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  • 99
    New England Biolabs dsdna fragmentase protocol
    Dsdna Fragmentase Protocol, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dsdna fragmentase protocol/product/New England Biolabs
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    99
    Thermo Fisher dsdna kit
    Dsdna Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dsdna kit/product/Thermo Fisher
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    93
    Millipore dsdna
    <t>A151</t> inhibits cGAS-STING signaling via both sequence and backbone specific binding to cGAS. (A) HEK cGAS low or HEK cGAS high cells were co-cultured with HEK STING cells for 24 h. Cultures were transfected with <t>dsDNA</t> (4 μg/ml) and/or treated with A151 (1 μM) where indicated. STING aggregate formation (mCherry clustering) in HEK STING cells was examined by fluorescence microscopy in three independent visual fields per well (containing > 100 cells/field) from four independent experiments. (B) To examine both sequence and backbone composition of A151-mediated cGAS inhibition, cells were pretreated with increasing amounts of A151, A151 (PD) or C151 and then stimulated with dsDNA or cGAMP (2 μg/ml). IFN-β mRNA levels were measured 6h later by qPCR using 18s RNA as an endogenous control. Results are shown as the percent inhibition of dsDNA stimulated cells from one representative of two independent experiments performed in triplicates (C) Lysates from immortalized THP-1 cells were subjected to pulldown analysis using dsDNA without (lane 1) or with (lane 2–7) 3 ’ -biotinylation. Increasing amounts of A151 (0.1, 0.3, 1, 3 and 10 μg) were included in lanes 3–7. 3 ’ -biotinylated A151 was run in lane 8 and whole lysate in lane 9. Immunoblots were probed for the presence of cGAS. Results show one representative experiment of two independent experiments. Statistical analysis: unpaired Student’s test, *** p
    Dsdna, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 785 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega quantifluor dsdna
    <t>A151</t> inhibits cGAS-STING signaling via both sequence and backbone specific binding to cGAS. (A) HEK cGAS low or HEK cGAS high cells were co-cultured with HEK STING cells for 24 h. Cultures were transfected with <t>dsDNA</t> (4 μg/ml) and/or treated with A151 (1 μM) where indicated. STING aggregate formation (mCherry clustering) in HEK STING cells was examined by fluorescence microscopy in three independent visual fields per well (containing > 100 cells/field) from four independent experiments. (B) To examine both sequence and backbone composition of A151-mediated cGAS inhibition, cells were pretreated with increasing amounts of A151, A151 (PD) or C151 and then stimulated with dsDNA or cGAMP (2 μg/ml). IFN-β mRNA levels were measured 6h later by qPCR using 18s RNA as an endogenous control. Results are shown as the percent inhibition of dsDNA stimulated cells from one representative of two independent experiments performed in triplicates (C) Lysates from immortalized THP-1 cells were subjected to pulldown analysis using dsDNA without (lane 1) or with (lane 2–7) 3 ’ -biotinylation. Increasing amounts of A151 (0.1, 0.3, 1, 3 and 10 μg) were included in lanes 3–7. 3 ’ -biotinylated A151 was run in lane 8 and whole lysate in lane 9. Immunoblots were probed for the presence of cGAS. Results show one representative experiment of two independent experiments. Statistical analysis: unpaired Student’s test, *** p
    Quantifluor Dsdna, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Fisher Scientific dsdna
