exonuclease iii  (Thermo Fisher)


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  • 94

    Structured Review

    Thermo Fisher exonuclease iii
    Optimising the protocol for <t>BrdU</t> detection. (A) HeLa cells were incubated with BrdU for 30 min, fixed with formaldehyde and BrdU was detected in DNA using 40 mM HCl. BrdU was detected either by the B44 or Bu20a anti-BrdU antibody with exonuclease <t>III.</t> The effect of formaldehyde post-fixation on the signal is shown as well. The data are presented as the mean ± SD. ( B-E) A comparison of various HCl concentrations on BrdU signal in HeLa cells labelled for 30 min with BrdU and fixed either with formaldehyde (B, D) or ethanol (C, E). The incorporated BrdU was detected using either B44 (B, C) or Bu20a (D, E) antibody clone with exonuclease III. The data are presented as the mean ± SD. ( F, G) A comparison of five monoclonal anti-BrdU antibody clones and one polyclonal antibody is shown. The HeLa cells were labelled with BrdU for 30 min and fixed either with formaldehyde (F) or ethanol (G). The impact of the post-fixation step is shown as well. The data are presented as the mean ± SD. ( H) The effect of the length of the washing step on the BrdU-derived signal. The HeLa cells were labelled with BrdU for 30 min and fixed with formaldehyde. After incubation with the primary antibody, the cells were washed for 5 s (0 min) or 5 or 25 min in 1× PBS and then post-fixed with formaldehyde. The data are normalised to the % of the average signal in samples washed for 5 s in 1× PBS and then immediately post-fixed with formaldehyde. The data are presented as the mean ± SD.
    Exonuclease Iii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Cell cycle profiling by image and flow cytometry: The optimised protocol for the detection of replicational activity using 5-Bromo-2′-deoxyuridine, low concentration of hydrochloric acid and exonuclease III"

    Article Title: Cell cycle profiling by image and flow cytometry: The optimised protocol for the detection of replicational activity using 5-Bromo-2′-deoxyuridine, low concentration of hydrochloric acid and exonuclease III

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0175880

    Optimising the protocol for BrdU detection. (A) HeLa cells were incubated with BrdU for 30 min, fixed with formaldehyde and BrdU was detected in DNA using 40 mM HCl. BrdU was detected either by the B44 or Bu20a anti-BrdU antibody with exonuclease III. The effect of formaldehyde post-fixation on the signal is shown as well. The data are presented as the mean ± SD. ( B-E) A comparison of various HCl concentrations on BrdU signal in HeLa cells labelled for 30 min with BrdU and fixed either with formaldehyde (B, D) or ethanol (C, E). The incorporated BrdU was detected using either B44 (B, C) or Bu20a (D, E) antibody clone with exonuclease III. The data are presented as the mean ± SD. ( F, G) A comparison of five monoclonal anti-BrdU antibody clones and one polyclonal antibody is shown. The HeLa cells were labelled with BrdU for 30 min and fixed either with formaldehyde (F) or ethanol (G). The impact of the post-fixation step is shown as well. The data are presented as the mean ± SD. ( H) The effect of the length of the washing step on the BrdU-derived signal. The HeLa cells were labelled with BrdU for 30 min and fixed with formaldehyde. After incubation with the primary antibody, the cells were washed for 5 s (0 min) or 5 or 25 min in 1× PBS and then post-fixed with formaldehyde. The data are normalised to the % of the average signal in samples washed for 5 s in 1× PBS and then immediately post-fixed with formaldehyde. The data are presented as the mean ± SD.
    Figure Legend Snippet: Optimising the protocol for BrdU detection. (A) HeLa cells were incubated with BrdU for 30 min, fixed with formaldehyde and BrdU was detected in DNA using 40 mM HCl. BrdU was detected either by the B44 or Bu20a anti-BrdU antibody with exonuclease III. The effect of formaldehyde post-fixation on the signal is shown as well. The data are presented as the mean ± SD. ( B-E) A comparison of various HCl concentrations on BrdU signal in HeLa cells labelled for 30 min with BrdU and fixed either with formaldehyde (B, D) or ethanol (C, E). The incorporated BrdU was detected using either B44 (B, C) or Bu20a (D, E) antibody clone with exonuclease III. The data are presented as the mean ± SD. ( F, G) A comparison of five monoclonal anti-BrdU antibody clones and one polyclonal antibody is shown. The HeLa cells were labelled with BrdU for 30 min and fixed either with formaldehyde (F) or ethanol (G). The impact of the post-fixation step is shown as well. The data are presented as the mean ± SD. ( H) The effect of the length of the washing step on the BrdU-derived signal. The HeLa cells were labelled with BrdU for 30 min and fixed with formaldehyde. After incubation with the primary antibody, the cells were washed for 5 s (0 min) or 5 or 25 min in 1× PBS and then post-fixed with formaldehyde. The data are normalised to the % of the average signal in samples washed for 5 s in 1× PBS and then immediately post-fixed with formaldehyde. The data are presented as the mean ± SD.

