfluoride (PMSF) and HisPur Ni-NTA resin were purchased from Fisher Scientific, and Roche Complete EDTA-free protease inhibitors were obtained from Roche (Penzberg, Germany). .. Pfu Turbo DNA polymerase and Pfu reaction buffer, used for all polymerase chain reaction (PCR) experiments, were purchased from Strat-agene/Agilent Technolog ies (Santa Clara, CA) along with E. coli BL21(DE3) competent expression cells.

Article Title: The Nla28S/Nla28 Two-Component Signal Transduction System Regulates Sporulation in Myxococcus xanthus
Article Snippet: PCR-generated DNA corresponding to the cytoplasmic domain of the E. coli EnvZ protein and the full-length E. coli OmpR protein were cloned into the vector pET28b (EMD Biosciences) to create N-terminal and C-terminal 6× histidine tags (His-EnvZ-cyt and His-OmpR, respectively). .. Cells were harvested, resuspended in lysis buffer (100 mM sodium phosphate [pH 8.0], 300 mM NaCl, 10% glycerol, 1 mg/ml of lysozyme, 5 U/ml of DNase I, 1 μg/ml of pepstatin, 1 μg/ml of leupeptin), and lysed by three 30-s sonication bursts using a model 100 Sonic Dismembrator (Fisher Scientific) set at an intensity of 4.

Article Title: Molecular tools enabling pennycress (Thlaspi arvense) as a model plant and oilseed cash cover crop
Article Snippet: .. For T7 endonuclease I analysis, 10 μL of each 20 μL PCR reaction was denatured by heating at 95 °C for 5 min in a thermocycler (Fisher) and annealed using gradual cooling: −2 °C per second decrease from 95 to 85 °C then −0.1 °C per second decrease from 85 to 25 °C, followed by T7 endonuclease I (Fisher Scientific cat. #50‐995‐224 to New England Biolabs cat. #M0302L, Ipswich, MA) digestion for 30 min. .. The digested product was electrophoresed in a 1% agarose gel to identify samples that partially digested, indicating an SpCas9‐induced edit in TaFAE1 , which were confirmed by Sanger sequence analyses.

Protein Extraction:

Article Title: Comprehensive Experimental and Computational Analysis of Binding Energy Hot Spots at the NF-?B Essential Modulator (NEMO)/IKK? Protein-Protein Interface
Article Snippet: .. Dithiothreitol (DTT), B-PER bacterial protein extraction reagent, lysozyme, DNase I, Gelcode Blue Safe Protein Stain, phenylmethanesulfonylfluoride (PMSF) and HisPur Ni-NTA resin were purchased from Fisher Scientific, and Roche Complete EDTA-free protease inhibitors were obtained from Roche (Penzberg, Germany). .. Pfu Turbo DNA polymerase and Pfu reaction buffer, used for all polymerase chain reaction (PCR) experiments, were purchased from Strat-agene/Agilent Technolog ies (Santa Clara, CA) along with E. coli BL21(DE3) competent expression cells.

FACS:

Article Title: Control of intestinal stem cell function and proliferation by mitochondrial pyruvate metabolism
Article Snippet: .. Single cell isolation was carried out as described previously by resuspending crypts in TrypLE Express (Invitrogen #1260413) with DNase I (Gold Biotechnology #D-300–1) for 10 minutes at 37 degree C. Dissociated cells were washed in culture media without growth factors, centrifuged at 700g , resuspended and passed through a 35-um strainer (Fisher Scientific #08–771-23) and analyzed by FACS. .. Neagtive staining was carried out with DAPI and single live cells were collected, pelleted, and snap frozen for analysis or resuspended for culture.

Fast Protein Liquid Chromatography:

Article Title: The Nla28S/Nla28 Two-Component Signal Transduction System Regulates Sporulation in Myxococcus xanthus
Article Snippet: Cells were harvested, resuspended in lysis buffer (100 mM sodium phosphate [pH 8.0], 300 mM NaCl, 10% glycerol, 1 mg/ml of lysozyme, 5 U/ml of DNase I, 1 μg/ml of pepstatin, 1 μg/ml of leupeptin), and lysed by three 30-s sonication bursts using a model 100 Sonic Dismembrator (Fisher Scientific) set at an intensity of 4. .. GST and the GST fusion proteins were purified using 5-ml GSTrapFF columns (GE Healthcare) on an AKTA purifier UPC 10 fast-performance liquid chromatography (FPLC) system (GE).