    <t>A151</t> inhibits cGAS-STING signaling via both sequence and backbone specific binding to cGAS. (A) HEK cGAS low or HEK cGAS high cells were co-cultured with HEK STING cells for 24 h. Cultures were transfected with <t>dsDNA</t> (4 μg/ml) and/or treated with A151 (1 μM) where indicated. STING aggregate formation (mCherry clustering) in HEK STING cells was examined by fluorescence microscopy in three independent visual fields per well (containing > 100 cells/field) from four independent experiments. (B) To examine both sequence and backbone composition of A151-mediated cGAS inhibition, cells were pretreated with increasing amounts of A151, A151 (PD) or C151 and then stimulated with dsDNA or cGAMP (2 μg/ml). IFN-β mRNA levels were measured 6h later by qPCR using 18s RNA as an endogenous control. Results are shown as the percent inhibition of dsDNA stimulated cells from one representative of two independent experiments performed in triplicates (C) Lysates from immortalized THP-1 cells were subjected to pulldown analysis using dsDNA without (lane 1) or with (lane 2–7) 3 ’ -biotinylation. Increasing amounts of A151 (0.1, 0.3, 1, 3 and 10 μg) were included in lanes 3–7. 3 ’ -biotinylated A151 was run in lane 8 and whole lysate in lane 9. Immunoblots were probed for the presence of cGAS. Results show one representative experiment of two independent experiments. Statistical analysis: unpaired Student’s test, *** p
    Dsdna, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega dsdna
    Influenza A infection increases <t>AIM2</t> and stimulates <t>dsDNA</t> release into BAL. ( A ) Cultured human primary ATII cells and AMs from six donors were infected with influenza virus PR8 or MOCK control (Ctrl) at moi of 1, and RNA was extracted at 24 hpi for evaluation
    Dsdna, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 436 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dsdna  (ALPCO)
    90
    ALPCO dsdna
    Influenza A infection increases <t>AIM2</t> and stimulates <t>dsDNA</t> release into BAL. ( A ) Cultured human primary ATII cells and AMs from six donors were infected with influenza virus PR8 or MOCK control (Ctrl) at moi of 1, and RNA was extracted at 24 hpi for evaluation
    Dsdna, supplied by ALPCO, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dsdna  (Roche)
    93
    Roche dsdna
    Influenza A infection increases <t>AIM2</t> and stimulates <t>dsDNA</t> release into BAL. ( A ) Cultured human primary ATII cells and AMs from six donors were infected with influenza virus PR8 or MOCK control (Ctrl) at moi of 1, and RNA was extracted at 24 hpi for evaluation
    Dsdna, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 257 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dsdna/product/Roche
    Average 93 stars, based on 257 article reviews
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    91
    Bioneer Corporation dsdna
    Influenza A infection increases <t>AIM2</t> and stimulates <t>dsDNA</t> release into BAL. ( A ) Cultured human primary ATII cells and AMs from six donors were infected with influenza virus PR8 or MOCK control (Ctrl) at moi of 1, and RNA was extracted at 24 hpi for evaluation
    Dsdna, supplied by Bioneer Corporation, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    New England Biolabs dsdna fragmentase
    Genome Coverage plots. Representation of the MPS relative coverage of both strands (rc+: relative coverage of the plus strand, rc-: relative coverage of the negative strand) of the pUC19 plasmid, or mtDNA molecules obtained from the Ion Torrent PGM or MiSeq sequencing system. The outer circle symbolizes the pUC19 (A) or mtDNA (B, C, D) gene structure, respectively. 1A: Use of the Ion Torrent PGM standard protocol on the pUC19 plasmid. 1B: Use of three different fragmentation methods in combination with the Ion Torrent sequencing protocol on the mtDNA: Ion Shear Plus Reagents (enzymatic), <t>NEBNext</t> <t>dsDNA</t> <t>Fragmentase</t> (enzymatic) and Covaris (physical). 1C: Use of an Ion Torrent PGM protocol without PCR amplification in the library construction on the mtDNA. 1D: LR-PCR products of the mtDNA were Covaris (physical) or NEBNext dsDNA Fragmentase (enzymatic) sheared, followed by a TruSeq DNA PCR free protocol on a MiSeq instrument. The same six samples were processed with a Nextera XT kit (enzymatic shearing and PCR amplification in library preparation) prior to MiSeq analysis.
    Dsdna Fragmentase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 247 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Dynatech Laboratories dsdna