    Techniques Used: Incubation, Clone Assay, Derivative Assay

    The effect of optimized procedure on the localisation of cellular proteins. HeLa cells were incubated with BrdU for 30 minutes and fixed with formaldehyde. BrdU was revealed using 20 mM HCl. The proteins SC35, mitochondrial protein MTCO2, histone H1.2 and coilin were concurrently detected with the incorporated BrdU. BrdU was detected using either chicken polyclonal antibody or B44 monoclonal antibody depending on the host producing the antibody for the particular cellular protein. The control cells were not labelled with BrdU and were not treated with HCl and exonuclease III. Proteins are in green, BrdU is in red and DAPI is in blue. Scale bar = 20 μm.
    Figure Legend Snippet: The effect of optimized procedure on the localisation of cellular proteins. HeLa cells were incubated with BrdU for 30 minutes and fixed with formaldehyde. BrdU was revealed using 20 mM HCl. The proteins SC35, mitochondrial protein MTCO2, histone H1.2 and coilin were concurrently detected with the incorporated BrdU. BrdU was detected using either chicken polyclonal antibody or B44 monoclonal antibody depending on the host producing the antibody for the particular cellular protein. The control cells were not labelled with BrdU and were not treated with HCl and exonuclease III. Proteins are in green, BrdU is in red and DAPI is in blue. Scale bar = 20 μm.

    Techniques Used: Incubation

    Related Articles

    Marker:

    Article Title: Structural basis for guide RNA processing and seed-dependent DNA targeting and cleavage by CRISPR-Cas12a
    Article Snippet: 1 μl of Exonuclease III (100 units/μl; Thermo Scientific) was added and the sample was incubated for 1h at 37 °C. .. For the marker, λ-TS and λ-NTS were incubated with S1 nuclease (1:100 dilution; Thermo Scientific) for 1 min at 37 °C.

    Incubation:

    Article Title: Structural basis for guide RNA processing and seed-dependent DNA targeting and cleavage by CRISPR-Cas12a
    Article Snippet: .. 1 μl of Exonuclease III (100 units/μl; Thermo Scientific) was added and the sample was incubated for 1h at 37 °C. .. For the marker, λ-TS and λ-NTS were incubated with S1 nuclease (1:100 dilution; Thermo Scientific) for 1 min at 37 °C.

    Article Title: Atomic Scissors: A New Method of Tracking the 5-Bromo-2?-Deoxyuridine-Labeled DNA In Situ
    Article Snippet: .. In this case, the cells were incubated for ten minutes in the above-described cleavage solution on the shaker followed by washing in 20 mM EDTA (30 minutes, RT) on the shaker and then by a 30-minute incubation with a mixture of antibody and exonuclease III (1 U/µl, Fermentas) and 1× buffer for exonuclease III at RT. .. For the detection of mitochondrial replication, only 60 seconds of incubation in the cleavage solution was used.

    Article Title: Most Anti-BrdU Antibodies React with 2?-Deoxy-5-Ethynyluridine -- The Method for the Effective Suppression of This Cross-Reactivity
    Article Snippet: BrdU Revelation The cells were incubated either with 4N HCl for 20 minutes at room temperature or in a freshly-prepared solution of 10 mM sodium ascorbate, and 4 mM copper(II) sulfate for 10 minutes and then in 20 mM EDTA for 30 minutes at room temperature. .. In the latter case, the primary antibodies were diluted in 1× buffer for exonuclease III (Fermentas) and supplemented by exonuclease III (0.1 U/µl; Fermentas).

