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  • 90
    Thermo Fisher quant it hs dsdna assay
    Quant It Hs Dsdna Assay, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher qubit dsdna hs assay kit
    Storage condition of purified ChIP DNA is important. Purified ChIP DNA was adjusted to a concentration of 1 ng/μL ( a ) or 0.1 ng/μL ( b ), aliquoted into 4 different types of microcentrifuge tubes in 15 μL volume, and stored at −20 °C. DNA was quantified using <t>Qubit</t> <t>dsDNA</t> High Sensitivity assay at the indicated time points and expressed as a percentage of the amount measured at day 0. Three independent DNA samples were used in the experiment and DNA concentration from five tubes were measured at each time point. MaxyClear, Axygen® 1.7 mL MaxyClear Snaplock Microcentrifuge Tube; LoBind, Eppendorf DNA LoBind Snap Cap PCR Tube; Siliconized, Fisherbrand™ Siliconized Low-Retention Microcentrifuge Tube; Premium, Fisherbrand™ Premium Microcentrifuge Tube
    Qubit Dsdna Hs Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 10725 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RSV triggers the release of NETs coated with NE and MPO. ( A – D ) Neutrophils (1 × 10 5 /300 μL) were stimulated with active RSV (10 4 PFU/mL) or UV-inactivated RSV (10 4 PFU/mL) for 180 min at 37 °C with 5% CO 2 in 8-chamber culture slides. Afterwards, cells were fixed with 4% PFA and stained with (A) Hoechst 33342 (1:2000), anti-RSV fusion protein (1:1000) followed by anti-mouse PE antibody (1:500); (B) Hoechst 33342 (1:2000), anti-elastase (NE; 1:1000) followed by anti-rabbit Cy3 antibody (1:500); (C) Hoechst 33342 (1:2000), anti-myeloperoxidase PE (MPO, 1:1000) antibody; (D) Hoechst 33342 (1:2000). Overlay of the fluorescence images are shown in the penultimate panels. Arrowheads indicate the presence of extracellular <t>DNA</t> lattices co-localized with RSV F protein, NE and MPO, respectively. NETs were magnified four times and are numbered (1, 2, 3 and 4) on the right side. Images are representative of 3 independent experiments. Images were taken in a Zeiss LSM 5 Exciter microscope. Scale bars = 5 μm. (E) Neutrophils (1 × 10 6 /mL) were stimulated with active RSV (10 4 PFU/mL) or UV-inactivated RSV (10 4 PFU/mL) for 180 min at 37 °C with 5% CO 2 . Afterwards, NETs were quantified in culture supernatants using Quant-iT <t>dsDNA</t> HS kit (Invitrogen). Data are representative of 2 independent experiments performed in triplicates and represent mean ± SEM. Data were analyzed with Mann Whitney test. *p
    Quant It Dsdna Hs Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 269 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RSV triggers the release of NETs coated with NE and MPO. ( A – D ) Neutrophils (1 × 10 5 /300 μL) were stimulated with active RSV (10 4 PFU/mL) or UV-inactivated RSV (10 4 PFU/mL) for 180 min at 37 °C with 5% CO 2 in 8-chamber culture slides. Afterwards, cells were fixed with 4% PFA and stained with (A) Hoechst 33342 (1:2000), anti-RSV fusion protein (1:1000) followed by anti-mouse PE antibody (1:500); (B) Hoechst 33342 (1:2000), anti-elastase (NE; 1:1000) followed by anti-rabbit Cy3 antibody (1:500); (C) Hoechst 33342 (1:2000), anti-myeloperoxidase PE (MPO, 1:1000) antibody; (D) Hoechst 33342 (1:2000). Overlay of the fluorescence images are shown in the penultimate panels. Arrowheads indicate the presence of extracellular <t>DNA</t> lattices co-localized with RSV F protein, NE and MPO, respectively. NETs were magnified four times and are numbered (1, 2, 3 and 4) on the right side. Images are representative of 3 independent experiments. Images were taken in a Zeiss LSM 5 Exciter microscope. Scale bars = 5 μm. (E) Neutrophils (1 × 10 6 /mL) were stimulated with active RSV (10 4 PFU/mL) or UV-inactivated RSV (10 4 PFU/mL) for 180 min at 37 °C with 5% CO 2 . Afterwards, NETs were quantified in culture supernatants using Quant-iT <t>dsDNA</t> HS kit (Invitrogen). Data are representative of 2 independent experiments performed in triplicates and represent mean ± SEM. Data were analyzed with Mann Whitney test. *p