CRISPR:

Article Title: Molecular tools enabling pennycress (Thlaspi arvense) as a model plant and oilseed cash cover crop
Article Snippet: Paragraph title: Screening for CRISPR‐Cas9 edits ... For T7 endonuclease I analysis, 10 μL of each 20 μL PCR reaction was denatured by heating at 95 °C for 5 min in a thermocycler (Fisher) and annealed using gradual cooling: −2 °C per second decrease from 95 to 85 °C then −0.1 °C per second decrease from 85 to 25 °C, followed by T7 endonuclease I (Fisher Scientific cat. #50‐995‐224 to New England Biolabs cat. #M0302L, Ipswich, MA) digestion for 30 min.

IA:

Article Title: Comprehensive Experimental and Computational Analysis of Binding Energy Hot Spots at the NF-?B Essential Modulator (NEMO)/IKK? Protein-Protein Interface
Article Snippet: Dithiothreitol (DTT), B-PER bacterial protein extraction reagent, lysozyme, DNase I, Gelcode Blue Safe Protein Stain, phenylmethanesulfonylfluoride (PMSF) and HisPur Ni-NTA resin were purchased from Fisher Scientific, and Roche Complete EDTA-free protease inhibitors were obtained from Roche (Penzberg, Germany). .. All oligonucleotide primers used for site-directed mutagenesis of IKKβ were obtained from Integrated DNA Technologies (Coralville, IA).

Liquid Chromatography:

Article Title: The Nla28S/Nla28 Two-Component Signal Transduction System Regulates Sporulation in Myxococcus xanthus
Article Snippet: Cells were harvested, resuspended in lysis buffer (100 mM sodium phosphate [pH 8.0], 300 mM NaCl, 10% glycerol, 1 mg/ml of lysozyme, 5 U/ml of DNase I, 1 μg/ml of pepstatin, 1 μg/ml of leupeptin), and lysed by three 30-s sonication bursts using a model 100 Sonic Dismembrator (Fisher Scientific) set at an intensity of 4. .. GST and the GST fusion proteins were purified using 5-ml GSTrapFF columns (GE Healthcare) on an AKTA purifier UPC 10 fast-performance liquid chromatography (FPLC) system (GE).

Plasmid Preparation:

Article Title: The Nla28S/Nla28 Two-Component Signal Transduction System Regulates Sporulation in Myxococcus xanthus
Article Snippet: PCR-generated DNA corresponding to the cytoplasmic domain of the E. coli EnvZ protein and the full-length E. coli OmpR protein were cloned into the vector pET28b (EMD Biosciences) to create N-terminal and C-terminal 6× histidine tags (His-EnvZ-cyt and His-OmpR, respectively). .. Cells were harvested, resuspended in lysis buffer (100 mM sodium phosphate [pH 8.0], 300 mM NaCl, 10% glycerol, 1 mg/ml of lysozyme, 5 U/ml of DNase I, 1 μg/ml of pepstatin, 1 μg/ml of leupeptin), and lysed by three 30-s sonication bursts using a model 100 Sonic Dismembrator (Fisher Scientific) set at an intensity of 4.

Agarose Gel Electrophoresis:

Article Title: Molecular tools enabling pennycress (Thlaspi arvense) as a model plant and oilseed cash cover crop
Article Snippet: For T7 endonuclease I analysis, 10 μL of each 20 μL PCR reaction was denatured by heating at 95 °C for 5 min in a thermocycler (Fisher) and annealed using gradual cooling: −2 °C per second decrease from 95 to 85 °C then −0.1 °C per second decrease from 85 to 25 °C, followed by T7 endonuclease I (Fisher Scientific cat. #50‐995‐224 to New England Biolabs cat. #M0302L, Ipswich, MA) digestion for 30 min. .. The digested product was electrophoresed in a 1% agarose gel to identify samples that partially digested, indicating an SpCas9‐induced edit in TaFAE1 , which were confirmed by Sanger sequence analyses.

Concentration Assay:

Article Title: The Third Transmembrane Domain of EscR Is Critical for Function of the Enteropathogenic Escherichia coli Type III Secretion System
Article Snippet: MgCl2 was then added to a final concentration of 20 mM, and samples were spun for 10 min at 5,000 × g . .. Ten micrograms of RNase A and DNase I per milliliter was added, and the samples were sonicated (Fisher Scientific; 3 times for 15 s each).

Article Title: Direct Assessment of Viral Diversity in Soils by Random PCR Amplification of Polymorphic DNA
Article Snippet: Prior to analysis by RAPD-PCR, viral concentrates were treated with a high concentration of DNase I to remove free, extraviral DNA ( ). .. Five units of DNase I (RQ1 RNase-free DNase; Fisher Scientific, Pittsburg, PA), 2 μl of 10× DNase buffer, and 20 μl of viral concentrate or viral extract were mixed, and the mixture was incubated at 37°C for 30 min.