    Article Title: Cell cycle profiling by image and flow cytometry: The optimised protocol for the detection of replicational activity using 5-Bromo-2′-deoxyuridine, low concentration of hydrochloric acid and exonuclease III
    Article Snippet: .. Then, BrdU was detected using the primary antibody supplemented with exonuclease III (1U/μl, Fermentas) in 1x buffer for exonuclease III (66 mM Tris-HCl, pH 8.0, 0.66 mM MgCl2 ) at 24°C for 30 min. Further cells were washed with 25 mM Tris-HCl, ~pH 7.5 and 150 mM NaCl and incubated with the secondary antibody and DAPI [ ]. .. Antibodies These primary antibodies were used: mouse anti-BrdU antibody clones: B44 and 3D4 (BD Biosciences), Bu20a and MoBu-1 (Exbio Praha), BMC9318 (Roche) and chicken polyclonal anti-BrdU antibody (Abcam).

    Activity Assay:

    Article Title: Most Anti-BrdU Antibodies React with 2?-Deoxy-5-Ethynyluridine -- The Method for the Effective Suppression of This Cross-Reactivity
    Article Snippet: In the latter case, the primary antibodies were diluted in 1× buffer for exonuclease III (Fermentas) and supplemented by exonuclease III (0.1 U/µl; Fermentas). .. The revelation of BrdU by means of copper(I) ions is based on the introduction of frequent gaps into DNA by oxidative attack at the deoxyribose moiety and subsequent prolongation of the gaps by exonuclease activity.

    Generated:

    Article Title: Structural basis for guide RNA processing and seed-dependent DNA targeting and cleavage by CRISPR-Cas12a
    Article Snippet: 2 μl double stranded λ-DNA (0.5 μM; generated by mixing 10 μM λ-TS and λ-NTS ( ) in a 1:1 ratio, incubating for 5 min at 95 °C, slowly cooling down to room temperature and diluting to 0.5 μM in H2 O) was added and the sample was incubated for 30 min at 37 °C to allow ternary complex formation (the molar ratio FnCas12a:crRNA:dsDNA was 50:50:1). .. 1 μl of Exonuclease III (100 units/μl; Thermo Scientific) was added and the sample was incubated for 1h at 37 °C.

    Size-exclusion Chromatography:

    Article Title: Structural basis for guide RNA processing and seed-dependent DNA targeting and cleavage by CRISPR-Cas12a
    Article Snippet: 10 μl FnCas12aDM (5μM in 0.5x SEC buffer) was mixed with 5 μl crRNA-λ (10 μM, ) and 2 μl MgCl2 (50 mM). .. 1 μl of Exonuclease III (100 units/μl; Thermo Scientific) was added and the sample was incubated for 1h at 37 °C.