    Correlation of autoAbs with <t>anti-dsDNA</t> and lupus nephritis The Pearson correlation coefficient of the 16 upregulated autoAbs with anti-dsDNA antibodies was analyzed by GraphPad Prism 6.0 with the levels of autoAbs measured using <t>ELISA</t> from the 35 SLE patients. Pearson's r represents the linear correlation between the levels of the selected autoAb and anti-dsDNA, where 1 means completely positive linear correlation, 0 means no linear correlation, and − 1 means completely negative linear correlation. Correlation with P
    Dsdna, supplied by Dynatech Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    InvivoGen dsdna
    NO 2 -FAs suppress STING signaling and release of type I IFN. ( A and B ) THP-1 cells and ( C and D ) BMMs (WT mice) were treated with indicated NO 2 -FAs (5–10 µM) or OA/LA (10 µM) 15 min before stimulation with <t>dsDNA</t> (4 µg/mL) or infection with <t>HSV-2</t> (MOI 1) or left untreated (Ut). After 20 h, supernatants were harvested and analyzed for type I IFN. Data represent one of two independent experiments and are presented as mean ± SEM. ( E – G ) THP-1 cells were treated with NO 2 -FAs (10 µM) or OA/LA (10 µM) 15 min before stimulation with cGAMP (4 µg/mL) or dsDNA (4 µg/mL) using Lipofectamine2000 (Lipo). After 3 h, lysates were separated by SDS/PAGE, and indicated proteins were detected by Western blotting using specific antibodies. STING and IRF3 dimers were detected using nondenaturing and nonreducing conditions. Vinculin was used as loading control.
    Dsdna, supplied by InvivoGen, used in various techniques. Bioz Stars score: 92/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dsdna
    RCA assay of pUC19 DNA plasmid. ( A ) Agarose gel of pUC19 RCA products. Lanes 1–7 RCA performed with increasing concentrations of T4 gene 32 protein (0,10, 20, 30, 50, 75, 100 ng/μl respectively); lane 8 negative control with no phi29 DNA polymerase in the reaction mixture; 1 kb plus DNA ladders (L). ( B ) Agarose gel of MlyI digestion test. RCA products in (A) were digested by MlyI restriction enzyme and the corresponding digestion products (9-16) were run on agarose gel. 1 kb plus DNA ladders (L). ( C ) <t>Picogreen</t> assay of pUC19 RCA. The amplification is expressed in percentage of relative fluorescence units (RFU) and the signal of the amplification product without T4 gene 32 is taken as 100%. Both MlyI digestion and picogreen assay confirm that rolling circle amplification makes mostly double-stranded DNA but they also suggest that T4 gene 32 SSB protein drastically reduces <t>dsDNA</t> production.
    Dsdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2684 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    A151 inhibits cGAS-STING signaling via both sequence and backbone specific binding to cGAS. (A) HEK cGAS low or HEK cGAS high cells were co-cultured with HEK STING cells for 24 h. Cultures were transfected with dsDNA (4 μg/ml) and/or treated with A151 (1 μM) where indicated. STING aggregate formation (mCherry clustering) in HEK STING cells was examined by fluorescence microscopy in three independent visual fields per well (containing > 100 cells/field) from four independent experiments. (B) To examine both sequence and backbone composition of A151-mediated cGAS inhibition, cells were pretreated with increasing amounts of A151, A151 (PD) or C151 and then stimulated with dsDNA or cGAMP (2 μg/ml). IFN-β mRNA levels were measured 6h later by qPCR using 18s RNA as an endogenous control. Results are shown as the percent inhibition of dsDNA stimulated cells from one representative of two independent experiments performed in triplicates (C) Lysates from immortalized THP-1 cells were subjected to pulldown analysis using dsDNA without (lane 1) or with (lane 2–7) 3 ’ -biotinylation. Increasing amounts of A151 (0.1, 0.3, 1, 3 and 10 μg) were included in lanes 3–7. 3 ’ -biotinylated A151 was run in lane 8 and whole lysate in lane 9. Immunoblots were probed for the presence of cGAS. Results show one representative experiment of two independent experiments. Statistical analysis: unpaired Student’s test, *** p