Article Title: The Ruler Protein EscP of the Enteropathogenic Escherichia coli Type III Secretion System Is Involved in Calcium Sensing and Secretion Hierarchy Regulation by Interacting with the Gatekeeper Protein SepL
Article Snippet: MgCl2 was then added to a final concentration of 20 mM, and samples were spun for 10 min at 5,000 × g . .. Ten micrograms of RNase A and DNase I per milliliter was added, and the samples were sonicated (Fisher Scientific, 3 × 15 s).

Article Title: EscE and EscG Are Cochaperones for the Type III Needle Protein EscF of Enteropathogenic Escherichia coli
Article Snippet: MgCl2 was then added to a final concentration of 20 mM, and samples were spun for 10 min at 5,000 × g . .. RNase A and DNase I (each at 10 μg/ml) were added, and the samples were sonicated (3 times, for 15 s each time; Fisher Scientific).

Article Title: Genome analysis of the staphylococcal temperate phage DW2 and functional studies on the endolysin and tail hydrolase
Article Snippet: .. Phage DNA preparation In order to remove contaminating host RNA and DNA from a filter sterilized lysate of phage DW2, prior to phage DNA extraction, DNase I was added to a final concentration of 3.3 U/µl (Fisher Scientific, 11873795) and RNase to a final concentration of 6.7 U/ml (Roche, 10109142001). .. The sample was then incubated at 37 °C for 10 min. To lyse the protein capsid 150 µl of lysis buffer (0.4 M EDTA, 1% SDS and 0.05 M TRIS-HCl pH 8) and 10 µl of 10 mg/ml proteinase K (Roche, 3115879001) was added to 750 µl aliquots of the phage lysate sample and they were incubated at 65 °C for 30 min. Proteins were removed by two chloroform: isoamylalcohol: phenol (24:1:25) and a chloroform: isoamylalcohol steps.

Article Title: The Role of the Small Export Apparatus Protein, SctS, in the Activity of the Type III Secretion System
Article Snippet: MgCl2 was then added to a final concentration of 20 mM, and samples were spun for 10 min at 5,000 × g . .. RNase A and DNase I (10 μg/mL) were added and the samples were sonicated (Fisher Scientific, 3 × 15 s).

Fractionation:

Article Title: The Third Transmembrane Domain of EscR Is Critical for Function of the Enteropathogenic Escherichia coli Type III Secretion System
Article Snippet: Paragraph title: Bacterial fractionation. ... Ten micrograms of RNase A and DNase I per milliliter was added, and the samples were sonicated (Fisher Scientific; 3 times for 15 s each).

Article Title: EscE and EscG Are Cochaperones for the Type III Needle Protein EscF of Enteropathogenic Escherichia coli
Article Snippet: Paragraph title: Bacterial fractionation. ... RNase A and DNase I (each at 10 μg/ml) were added, and the samples were sonicated (3 times, for 15 s each time; Fisher Scientific).

Article Title: The Role of the Small Export Apparatus Protein, SctS, in the Activity of the Type III Secretion System
Article Snippet: Paragraph title: Bacterial Fractionation ... RNase A and DNase I (10 μg/mL) were added and the samples were sonicated (Fisher Scientific, 3 × 15 s).

Article Title: The BAF (BRG1/BRM-Associated Factor) Chromatin-Remodeling Complex Exhibits Ethanol Sensitivity in Fetal Neural Progenitor Cells and Regulates Transcription at the Mir-9-2 Encoding Gene Locus
Article Snippet: As described in , nuclear pellets were treated with either 0, 20, 60 or 120U of DNAse I (Ambion/Thermo Fisher Scientific) for one hour on ice. .. Digestion into 200–500bp fragments was confirmed by size fractionation on an agarose DNA gel, and fragmented DNA was then blunt ended using T4 polymerase, followed by ligation to a double stranded linker DNA.

Lysis:

Article Title: The Third Transmembrane Domain of EscR Is Critical for Function of the Enteropathogenic Escherichia coli Type III Secretion System
Article Snippet: The pellets, which contained the cytoplasm and the membrane fractions, were resuspended in 1 ml lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 3 mM MgCl2 , 1 mM CaCl2 , and 2 mM β-mercaptoethanol with protease inhibitors). .. Ten micrograms of RNase A and DNase I per milliliter was added, and the samples were sonicated (Fisher Scientific; 3 times for 15 s each).