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    Thermo Fisher exonuclease iii
    Most extracellular DNA is packaged into <t>L-EVs</t> . (a) Tunable resistive pulse sensing (TRPS, qNano) using two different pore membranes (NP4000 and NP200) identified as L-EVs (left) and S-EVs (right) derived from PC3 cells. NP4000 membrane, which can detect particles with a diameter between 1.0 and 6.0 μm, was used for quantitation of L-EVs, while NP200 membrane, which can detect particles with a diameter between 60 and 400 nm, was used for quantitation of S-EVs. (b) Protein lysates from L-EVs and S-EVs purified by iodixanol density gradient (at 1.10 and 1.15 g/ml) were blotted with LO markers HSPA5 and CK18, and with Exo marker CD81. (c) Total DNA was quantified by Qubit Fluorometer in L-EVs and S-EVs isolated from PC3 and U87 cell lines. The plot shows the DNA ratio between L-EVs and S-EVs. (d) Double stranded (ds)DNA was quantified by High Sensitivity (HS) dsDNA Qubit Assay in L-EVs and S-EVs isolated from 1 ml of plasma from patients with mCRPC ( n = 40) and cancer-free individuals ( n = 6). (e) Quantification of both protein and DNA content in L-EVs and S-EVs isolated from conditioned media of 12.6 × 10 7 PC3 cells. (f) Single stranded (ss) and dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Exonuclease <t>III,</t> were quantified by Qubit. (g) Chip-based capillary electrophoresis (Bioanalyzer) showing the presence of dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Endonuclease III. L-EVs contain abundant DNA with a large peak around 10 kbp. Conversely, the amount of DNA in S-EVs is negligible. (h) ss- and dsDNA in PC3-derived L-EVs and S-EVs were quantified by Qubit after treatment with nucleases (DNase I and Exonuclease III) with or without addition of a detergent (Triton X-100) prior to nuclease treatment. (i) Chip-based capillary electrophoresis (Bioanalyzer) showing that only miniscule amounts of dsDNA could be detected after EV lysis using a detergent prior to treatment with nucleases.
    Exonuclease Iii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    Thermo Fisher exo iii
    Optimization of experimental conditions: (a) effect of the incubation time of <t>Exo</t> <t>III</t> digestion; (b) effect of Exo III concentration; (c) effect of signal DNA concentration; (d) effect of the incubation temperature; (e) effect of the signal DNA/capture DNA ratio; (f) effect of the pH value. Experimental conditions: 10 nmol/L oligo-1, 40 nmol/L oligo-2, 10 U Exo III, 50 mmol/L K + , and 160 nmol/L NMM. Error bars represent the standard deviation of three independent experiments.
    Exo Iii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 83/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gene exp trex2 mm04210320 m1
    DNA-driven gene expression signature is not induced in the tongue of the <t>Trex2</t> −/− Dnase1l2 −/− mice. Expression of the indicated genes in the tongue from wt, Trex2 −/− , Dnase1l2 −/− and Trex2 −/− Dnase1l2 −/− mice, as determined by RT-qPCR. Each dot indicates a sample from an individual mouse, and horizontal lines represent the mean value. The results from one of two independent experiments with similar results are shown. Significant differences, by the Mann-Whitney test between genotypes: *P
    Gene Exp Trex2 Mm04210320 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 82/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Most extracellular DNA is packaged into L-EVs . (a) Tunable resistive pulse sensing (TRPS, qNano) using two different pore membranes (NP4000 and NP200) identified as L-EVs (left) and S-EVs (right) derived from PC3 cells. NP4000 membrane, which can detect particles with a diameter between 1.0 and 6.0 μm, was used for quantitation of L-EVs, while NP200 membrane, which can detect particles with a diameter between 60 and 400 nm, was used for quantitation of S-EVs. (b) Protein lysates from L-EVs and S-EVs purified by iodixanol density gradient (at 1.10 and 1.15 g/ml) were blotted with LO markers HSPA5 and CK18, and with Exo marker CD81. (c) Total DNA was quantified by Qubit Fluorometer in L-EVs and S-EVs isolated from PC3 and U87 cell lines. The plot shows the DNA ratio between L-EVs and S-EVs. (d) Double stranded (ds)DNA was quantified by High Sensitivity (HS) dsDNA Qubit Assay in L-EVs and S-EVs isolated from 1 ml of plasma from patients with mCRPC ( n = 40) and cancer-free individuals ( n = 6). (e) Quantification of both protein and DNA content in L-EVs and S-EVs isolated from conditioned media of 12.6 × 10 7 PC3 cells. (f) Single stranded (ss) and dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Exonuclease III, were quantified by Qubit. (g) Chip-based capillary electrophoresis (Bioanalyzer) showing the presence of dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Endonuclease III. L-EVs contain abundant DNA with a large peak around 10 kbp. Conversely, the amount of DNA in S-EVs is negligible. (h) ss- and dsDNA in PC3-derived L-EVs and S-EVs were quantified by Qubit after treatment with nucleases (DNase I and Exonuclease III) with or without addition of a detergent (Triton X-100) prior to nuclease treatment. (i) Chip-based capillary electrophoresis (Bioanalyzer) showing that only miniscule amounts of dsDNA could be detected after EV lysis using a detergent prior to treatment with nucleases.