    Journal: European journal of immunology

    Article Title: Suppressive oligodeoxynucleotides containing TTAGGG motifs inhibit cGAS activation in human monocytes

    doi: 10.1002/eji.201747338

    Figure Lengend Snippet: A151 inhibits cGAS-STING signaling via both sequence and backbone specific binding to cGAS. (A) HEK cGAS low or HEK cGAS high cells were co-cultured with HEK STING cells for 24 h. Cultures were transfected with dsDNA (4 μg/ml) and/or treated with A151 (1 μM) where indicated. STING aggregate formation (mCherry clustering) in HEK STING cells was examined by fluorescence microscopy in three independent visual fields per well (containing > 100 cells/field) from four independent experiments. (B) To examine both sequence and backbone composition of A151-mediated cGAS inhibition, cells were pretreated with increasing amounts of A151, A151 (PD) or C151 and then stimulated with dsDNA or cGAMP (2 μg/ml). IFN-β mRNA levels were measured 6h later by qPCR using 18s RNA as an endogenous control. Results are shown as the percent inhibition of dsDNA stimulated cells from one representative of two independent experiments performed in triplicates (C) Lysates from immortalized THP-1 cells were subjected to pulldown analysis using dsDNA without (lane 1) or with (lane 2–7) 3 ’ -biotinylation. Increasing amounts of A151 (0.1, 0.3, 1, 3 and 10 μg) were included in lanes 3–7. 3 ’ -biotinylated A151 was run in lane 8 and whole lysate in lane 9. Immunoblots were probed for the presence of cGAS. Results show one representative experiment of two independent experiments. Statistical analysis: unpaired Student’s test, *** p

    Article Snippet: A 3’ -biotin tag was added to A151 or dsDNA for pulldowns. dsDNA isolated from herring sperm was purchased from Sigma-Aldrich (St. Louis, MO, USA). cGAMP (cyclic [G(2’,5’)pA(3’,5’)p]) was purchased from Invivogen (San Diego, CA, USA). mtDNA was isolated from human hepatoma cells (HepG2) using the Mitochondrial Isolation Kit for mammalian cells (Thermo Fisher Scientific, Darmstadt, Germany) according to the manufacturer’s instruction.

    Techniques: Sequencing, Binding Assay, Cell Culture, Transfection, Fluorescence, Microscopy, Inhibition, Real-time Polymerase Chain Reaction, Western Blot

    A151 inhibits cytosolic DNA-mediated type I IFN response in human monocytes. (A) Human THP1 cells were transfected with increasing concentrations (0.5, 1, 2 and 4 μg/ml) of dsDNA, mtDNA or cGAMP for 6 h. Additionally, cells were treated with all three ligands (4 μg/ml) in the absence of Lipofectamin (nL). (B) Wild-type (WT) and cGAS kockout (KO) THP-1 cells were transfected with dsDNA, mtDNA or cGAMP for 6 h. (C) THP-1 cells were transfected with dsDNA, mtDNA or cGAMP (2 μg/ml) for 6h and pretreated with A151 or C151 (0.3 μM). IFN-β mRNA levels were determined by qPCR using 18s RNA as an endogenous control. The relative rise in IFN-β levels compared to untreated controls is shown. Results show (A) one of three independent experiments and (B) the mean + SEM from three or (C) four independent experiments performed in triplicates.(D) THP-1 cells were transfected with 2 μg/ml dsDNA, mtDNA or cGAMP for 6 h and pretreated (+) with A151 (0.3 μM) or left untreated (−). Lysates were studied for IRF-3 phosphorylation (pIRF3) by immunoblot. Results show one representative experiment of three independent experiments.(E) THP-1 cells were transfected with dsDNA (2 μg/ml) and pretreated with increasing concentrations of A151. IP-10 protein levels were measured by ELISA in supernatants collected after 24h of stimulation. Results show the mean +SEM from four independent experiments performed in duplicates. Statistical analysis: unpaired Student’s test, * p