Article Title: The Nla28S/Nla28 Two-Component Signal Transduction System Regulates Sporulation in Myxococcus xanthus
Article Snippet: .. Cells were harvested, resuspended in lysis buffer (100 mM sodium phosphate [pH 8.0], 300 mM NaCl, 10% glycerol, 1 mg/ml of lysozyme, 5 U/ml of DNase I, 1 μg/ml of pepstatin, 1 μg/ml of leupeptin), and lysed by three 30-s sonication bursts using a model 100 Sonic Dismembrator (Fisher Scientific) set at an intensity of 4. .. GST and the GST fusion proteins were purified using 5-ml GSTrapFF columns (GE Healthcare) on an AKTA purifier UPC 10 fast-performance liquid chromatography (FPLC) system (GE).

Article Title: The Ruler Protein EscP of the Enteropathogenic Escherichia coli Type III Secretion System Is Involved in Calcium Sensing and Secretion Hierarchy Regulation by Interacting with the Gatekeeper Protein SepL
Article Snippet: The pellets, which contained the cytoplasm and membrane fractions, were resuspended in 1 ml of lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 3 mM MgCl2 , 1 mM CaCl2 , and 2 mM β-mercaptoethanol with protease inhibitors). .. Ten micrograms of RNase A and DNase I per milliliter was added, and the samples were sonicated (Fisher Scientific, 3 × 15 s).

Article Title: EscE and EscG Are Cochaperones for the Type III Needle Protein EscF of Enteropathogenic Escherichia coli
Article Snippet: The pellet containing the cytoplasm and the membrane fractions was resuspended in 1 ml lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 3 mM MgCl2 , 1 mM CaCl2 , and 2 mM β-mercaptoethanol with protease inhibitors). .. RNase A and DNase I (each at 10 μg/ml) were added, and the samples were sonicated (3 times, for 15 s each time; Fisher Scientific).

Article Title: Genome analysis of the staphylococcal temperate phage DW2 and functional studies on the endolysin and tail hydrolase
Article Snippet: Phage DNA preparation In order to remove contaminating host RNA and DNA from a filter sterilized lysate of phage DW2, prior to phage DNA extraction, DNase I was added to a final concentration of 3.3 U/µl (Fisher Scientific, 11873795) and RNase to a final concentration of 6.7 U/ml (Roche, 10109142001). .. The sample was then incubated at 37 °C for 10 min. To lyse the protein capsid 150 µl of lysis buffer (0.4 M EDTA, 1% SDS and 0.05 M TRIS-HCl pH 8) and 10 µl of 10 mg/ml proteinase K (Roche, 3115879001) was added to 750 µl aliquots of the phage lysate sample and they were incubated at 65 °C for 30 min. Proteins were removed by two chloroform: isoamylalcohol: phenol (24:1:25) and a chloroform: isoamylalcohol steps.

Article Title: The Role of the Small Export Apparatus Protein, SctS, in the Activity of the Type III Secretion System
Article Snippet: The pellets, which contained the cytoplasm and the membrane fractions, were resuspended in 1 mL lysis buffer (20 mM Tris/HCl pH 7.5, 150 mM NaCl, 3 mM MgCl2 , 1 mM CaCl2 , and 2 mM β-mercaptoethanol with protease inhibitors). .. RNase A and DNase I (10 μg/mL) were added and the samples were sonicated (Fisher Scientific, 3 × 15 s).