    Journal: Journal of Extracellular Vesicles

    Article Title: Large extracellular vesicles carry most of the tumour DNA circulating in prostate cancer patient plasma

    doi: 10.1080/20013078.2018.1505403

    Figure Lengend Snippet: Most extracellular DNA is packaged into L-EVs . (a) Tunable resistive pulse sensing (TRPS, qNano) using two different pore membranes (NP4000 and NP200) identified as L-EVs (left) and S-EVs (right) derived from PC3 cells. NP4000 membrane, which can detect particles with a diameter between 1.0 and 6.0 μm, was used for quantitation of L-EVs, while NP200 membrane, which can detect particles with a diameter between 60 and 400 nm, was used for quantitation of S-EVs. (b) Protein lysates from L-EVs and S-EVs purified by iodixanol density gradient (at 1.10 and 1.15 g/ml) were blotted with LO markers HSPA5 and CK18, and with Exo marker CD81. (c) Total DNA was quantified by Qubit Fluorometer in L-EVs and S-EVs isolated from PC3 and U87 cell lines. The plot shows the DNA ratio between L-EVs and S-EVs. (d) Double stranded (ds)DNA was quantified by High Sensitivity (HS) dsDNA Qubit Assay in L-EVs and S-EVs isolated from 1 ml of plasma from patients with mCRPC ( n = 40) and cancer-free individuals ( n = 6). (e) Quantification of both protein and DNA content in L-EVs and S-EVs isolated from conditioned media of 12.6 × 10 7 PC3 cells. (f) Single stranded (ss) and dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Exonuclease III, were quantified by Qubit. (g) Chip-based capillary electrophoresis (Bioanalyzer) showing the presence of dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Endonuclease III. L-EVs contain abundant DNA with a large peak around 10 kbp. Conversely, the amount of DNA in S-EVs is negligible. (h) ss- and dsDNA in PC3-derived L-EVs and S-EVs were quantified by Qubit after treatment with nucleases (DNase I and Exonuclease III) with or without addition of a detergent (Triton X-100) prior to nuclease treatment. (i) Chip-based capillary electrophoresis (Bioanalyzer) showing that only miniscule amounts of dsDNA could be detected after EV lysis using a detergent prior to treatment with nucleases.

    Article Snippet: DNA extraction and characterization L- and S-EVs were treated with rDNase I (2 U/μl, DNA-Free Kit, Ambion) and Exonuclease III (200 U/μl, Thermofisher).

    Techniques: Tunable Resistive Pulse Sensing, Derivative Assay, Quantitation Assay, Purification, Marker, Isolation, HS DSDNA Qubit Assay, Chromatin Immunoprecipitation, Electrophoresis, Lysis

    Optimization of experimental conditions: (a) effect of the incubation time of Exo III digestion; (b) effect of Exo III concentration; (c) effect of signal DNA concentration; (d) effect of the incubation temperature; (e) effect of the signal DNA/capture DNA ratio; (f) effect of the pH value. Experimental conditions: 10 nmol/L oligo-1, 40 nmol/L oligo-2, 10 U Exo III, 50 mmol/L K + , and 160 nmol/L NMM. Error bars represent the standard deviation of three independent experiments.

    Journal: Journal of Analytical Methods in Chemistry

    Article Title: A Sensitive Fluorescence Biosensor for Silver Ions (Ag+) Detection Based on C-Ag+-C Structure and Exonuclease III-Assisted Dual-Recycling Amplification

    doi: 10.1155/2019/3712032

    Figure Lengend Snippet: Optimization of experimental conditions: (a) effect of the incubation time of Exo III digestion; (b) effect of Exo III concentration; (c) effect of signal DNA concentration; (d) effect of the incubation temperature; (e) effect of the signal DNA/capture DNA ratio; (f) effect of the pH value. Experimental conditions: 10 nmol/L oligo-1, 40 nmol/L oligo-2, 10 U Exo III, 50 mmol/L K + , and 160 nmol/L NMM. Error bars represent the standard deviation of three independent experiments.

    Article Snippet: Exo III was purchased from the Thermo Fisher Scientific Inc. (USA).

    Techniques: Incubation, Concentration Assay, Standard Deviation

    Fluorescence spectra (a) and calibration plot (b) for Ag + . Experimental conditions: 10 nmol/L oligo-1, 40 nmol/L oligo-2, 10 U Exo III, 50 mmol/L K + , 160 nmol/L NMM, and 50 min incubation time. Error bars represent the standard deviation of three independent experiments.