    Journal: European journal of immunology

    Article Title: Suppressive oligodeoxynucleotides containing TTAGGG motifs inhibit cGAS activation in human monocytes

    doi: 10.1002/eji.201747338

    Figure Lengend Snippet: A151 inhibits cytosolic DNA-mediated type I IFN response in human monocytes. (A) Human THP1 cells were transfected with increasing concentrations (0.5, 1, 2 and 4 μg/ml) of dsDNA, mtDNA or cGAMP for 6 h. Additionally, cells were treated with all three ligands (4 μg/ml) in the absence of Lipofectamin (nL). (B) Wild-type (WT) and cGAS kockout (KO) THP-1 cells were transfected with dsDNA, mtDNA or cGAMP for 6 h. (C) THP-1 cells were transfected with dsDNA, mtDNA or cGAMP (2 μg/ml) for 6h and pretreated with A151 or C151 (0.3 μM). IFN-β mRNA levels were determined by qPCR using 18s RNA as an endogenous control. The relative rise in IFN-β levels compared to untreated controls is shown. Results show (A) one of three independent experiments and (B) the mean + SEM from three or (C) four independent experiments performed in triplicates.(D) THP-1 cells were transfected with 2 μg/ml dsDNA, mtDNA or cGAMP for 6 h and pretreated (+) with A151 (0.3 μM) or left untreated (−). Lysates were studied for IRF-3 phosphorylation (pIRF3) by immunoblot. Results show one representative experiment of three independent experiments.(E) THP-1 cells were transfected with dsDNA (2 μg/ml) and pretreated with increasing concentrations of A151. IP-10 protein levels were measured by ELISA in supernatants collected after 24h of stimulation. Results show the mean +SEM from four independent experiments performed in duplicates. Statistical analysis: unpaired Student’s test, * p

    Article Snippet: A 3’ -biotin tag was added to A151 or dsDNA for pulldowns. dsDNA isolated from herring sperm was purchased from Sigma-Aldrich (St. Louis, MO, USA). cGAMP (cyclic [G(2’,5’)pA(3’,5’)p]) was purchased from Invivogen (San Diego, CA, USA). mtDNA was isolated from human hepatoma cells (HepG2) using the Mitochondrial Isolation Kit for mammalian cells (Thermo Fisher Scientific, Darmstadt, Germany) according to the manufacturer’s instruction.

    Techniques: Transfection, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Influenza A infection increases AIM2 and stimulates dsDNA release into BAL. ( A ) Cultured human primary ATII cells and AMs from six donors were infected with influenza virus PR8 or MOCK control (Ctrl) at moi of 1, and RNA was extracted at 24 hpi for evaluation

    Journal: The Journal of Immunology Author Choice

    Article Title: AIM2 Inflammasome Is Critical for Influenza-Induced Lung Injury and Mortality

    doi: 10.4049/jimmunol.1600714

    Figure Lengend Snippet: Influenza A infection increases AIM2 and stimulates dsDNA release into BAL. ( A ) Cultured human primary ATII cells and AMs from six donors were infected with influenza virus PR8 or MOCK control (Ctrl) at moi of 1, and RNA was extracted at 24 hpi for evaluation

    Article Snippet: We infected both WT and AIM2−/− mice with PR8, collected BALF, and determined the level of dsDNA using QuantiFluor dsDNA kit (Promega). confirmed the presence of dsDNA in cell-free BALF as early as 1 day postinfection (dpi) in both WT and AIM2−/− mice, and the amount of dsDNA was further increased at 3 dpi.

    Techniques: Infection, Cell Culture, Affinity Magnetic Separation

    Impact of DNase I treatment on B . pseudomallei biofilm formation. Static biofilms of B . pseudomallei strains L1, P1 and H777 in LB were treated with DNase I (0.01, 0.1 and 1 U/mL) at 0 h, 24 h or 45 h after inoculation and maintained for up to 48 h. Biofilm formation and eDNA concentration of the 2-day biofilms were assessed using crystal-violet absorbance (OD 620 ) and the QuantiFluor dsDNA System, respectively. DNase I buffer acted as control. Biofilm formation of each strain was examined in eight replicates and eDNA was quantified in duplicates, on three independent occasions. Data represents mean ± SD. * p