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    Fisher Scientific dnase i
    Dirofilaria immitis -induced dose- and viability- and diphenyleneiodonium (DPI)-independent neutrophil extracellular trap (NET) formation. (A) D. immitis microfilariae (MF; 100) were cocultured with canine polymorphonuclear neutrophils (PMN) for 180 min. For NADPH oxidase inhibition, DPI pre-treatment was used. To resolve NET formation, <t>DNase</t> I was added to coculture. (B) Canine PMN were cocultured with vital or heat-inactivated microfilariae (MF-HI). (C) Vital and heat-inactivated D. immitis L3 were exposed to canine PMN. Sytox Orange-derived fluorescence intensities were analyzed by spectrofluorometric analysis at an excitation wavelength of 547 nm and emission wavelength 570 nm using an automated plate monochrome reader. As negative control, PMN in plain medium were used. PMN stimulated with zymosan served as positive control.
    Dnase I, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase i/product/Fisher Scientific
    Average 99 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    dnase i - by Bioz Stars, 2020-03
    99/100 stars
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    78
    Fisher Scientific dnase i protection assay dna fragments
    Transposase binding to the Herves -right (R) end . (a) Herves transposase binds to Herves -R bp 1-30, bp 5-45 and bp 61-90. Electrophoretic mobility shift assay (EMSA) analysis of transposase binding to the overlapping 30 bp fragments (bp 1-30, bp 31-60, bp 61-90, bp 91-110, bp 15-45 and bp 46-75). The asterisk (*) indicates various protein <t>DNA</t> complexes. (b) Herves transposase binding to Herves -R bp 15-45 and bp 61-90. The Herves -R bp 61-90 fragment was used as a probe in EMSAs. The fraction of the transposase-bound probe was quantified using a phosphoimager. A homologous fragment was used as specific competitor at a molar excess of 50-fold, 100-fold and 200-fold, whereas non-specific competition was used as described for Figure 2b. Overlapping fragments (bp 1-30, bp 31-60, bp 91-110, bp 15-45, and bp 46-75) were used as competitors of transposase binding to the probe, at 200-fold molar excess. (c) <t>DNase</t> I protection assay of the Herves -R end. The single-end-labeled Herves -R 1-100 bp fragment (100 nM) was incubated in presence (+, ++) or absence (-) of DNase I or the transposase. The ++ indicates 1.4 μM of transposase or 0.5 units of DNase I, whereas + indicates 850 nM of transposase or 0.25 units of DNase I. 32 P indicates the end of the probe that was labeled. The solid bars on the sides indicate the region of the probe protected by the transposase. The asterisk (*) indicates hypersensitive sites.
    Dnase I Protection Assay Dna Fragments, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase i protection assay dna fragments/product/Fisher Scientific
    Average 78 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dnase i protection assay dna fragments - by Bioz Stars, 2020-03
    78/100 stars
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    Dirofilaria immitis -induced dose- and viability- and diphenyleneiodonium (DPI)-independent neutrophil extracellular trap (NET) formation. (A) D. immitis microfilariae (MF; 100) were cocultured with canine polymorphonuclear neutrophils (PMN) for 180 min. For NADPH oxidase inhibition, DPI pre-treatment was used. To resolve NET formation, DNase I was added to coculture. (B) Canine PMN were cocultured with vital or heat-inactivated microfilariae (MF-HI). (C) Vital and heat-inactivated D. immitis L3 were exposed to canine PMN. Sytox Orange-derived fluorescence intensities were analyzed by spectrofluorometric analysis at an excitation wavelength of 547 nm and emission wavelength 570 nm using an automated plate monochrome reader. As negative control, PMN in plain medium were used. PMN stimulated with zymosan served as positive control.

    Journal: Frontiers in Immunology

    Article Title: Dirofilaria immitis Microfilariae and Third-Stage Larvae Induce Canine NETosis Resulting in Different Types of Neutrophil Extracellular Traps

    doi: 10.3389/fimmu.2018.00968

    Figure Lengend Snippet: Dirofilaria immitis -induced dose- and viability- and diphenyleneiodonium (DPI)-independent neutrophil extracellular trap (NET) formation. (A) D. immitis microfilariae (MF; 100) were cocultured with canine polymorphonuclear neutrophils (PMN) for 180 min. For NADPH oxidase inhibition, DPI pre-treatment was used. To resolve NET formation, DNase I was added to coculture. (B) Canine PMN were cocultured with vital or heat-inactivated microfilariae (MF-HI). (C) Vital and heat-inactivated D. immitis L3 were exposed to canine PMN. Sytox Orange-derived fluorescence intensities were analyzed by spectrofluorometric analysis at an excitation wavelength of 547 nm and emission wavelength 570 nm using an automated plate monochrome reader. As negative control, PMN in plain medium were used. PMN stimulated with zymosan served as positive control.

    Article Snippet: Treatments of PMN with 90U DNase I (Fisher Scientific) at the moment of parasite exposure were used to dissolve NETs.

    Techniques: Inhibition, Hi-C, Derivative Assay, Fluorescence, Negative Control, Positive Control

    Dirofilaria immitis- induced parasite entrapment. Canine polymorphonuclear neutrophils (PMN) were exposed to vital D. immitis microfilariae (A) or L3 (B,C) for 60 and 180 min. (D) In parallel settings, the same number of PMN was incubated either with heat-inactivated (HI) microfilariae (HI MF) or pretreated with diphenyleneiodonium (DPI) before exposure to vital microfilariae. Furthermore, DNase I was added at the moment of exposure to vital microfilariae. Larvae were considered as entrapped when PMN and/or PMN-derived neutrophil extracellular traps (NETs) were in contact with larvae. The data are expressed as percentage of entrapped larvae relative to the total amount of larvae per condition. The formation of “clasp”-like NET structures sticking mainly to the anterior part of the larvae is displayed in white arrow: image [ (C) , 2].