    Journal: Journal of Analytical Methods in Chemistry

    Article Title: A Sensitive Fluorescence Biosensor for Silver Ions (Ag+) Detection Based on C-Ag+-C Structure and Exonuclease III-Assisted Dual-Recycling Amplification

    doi: 10.1155/2019/3712032

    Figure Lengend Snippet: Fluorescence spectra (a) and calibration plot (b) for Ag + . Experimental conditions: 10 nmol/L oligo-1, 40 nmol/L oligo-2, 10 U Exo III, 50 mmol/L K + , 160 nmol/L NMM, and 50 min incubation time. Error bars represent the standard deviation of three independent experiments.

    Article Snippet: Exo III was purchased from the Thermo Fisher Scientific Inc. (USA).

    Techniques: Fluorescence, Incubation, Standard Deviation

    Selectivity of the biosensor. Experimental conditions: 10 nmol/L oligo-1, 40 nmol/L oligo-2, 10 U Exo III, 50 mmol/L K + , 160 nmol/L NMM, and 50 min incubation time. The concentration of Ag + was 1.5 nmol/L, and the concentrations of other metal ions were 15 nmol/L. Error bars represent the standard deviation of three independent experiments.

    Journal: Journal of Analytical Methods in Chemistry

    Article Title: A Sensitive Fluorescence Biosensor for Silver Ions (Ag+) Detection Based on C-Ag+-C Structure and Exonuclease III-Assisted Dual-Recycling Amplification

    doi: 10.1155/2019/3712032

    Figure Lengend Snippet: Selectivity of the biosensor. Experimental conditions: 10 nmol/L oligo-1, 40 nmol/L oligo-2, 10 U Exo III, 50 mmol/L K + , 160 nmol/L NMM, and 50 min incubation time. The concentration of Ag + was 1.5 nmol/L, and the concentrations of other metal ions were 15 nmol/L. Error bars represent the standard deviation of three independent experiments.

    Article Snippet: Exo III was purchased from the Thermo Fisher Scientific Inc. (USA).

    Techniques: Incubation, Concentration Assay, Standard Deviation

    Fluorescence emission spectra of different solutions: (a) 10 nmol/L oligo-1 + 1500 pmol/L Ag + ; (b) 40 nmol/L oligo-2 + 1500 pmol/L Ag + ; (c) 10 nmol/L oligo-1 + 40 nmol/L oligo-2; (d) 10 nmol/L oligo-1 + 40 nmol/L oligo-2 + 10 U Exo III; (e) 10 nmol/L oligo-1 + 40 nmol/L oligo-2 + 10 U Exo III + 1500 pmol/L Ag + . Experimental conditions: 50 mmol/L K + and 160 nmol/L NMM.

    Journal: Journal of Analytical Methods in Chemistry

    Article Title: A Sensitive Fluorescence Biosensor for Silver Ions (Ag+) Detection Based on C-Ag+-C Structure and Exonuclease III-Assisted Dual-Recycling Amplification

    doi: 10.1155/2019/3712032

    Figure Lengend Snippet: Fluorescence emission spectra of different solutions: (a) 10 nmol/L oligo-1 + 1500 pmol/L Ag + ; (b) 40 nmol/L oligo-2 + 1500 pmol/L Ag + ; (c) 10 nmol/L oligo-1 + 40 nmol/L oligo-2; (d) 10 nmol/L oligo-1 + 40 nmol/L oligo-2 + 10 U Exo III; (e) 10 nmol/L oligo-1 + 40 nmol/L oligo-2 + 10 U Exo III + 1500 pmol/L Ag + . Experimental conditions: 50 mmol/L K + and 160 nmol/L NMM.

    Article Snippet: Exo III was purchased from the Thermo Fisher Scientific Inc. (USA).