    Journal: PLoS ONE

    Article Title: Extracellular DNA facilitates bacterial adhesion during Burkholderia pseudomallei biofilm formation

    doi: 10.1371/journal.pone.0213288

    Figure Lengend Snippet: Impact of DNase I treatment on B . pseudomallei biofilm formation. Static biofilms of B . pseudomallei strains L1, P1 and H777 in LB were treated with DNase I (0.01, 0.1 and 1 U/mL) at 0 h, 24 h or 45 h after inoculation and maintained for up to 48 h. Biofilm formation and eDNA concentration of the 2-day biofilms were assessed using crystal-violet absorbance (OD 620 ) and the QuantiFluor dsDNA System, respectively. DNase I buffer acted as control. Biofilm formation of each strain was examined in eight replicates and eDNA was quantified in duplicates, on three independent occasions. Data represents mean ± SD. * p

    Article Snippet: The 2-day biofilm culture was rinsed three times with sterile distilled water. eDNA in each well was mixed with 200 μL of freshly prepared QuantiFluor dsDNA dye in TE buffer for 5 min (QuantiFluor dsDNA System, Promega, Madison, WI, USA) before measuring the fluorescence intensity (excitation 504 nm/emission 531 nm) using a fluorometer (Varioskan Flash Multimode Reader, Singapore) with SkanIt Software 2.4.3 RE for Varioskan Flash.

    Techniques: Concentration Assay

    Variation in B . pseudomallei biofilm formation and eDNA quantity. (A) Degree of biofilm formation by 10 B . pseudomallei isolates grown in LB in 96-well plates at 37°C for 2 days was assessed using crystal-violet absorbance (OD 620 ). (B) eDNA concentration in 2-day biofilm of 10 B . pseudomallei isolates was assessed using the QuantiFluor dsDNA System. Controls were LB medium lacking bacteria. Data are represented as mean ± SD from at least three independent experiments.

    Journal: PLoS ONE

    Article Title: Extracellular DNA facilitates bacterial adhesion during Burkholderia pseudomallei biofilm formation

    doi: 10.1371/journal.pone.0213288

    Figure Lengend Snippet: Variation in B . pseudomallei biofilm formation and eDNA quantity. (A) Degree of biofilm formation by 10 B . pseudomallei isolates grown in LB in 96-well plates at 37°C for 2 days was assessed using crystal-violet absorbance (OD 620 ). (B) eDNA concentration in 2-day biofilm of 10 B . pseudomallei isolates was assessed using the QuantiFluor dsDNA System. Controls were LB medium lacking bacteria. Data are represented as mean ± SD from at least three independent experiments.

    Article Snippet: The 2-day biofilm culture was rinsed three times with sterile distilled water. eDNA in each well was mixed with 200 μL of freshly prepared QuantiFluor dsDNA dye in TE buffer for 5 min (QuantiFluor dsDNA System, Promega, Madison, WI, USA) before measuring the fluorescence intensity (excitation 504 nm/emission 531 nm) using a fluorometer (Varioskan Flash Multimode Reader, Singapore) with SkanIt Software 2.4.3 RE for Varioskan Flash.

    Techniques: Concentration Assay

    Genome Coverage plots. Representation of the MPS relative coverage of both strands (rc+: relative coverage of the plus strand, rc-: relative coverage of the negative strand) of the pUC19 plasmid, or mtDNA molecules obtained from the Ion Torrent PGM or MiSeq sequencing system. The outer circle symbolizes the pUC19 (A) or mtDNA (B, C, D) gene structure, respectively. 1A: Use of the Ion Torrent PGM standard protocol on the pUC19 plasmid. 1B: Use of three different fragmentation methods in combination with the Ion Torrent sequencing protocol on the mtDNA: Ion Shear Plus Reagents (enzymatic), NEBNext dsDNA Fragmentase (enzymatic) and Covaris (physical). 1C: Use of an Ion Torrent PGM protocol without PCR amplification in the library construction on the mtDNA. 1D: LR-PCR products of the mtDNA were Covaris (physical) or NEBNext dsDNA Fragmentase (enzymatic) sheared, followed by a TruSeq DNA PCR free protocol on a MiSeq instrument. The same six samples were processed with a Nextera XT kit (enzymatic shearing and PCR amplification in library preparation) prior to MiSeq analysis.