    Journal: Frontiers in Immunology

    Article Title: Dirofilaria immitis Microfilariae and Third-Stage Larvae Induce Canine NETosis Resulting in Different Types of Neutrophil Extracellular Traps

    doi: 10.3389/fimmu.2018.00968

    Figure Lengend Snippet: Dirofilaria immitis- induced parasite entrapment. Canine polymorphonuclear neutrophils (PMN) were exposed to vital D. immitis microfilariae (A) or L3 (B,C) for 60 and 180 min. (D) In parallel settings, the same number of PMN was incubated either with heat-inactivated (HI) microfilariae (HI MF) or pretreated with diphenyleneiodonium (DPI) before exposure to vital microfilariae. Furthermore, DNase I was added at the moment of exposure to vital microfilariae. Larvae were considered as entrapped when PMN and/or PMN-derived neutrophil extracellular traps (NETs) were in contact with larvae. The data are expressed as percentage of entrapped larvae relative to the total amount of larvae per condition. The formation of “clasp”-like NET structures sticking mainly to the anterior part of the larvae is displayed in white arrow: image [ (C) , 2].

    Article Snippet: Treatments of PMN with 90U DNase I (Fisher Scientific) at the moment of parasite exposure were used to dissolve NETs.

    Techniques: Incubation, Derivative Assay

    ChIP analysis indicates that ethanol exposure increases BRG1 binding to DNAse I-resistant/CpG island containing region 3 and the pre-miR-9-2 coding exon Bar graph shows the effect of ethanol on BRG1-association with regions 1 to 5 and the pre-miR-9-2 exon-coding region of the primary (pri)-miR-9-2 coding locus. Primers for a gene desert on chromosome 6 (not predicted to bind any transcription factors) shows the specificity of the immuno-precipitation. Vertical axis shows the fold change of the specific associated DNA region to BRG1 in ethanol treated group relative to control group. Error bars indicate standard error of the mean.

    Journal: Alcohol (Fayetteville, N.Y.)

    Article Title: The BAF (BRG1/BRM-Associated Factor) Chromatin-Remodeling Complex Exhibits Ethanol Sensitivity in Fetal Neural Progenitor Cells and Regulates Transcription at the Mir-9-2 Encoding Gene Locus

    doi: 10.1016/j.alcohol.2017.01.003

    Figure Lengend Snippet: ChIP analysis indicates that ethanol exposure increases BRG1 binding to DNAse I-resistant/CpG island containing region 3 and the pre-miR-9-2 coding exon Bar graph shows the effect of ethanol on BRG1-association with regions 1 to 5 and the pre-miR-9-2 exon-coding region of the primary (pri)-miR-9-2 coding locus. Primers for a gene desert on chromosome 6 (not predicted to bind any transcription factors) shows the specificity of the immuno-precipitation. Vertical axis shows the fold change of the specific associated DNA region to BRG1 in ethanol treated group relative to control group. Error bars indicate standard error of the mean.

    Article Snippet: As described in , nuclear pellets were treated with either 0, 20, 60 or 120U of DNAse I (Ambion/Thermo Fisher Scientific) for one hour on ice.

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation

    BRG1-containing complexes associate with both DNAse I-hypersensitive and insensitive sites on the miR-9-2 gene locus (a) Positional representation of POU5F1/Oct4, c-myc and REST transcription regulatory factor binding sites along the human pre-miR-9-2 gene locus identified with the UCSC genome browser ENCODE analysis hub track. Colored circles indicate locations for primer pairs for pri-miR-9-2 regions 1,2,3,4 and 5 and the pre-miR-9-2 coding region. (b) qPCR results of BRG1-ChIP from neurospheres. Primers were used to amplify 6 distinct positions along the pri-miR-9-2 gene locus and a genetically sparse region on mouse chromosome 6. Vertical axis shows fold change relative to an IgG pulldown control. Error bars indicate standard error of the mean. (c) qPCR of neurosphere DNA following digestion with 20, 60 and 120U of DNAse I at the 6 positions along the pri-miR-9-2 gene locus. The vertical axis shows fold amplification relative to DNAse I-untreated neurosphere DNA.

    Journal: Alcohol (Fayetteville, N.Y.)