    Techniques: Fluorescence

    DNA-driven gene expression signature is not induced in the tongue of the Trex2 −/− Dnase1l2 −/− mice. Expression of the indicated genes in the tongue from wt, Trex2 −/− , Dnase1l2 −/− and Trex2 −/− Dnase1l2 −/− mice, as determined by RT-qPCR. Each dot indicates a sample from an individual mouse, and horizontal lines represent the mean value. The results from one of two independent experiments with similar results are shown. Significant differences, by the Mann-Whitney test between genotypes: *P

    Journal: Scientific Reports

    Article Title: Double deficiency of Trex2 and DNase1L2 nucleases leads to accumulation of DNA in lingual cornifying keratinocytes without activating inflammatory responses

    doi: 10.1038/s41598-017-12308-4

    Figure Lengend Snippet: DNA-driven gene expression signature is not induced in the tongue of the Trex2 −/− Dnase1l2 −/− mice. Expression of the indicated genes in the tongue from wt, Trex2 −/− , Dnase1l2 −/− and Trex2 −/− Dnase1l2 −/− mice, as determined by RT-qPCR. Each dot indicates a sample from an individual mouse, and horizontal lines represent the mean value. The results from one of two independent experiments with similar results are shown. Significant differences, by the Mann-Whitney test between genotypes: *P

    Article Snippet: TaqMan Gene Expression Master Mix and predesigned probes (Applied Biosystems) were used to quantify mRNA expression of the following genes: Trex2 (Mm04210320_m1), Dnase1l2 (Mm00481868_g1), Ifnβ (Mm00439552_s1), Tnf α (Mm00443258_m1), Cxcl10 (Mm00445235_m1), Mx1 (Mm00487796_m1), Aim2 (Mm01295719_m1), IL6 (Mm00446190_m1), IL1β (Mm00434228_m1), and Sdha (Mm01352366_m1).

    Techniques: Expressing, Mouse Assay, Quantitative RT-PCR, MANN-WHITNEY

    Double Trex2 and Dnase1l2 deficiency does not trigger parakeratosis in the skin. H E (lanes 1 and 2) staining and Hoechst DNA labelling (lanes 3 and 4) in back skin and tongue sections from wt, Trex2 −/− , Dnase1l2 −/− and Trex2 −/− Dnase1l2 −/− mice. Back skin samples were from 6 days-old mice which are consistently in the anagen phase of the hair cycle. Likewise, adult back skin was free from parakeratosis in all samples investigated. Tongue samples were from adult 7–9 weeks-old mice. The images are representative for at least five mice from each genotype. Scale bars = 25 μm. Dashed lines, epidermal-dermal border; continuous lines, bottom of the stratum corneum; dotted lines, upper border of the stratum corneum.

    Journal: Scientific Reports

    Article Title: Double deficiency of Trex2 and DNase1L2 nucleases leads to accumulation of DNA in lingual cornifying keratinocytes without activating inflammatory responses

    doi: 10.1038/s41598-017-12308-4

    Figure Lengend Snippet: Double Trex2 and Dnase1l2 deficiency does not trigger parakeratosis in the skin. H E (lanes 1 and 2) staining and Hoechst DNA labelling (lanes 3 and 4) in back skin and tongue sections from wt, Trex2 −/− , Dnase1l2 −/− and Trex2 −/− Dnase1l2 −/− mice. Back skin samples were from 6 days-old mice which are consistently in the anagen phase of the hair cycle. Likewise, adult back skin was free from parakeratosis in all samples investigated. Tongue samples were from adult 7–9 weeks-old mice. The images are representative for at least five mice from each genotype. Scale bars = 25 μm. Dashed lines, epidermal-dermal border; continuous lines, bottom of the stratum corneum; dotted lines, upper border of the stratum corneum.

    Article Snippet: TaqMan Gene Expression Master Mix and predesigned probes (Applied Biosystems) were used to quantify mRNA expression of the following genes: Trex2 (Mm04210320_m1), Dnase1l2 (Mm00481868_g1), Ifnβ (Mm00439552_s1), Tnf α (Mm00443258_m1), Cxcl10 (Mm00445235_m1), Mx1 (Mm00487796_m1), Aim2 (Mm01295719_m1), IL6 (Mm00446190_m1), IL1β (Mm00434228_m1), and Sdha (Mm01352366_m1).

    Techniques: Staining, Mouse Assay

    Double Trex2 and Dnase1l2 deficiency leads to an increase of fragmented DNA in the upper keratinocyte layers of the tongue, but not of the epidermis. TUNEL labelling of free 3′-OH DNA ends (red) and nuclei (blue) counterstained with Hoechst of back and ear skin, and the filiform papillae and back side tongue sections from wt, Trex2 −/− , Dnase1l2 −/− and Trex2 −/− Dnase1l2 −/− mice. Back and ear skin samples were from 6 days-old mice. Tongue samples were from 7–9 weeks-old mice. The images are representative for at least five mice from each genotype. Scale bars = 25 μm. Dashed lines, epidermal-dermal border; continuous lines, bottom of the stratum corneum; dotted lines, upper border of the stratum corneum.