    Journal: PLoS ONE

    Article Title: A Bumpy Ride on the Diagnostic Bench of Massive Parallel Sequencing, the Case of the Mitochondrial Genome

    doi: 10.1371/journal.pone.0112950

    Figure Lengend Snippet: Genome Coverage plots. Representation of the MPS relative coverage of both strands (rc+: relative coverage of the plus strand, rc-: relative coverage of the negative strand) of the pUC19 plasmid, or mtDNA molecules obtained from the Ion Torrent PGM or MiSeq sequencing system. The outer circle symbolizes the pUC19 (A) or mtDNA (B, C, D) gene structure, respectively. 1A: Use of the Ion Torrent PGM standard protocol on the pUC19 plasmid. 1B: Use of three different fragmentation methods in combination with the Ion Torrent sequencing protocol on the mtDNA: Ion Shear Plus Reagents (enzymatic), NEBNext dsDNA Fragmentase (enzymatic) and Covaris (physical). 1C: Use of an Ion Torrent PGM protocol without PCR amplification in the library construction on the mtDNA. 1D: LR-PCR products of the mtDNA were Covaris (physical) or NEBNext dsDNA Fragmentase (enzymatic) sheared, followed by a TruSeq DNA PCR free protocol on a MiSeq instrument. The same six samples were processed with a Nextera XT kit (enzymatic shearing and PCR amplification in library preparation) prior to MiSeq analysis.

    Article Snippet: One µg of pUC19 plasmid DNA (Thermo Fisher, Erembodegem-Aalst, Belgium) was sheared by the Covaris or NEBNext dsDNA Fragmentase.

    Techniques: Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Amplification

    Correlation of autoAbs with anti-dsDNA and lupus nephritis The Pearson correlation coefficient of the 16 upregulated autoAbs with anti-dsDNA antibodies was analyzed by GraphPad Prism 6.0 with the levels of autoAbs measured using ELISA from the 35 SLE patients. Pearson's r represents the linear correlation between the levels of the selected autoAb and anti-dsDNA, where 1 means completely positive linear correlation, 0 means no linear correlation, and − 1 means completely negative linear correlation. Correlation with P

    Journal: Genomics, Proteomics & Bioinformatics

    Article Title: Novel Autoantibodies Related to Cell Death and DNA Repair Pathways in Systemic Lupus Erythematosus

    doi: 10.1016/j.gpb.2018.11.004

    Figure Lengend Snippet: Correlation of autoAbs with anti-dsDNA and lupus nephritis The Pearson correlation coefficient of the 16 upregulated autoAbs with anti-dsDNA antibodies was analyzed by GraphPad Prism 6.0 with the levels of autoAbs measured using ELISA from the 35 SLE patients. Pearson's r represents the linear correlation between the levels of the selected autoAb and anti-dsDNA, where 1 means completely positive linear correlation, 0 means no linear correlation, and − 1 means completely negative linear correlation. Correlation with P

    Article Snippet: The titer of anti-dsDNA autoAb in each serum was also measured by ELISA using the Immulon II plates coated with dsDNA (50 μg/ml) (Dynatech Laboratories, Chantilly, VA) as previously described .

    Techniques: Enzyme-linked Immunosorbent Assay

    NO 2 -FAs suppress STING signaling and release of type I IFN. ( A and B ) THP-1 cells and ( C and D ) BMMs (WT mice) were treated with indicated NO 2 -FAs (5–10 µM) or OA/LA (10 µM) 15 min before stimulation with dsDNA (4 µg/mL) or infection with HSV-2 (MOI 1) or left untreated (Ut). After 20 h, supernatants were harvested and analyzed for type I IFN. Data represent one of two independent experiments and are presented as mean ± SEM. ( E – G ) THP-1 cells were treated with NO 2 -FAs (10 µM) or OA/LA (10 µM) 15 min before stimulation with cGAMP (4 µg/mL) or dsDNA (4 µg/mL) using Lipofectamine2000 (Lipo). After 3 h, lysates were separated by SDS/PAGE, and indicated proteins were detected by Western blotting using specific antibodies. STING and IRF3 dimers were detected using nondenaturing and nonreducing conditions. Vinculin was used as loading control.