    Article Title: The BAF (BRG1/BRM-Associated Factor) Chromatin-Remodeling Complex Exhibits Ethanol Sensitivity in Fetal Neural Progenitor Cells and Regulates Transcription at the Mir-9-2 Encoding Gene Locus

    doi: 10.1016/j.alcohol.2017.01.003

    Figure Lengend Snippet: BRG1-containing complexes associate with both DNAse I-hypersensitive and insensitive sites on the miR-9-2 gene locus (a) Positional representation of POU5F1/Oct4, c-myc and REST transcription regulatory factor binding sites along the human pre-miR-9-2 gene locus identified with the UCSC genome browser ENCODE analysis hub track. Colored circles indicate locations for primer pairs for pri-miR-9-2 regions 1,2,3,4 and 5 and the pre-miR-9-2 coding region. (b) qPCR results of BRG1-ChIP from neurospheres. Primers were used to amplify 6 distinct positions along the pri-miR-9-2 gene locus and a genetically sparse region on mouse chromosome 6. Vertical axis shows fold change relative to an IgG pulldown control. Error bars indicate standard error of the mean. (c) qPCR of neurosphere DNA following digestion with 20, 60 and 120U of DNAse I at the 6 positions along the pri-miR-9-2 gene locus. The vertical axis shows fold amplification relative to DNAse I-untreated neurosphere DNA.

    Article Snippet: As described in , nuclear pellets were treated with either 0, 20, 60 or 120U of DNAse I (Ambion/Thermo Fisher Scientific) for one hour on ice.

    Techniques: Binding Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Amplification

    Dirofilaria immitis -induced dose- and viability- and diphenyleneiodonium (DPI)-independent neutrophil extracellular trap (NET) formation. (A) D. immitis microfilariae (MF; 100) were cocultured with canine polymorphonuclear neutrophils (PMN) for 180 min. For NADPH oxidase inhibition, DPI pre-treatment was used. To resolve NET formation, DNase I was added to coculture. (B) Canine PMN were cocultured with vital or heat-inactivated microfilariae (MF-HI). (C) Vital and heat-inactivated D. immitis L3 were exposed to canine PMN. Sytox Orange-derived fluorescence intensities were analyzed by spectrofluorometric analysis at an excitation wavelength of 547 nm and emission wavelength 570 nm using an automated plate monochrome reader. As negative control, PMN in plain medium were used. PMN stimulated with zymosan served as positive control.

    Journal: Frontiers in Immunology

    Article Title: Dirofilaria immitis Microfilariae and Third-Stage Larvae Induce Canine NETosis Resulting in Different Types of Neutrophil Extracellular Traps

    doi: 10.3389/fimmu.2018.00968

    Figure Lengend Snippet: Dirofilaria immitis -induced dose- and viability- and diphenyleneiodonium (DPI)-independent neutrophil extracellular trap (NET) formation. (A) D. immitis microfilariae (MF; 100) were cocultured with canine polymorphonuclear neutrophils (PMN) for 180 min. For NADPH oxidase inhibition, DPI pre-treatment was used. To resolve NET formation, DNase I was added to coculture. (B) Canine PMN were cocultured with vital or heat-inactivated microfilariae (MF-HI). (C) Vital and heat-inactivated D. immitis L3 were exposed to canine PMN. Sytox Orange-derived fluorescence intensities were analyzed by spectrofluorometric analysis at an excitation wavelength of 547 nm and emission wavelength 570 nm using an automated plate monochrome reader. As negative control, PMN in plain medium were used. PMN stimulated with zymosan served as positive control.

    Article Snippet: Treatments of PMN with 90U DNase I (Fisher Scientific) at the moment of parasite exposure were used to dissolve NETs.

    Techniques: Inhibition, Hi-C, Derivative Assay, Fluorescence, Negative Control, Positive Control

    Dirofilaria immitis- induced parasite entrapment. Canine polymorphonuclear neutrophils (PMN) were exposed to vital D. immitis microfilariae (A) or L3 (B,C) for 60 and 180 min. (D) In parallel settings, the same number of PMN was incubated either with heat-inactivated (HI) microfilariae (HI MF) or pretreated with diphenyleneiodonium (DPI) before exposure to vital microfilariae. Furthermore, DNase I was added at the moment of exposure to vital microfilariae. Larvae were considered as entrapped when PMN and/or PMN-derived neutrophil extracellular traps (NETs) were in contact with larvae. The data are expressed as percentage of entrapped larvae relative to the total amount of larvae per condition. The formation of “clasp”-like NET structures sticking mainly to the anterior part of the larvae is displayed in white arrow: image [ (C) , 2].