    Journal: Scientific Reports

    Article Title: Double deficiency of Trex2 and DNase1L2 nucleases leads to accumulation of DNA in lingual cornifying keratinocytes without activating inflammatory responses

    doi: 10.1038/s41598-017-12308-4

    Figure Lengend Snippet: Double Trex2 and Dnase1l2 deficiency leads to an increase of fragmented DNA in the upper keratinocyte layers of the tongue, but not of the epidermis. TUNEL labelling of free 3′-OH DNA ends (red) and nuclei (blue) counterstained with Hoechst of back and ear skin, and the filiform papillae and back side tongue sections from wt, Trex2 −/− , Dnase1l2 −/− and Trex2 −/− Dnase1l2 −/− mice. Back and ear skin samples were from 6 days-old mice. Tongue samples were from 7–9 weeks-old mice. The images are representative for at least five mice from each genotype. Scale bars = 25 μm. Dashed lines, epidermal-dermal border; continuous lines, bottom of the stratum corneum; dotted lines, upper border of the stratum corneum.

    Article Snippet: TaqMan Gene Expression Master Mix and predesigned probes (Applied Biosystems) were used to quantify mRNA expression of the following genes: Trex2 (Mm04210320_m1), Dnase1l2 (Mm00481868_g1), Ifnβ (Mm00439552_s1), Tnf α (Mm00443258_m1), Cxcl10 (Mm00445235_m1), Mx1 (Mm00487796_m1), Aim2 (Mm01295719_m1), IL6 (Mm00446190_m1), IL1β (Mm00434228_m1), and Sdha (Mm01352366_m1).

    Techniques: TUNEL Assay, Mouse Assay

    The nucleases Trex2 and DNase1L2 are mostly expressed in the keratinocyte upper layers of the skin and tongue epithelia. ( a ) Immunofluorescence images showing DNase1L2 (red) and DNA (blue) staining in the back skin and tongue sections from wild-type (wt) and Dnase1l2 knockout ( Dnase1l2 −/− ) mice. ( b ) Immunofluorescence images illustrating Trex2 (red) and DNA (blue) staining in back skin and tongue sections from wt and Trex2 knockout ( Trex2 −/− ) mice. Scale bars = 25 μm. Dashed lines, epidermal-dermal border; continuous lines, bottom of the stratum corneum; dotted lines, upper border of the stratum corneum.

    Journal: Scientific Reports

    Article Title: Double deficiency of Trex2 and DNase1L2 nucleases leads to accumulation of DNA in lingual cornifying keratinocytes without activating inflammatory responses

    doi: 10.1038/s41598-017-12308-4

    Figure Lengend Snippet: The nucleases Trex2 and DNase1L2 are mostly expressed in the keratinocyte upper layers of the skin and tongue epithelia. ( a ) Immunofluorescence images showing DNase1L2 (red) and DNA (blue) staining in the back skin and tongue sections from wild-type (wt) and Dnase1l2 knockout ( Dnase1l2 −/− ) mice. ( b ) Immunofluorescence images illustrating Trex2 (red) and DNA (blue) staining in back skin and tongue sections from wt and Trex2 knockout ( Trex2 −/− ) mice. Scale bars = 25 μm. Dashed lines, epidermal-dermal border; continuous lines, bottom of the stratum corneum; dotted lines, upper border of the stratum corneum.

    Article Snippet: TaqMan Gene Expression Master Mix and predesigned probes (Applied Biosystems) were used to quantify mRNA expression of the following genes: Trex2 (Mm04210320_m1), Dnase1l2 (Mm00481868_g1), Ifnβ (Mm00439552_s1), Tnf α (Mm00443258_m1), Cxcl10 (Mm00445235_m1), Mx1 (Mm00487796_m1), Aim2 (Mm01295719_m1), IL6 (Mm00446190_m1), IL1β (Mm00434228_m1), and Sdha (Mm01352366_m1).

    Techniques: Immunofluorescence, Staining, Knock-Out, Mouse Assay