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

    Article Title: Nitro-fatty acids are formed in response to virus infection and are potent inhibitors of STING palmitoylation and signaling

    doi: 10.1073/pnas.1806239115

    Figure Lengend Snippet: NO 2 -FAs suppress STING signaling and release of type I IFN. ( A and B ) THP-1 cells and ( C and D ) BMMs (WT mice) were treated with indicated NO 2 -FAs (5–10 µM) or OA/LA (10 µM) 15 min before stimulation with dsDNA (4 µg/mL) or infection with HSV-2 (MOI 1) or left untreated (Ut). After 20 h, supernatants were harvested and analyzed for type I IFN. Data represent one of two independent experiments and are presented as mean ± SEM. ( E – G ) THP-1 cells were treated with NO 2 -FAs (10 µM) or OA/LA (10 µM) 15 min before stimulation with cGAMP (4 µg/mL) or dsDNA (4 µg/mL) using Lipofectamine2000 (Lipo). After 3 h, lysates were separated by SDS/PAGE, and indicated proteins were detected by Western blotting using specific antibodies. STING and IRF3 dimers were detected using nondenaturing and nonreducing conditions. Vinculin was used as loading control.

    Article Snippet: For transfection setups, 4 µg/mL dsDNA (HSV-60; InvivoGen) and 4 µL/mL Lipofectamine2000 (Invitrogen) were used according to the manufacturer’s instructions.

    Techniques: Mouse Assay, Infection, SDS Page, Western Blot

    RCA assay of pUC19 DNA plasmid. ( A ) Agarose gel of pUC19 RCA products. Lanes 1–7 RCA performed with increasing concentrations of T4 gene 32 protein (0,10, 20, 30, 50, 75, 100 ng/μl respectively); lane 8 negative control with no phi29 DNA polymerase in the reaction mixture; 1 kb plus DNA ladders (L). ( B ) Agarose gel of MlyI digestion test. RCA products in (A) were digested by MlyI restriction enzyme and the corresponding digestion products (9-16) were run on agarose gel. 1 kb plus DNA ladders (L). ( C ) Picogreen assay of pUC19 RCA. The amplification is expressed in percentage of relative fluorescence units (RFU) and the signal of the amplification product without T4 gene 32 is taken as 100%. Both MlyI digestion and picogreen assay confirm that rolling circle amplification makes mostly double-stranded DNA but they also suggest that T4 gene 32 SSB protein drastically reduces dsDNA production.

    Journal: Nucleic Acids Research

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA

    doi: 10.1093/nar/gku737

    Figure Lengend Snippet: RCA assay of pUC19 DNA plasmid. ( A ) Agarose gel of pUC19 RCA products. Lanes 1–7 RCA performed with increasing concentrations of T4 gene 32 protein (0,10, 20, 30, 50, 75, 100 ng/μl respectively); lane 8 negative control with no phi29 DNA polymerase in the reaction mixture; 1 kb plus DNA ladders (L). ( B ) Agarose gel of MlyI digestion test. RCA products in (A) were digested by MlyI restriction enzyme and the corresponding digestion products (9-16) were run on agarose gel. 1 kb plus DNA ladders (L). ( C ) Picogreen assay of pUC19 RCA. The amplification is expressed in percentage of relative fluorescence units (RFU) and the signal of the amplification product without T4 gene 32 is taken as 100%. Both MlyI digestion and picogreen assay confirm that rolling circle amplification makes mostly double-stranded DNA but they also suggest that T4 gene 32 SSB protein drastically reduces dsDNA production.

    Article Snippet: We mixed in a microtiter plate (Nucleon Delta Surface, Thermoscientific) 8 μl of each reaction with 50 μl of a 200× diluted dsDNA specific dye (picogreen, Invitrogen) in 1× TE buffer.

    Techniques: Plasmid Preparation, Agarose Gel Electrophoresis, Negative Control, Picogreen Assay, Amplification, Fluorescence