    Journal: Frontiers in Immunology

    Article Title: Dirofilaria immitis Microfilariae and Third-Stage Larvae Induce Canine NETosis Resulting in Different Types of Neutrophil Extracellular Traps

    doi: 10.3389/fimmu.2018.00968

    Figure Lengend Snippet: Dirofilaria immitis- induced parasite entrapment. Canine polymorphonuclear neutrophils (PMN) were exposed to vital D. immitis microfilariae (A) or L3 (B,C) for 60 and 180 min. (D) In parallel settings, the same number of PMN was incubated either with heat-inactivated (HI) microfilariae (HI MF) or pretreated with diphenyleneiodonium (DPI) before exposure to vital microfilariae. Furthermore, DNase I was added at the moment of exposure to vital microfilariae. Larvae were considered as entrapped when PMN and/or PMN-derived neutrophil extracellular traps (NETs) were in contact with larvae. The data are expressed as percentage of entrapped larvae relative to the total amount of larvae per condition. The formation of “clasp”-like NET structures sticking mainly to the anterior part of the larvae is displayed in white arrow: image [ (C) , 2].

    Article Snippet: Treatments of PMN with 90U DNase I (Fisher Scientific) at the moment of parasite exposure were used to dissolve NETs.

    Techniques: Incubation, Derivative Assay

    Transposase binding to the Herves -right (R) end . (a) Herves transposase binds to Herves -R bp 1-30, bp 5-45 and bp 61-90. Electrophoretic mobility shift assay (EMSA) analysis of transposase binding to the overlapping 30 bp fragments (bp 1-30, bp 31-60, bp 61-90, bp 91-110, bp 15-45 and bp 46-75). The asterisk (*) indicates various protein DNA complexes. (b) Herves transposase binding to Herves -R bp 15-45 and bp 61-90. The Herves -R bp 61-90 fragment was used as a probe in EMSAs. The fraction of the transposase-bound probe was quantified using a phosphoimager. A homologous fragment was used as specific competitor at a molar excess of 50-fold, 100-fold and 200-fold, whereas non-specific competition was used as described for Figure 2b. Overlapping fragments (bp 1-30, bp 31-60, bp 91-110, bp 15-45, and bp 46-75) were used as competitors of transposase binding to the probe, at 200-fold molar excess. (c) DNase I protection assay of the Herves -R end. The single-end-labeled Herves -R 1-100 bp fragment (100 nM) was incubated in presence (+, ++) or absence (-) of DNase I or the transposase. The ++ indicates 1.4 μM of transposase or 0.5 units of DNase I, whereas + indicates 850 nM of transposase or 0.25 units of DNase I. 32 P indicates the end of the probe that was labeled. The solid bars on the sides indicate the region of the probe protected by the transposase. The asterisk (*) indicates hypersensitive sites.

    Journal: Mobile DNA

    Article Title: DNA binding activities of the Herves transposase from the mosquito Anopheles gambiae

    doi: 10.1186/1759-8753-2-9

    Figure Lengend Snippet: Transposase binding to the Herves -right (R) end . (a) Herves transposase binds to Herves -R bp 1-30, bp 5-45 and bp 61-90. Electrophoretic mobility shift assay (EMSA) analysis of transposase binding to the overlapping 30 bp fragments (bp 1-30, bp 31-60, bp 61-90, bp 91-110, bp 15-45 and bp 46-75). The asterisk (*) indicates various protein DNA complexes. (b) Herves transposase binding to Herves -R bp 15-45 and bp 61-90. The Herves -R bp 61-90 fragment was used as a probe in EMSAs. The fraction of the transposase-bound probe was quantified using a phosphoimager. A homologous fragment was used as specific competitor at a molar excess of 50-fold, 100-fold and 200-fold, whereas non-specific competition was used as described for Figure 2b. Overlapping fragments (bp 1-30, bp 31-60, bp 91-110, bp 15-45, and bp 46-75) were used as competitors of transposase binding to the probe, at 200-fold molar excess. (c) DNase I protection assay of the Herves -R end. The single-end-labeled Herves -R 1-100 bp fragment (100 nM) was incubated in presence (+, ++) or absence (-) of DNase I or the transposase. The ++ indicates 1.4 μM of transposase or 0.5 units of DNase I, whereas + indicates 850 nM of transposase or 0.25 units of DNase I. 32 P indicates the end of the probe that was labeled. The solid bars on the sides indicate the region of the probe protected by the transposase. The asterisk (*) indicates hypersensitive sites.

    Article Snippet: DNase I protection assay DNA fragments (100 bp each) from the Herves -L and Herves -R ends, containing an Eco RV restriction site at the L-end or R-end, were cloned into pJET 1.2 (Fermentas/Thermo Fisher Scientific, Piscataway, NJ) to generate pL5'Eco RV, pL3'Eco RV, pR5'Eco RV, and pR3'Eco RV.

    Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Labeling, Incubation