    Quant It Picogreen Dsdna Hs Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher quant it picogreen dsdna hs assay
    RSV triggers the release of NETs coated with NE and MPO. ( A – D ) Neutrophils (1 × 10 5 /300 μL) were stimulated with active RSV (10 4 PFU/mL) or UV-inactivated RSV (10 4 PFU/mL) for 180 min at 37 °C with 5% CO 2 in 8-chamber culture slides. Afterwards, cells were fixed with 4% PFA and stained with (A) Hoechst 33342 (1:2000), anti-RSV fusion protein (1:1000) followed by anti-mouse PE antibody (1:500); (B) Hoechst 33342 (1:2000), anti-elastase (NE; 1:1000) followed by anti-rabbit Cy3 antibody (1:500); (C) Hoechst 33342 (1:2000), anti-myeloperoxidase PE (MPO, 1:1000) antibody; (D) Hoechst 33342 (1:2000). Overlay of the fluorescence images are shown in the penultimate panels. Arrowheads indicate the presence of extracellular <t>DNA</t> lattices co-localized with RSV F protein, NE and MPO, respectively. NETs were magnified four times and are numbered (1, 2, 3 and 4) on the right side. Images are representative of 3 independent experiments. Images were taken in a Zeiss LSM 5 Exciter microscope. Scale bars = 5 μm. (E) Neutrophils (1 × 10 6 /mL) were stimulated with active RSV (10 4 PFU/mL) or UV-inactivated RSV (10 4 PFU/mL) for 180 min at 37 °C with 5% CO 2 . Afterwards, NETs were quantified in culture supernatants using Quant-iT <t>dsDNA</t> HS kit (Invitrogen). Data are representative of 2 independent experiments performed in triplicates and represent mean ± SEM. Data were analyzed with Mann Whitney test. *p
    Quant It Picogreen Dsdna Hs Assay, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RSV triggers the release of NETs coated with NE and MPO. ( A – D ) Neutrophils (1 × 10 5 /300 μL) were stimulated with active RSV (10 4 PFU/mL) or UV-inactivated RSV (10 4 PFU/mL) for 180 min at 37 °C with 5% CO 2 in 8-chamber culture slides. Afterwards, cells were fixed with 4% PFA and stained with (A) Hoechst 33342 (1:2000), anti-RSV fusion protein (1:1000) followed by anti-mouse PE antibody (1:500); (B) Hoechst 33342 (1:2000), anti-elastase (NE; 1:1000) followed by anti-rabbit Cy3 antibody (1:500); (C) Hoechst 33342 (1:2000), anti-myeloperoxidase PE (MPO, 1:1000) antibody; (D) Hoechst 33342 (1:2000). Overlay of the fluorescence images are shown in the penultimate panels. Arrowheads indicate the presence of extracellular <t>DNA</t> lattices co-localized with RSV F protein, NE and MPO, respectively. NETs were magnified four times and are numbered (1, 2, 3 and 4) on the right side. Images are representative of 3 independent experiments. Images were taken in a Zeiss LSM 5 Exciter microscope. Scale bars = 5 μm. (E) Neutrophils (1 × 10 6 /mL) were stimulated with active RSV (10 4 PFU/mL) or UV-inactivated RSV (10 4 PFU/mL) for 180 min at 37 °C with 5% CO 2 . Afterwards, NETs were quantified in culture supernatants using Quant-iT <t>dsDNA</t> HS kit (Invitrogen). Data are representative of 2 independent experiments performed in triplicates and represent mean ± SEM. Data were analyzed with Mann Whitney test. *p
    Quant Ittm Dsdna Hs Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 109 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RSV triggers the release of NETs coated with NE and MPO. ( A – D ) Neutrophils (1 × 10 5 /300 μL) were stimulated with active RSV (10 4 PFU/mL) or UV-inactivated RSV (10 4 PFU/mL) for 180 min at 37 °C with 5% CO 2 in 8-chamber culture slides. Afterwards, cells were fixed with 4% PFA and stained with (A) Hoechst 33342 (1:2000), anti-RSV fusion protein (1:1000) followed by anti-mouse PE antibody (1:500); (B) Hoechst 33342 (1:2000), anti-elastase (NE; 1:1000) followed by anti-rabbit Cy3 antibody (1:500); (C) Hoechst 33342 (1:2000), anti-myeloperoxidase PE (MPO, 1:1000) antibody; (D) Hoechst 33342 (1:2000). Overlay of the fluorescence images are shown in the penultimate panels. Arrowheads indicate the presence of extracellular <t>DNA</t> lattices co-localized with RSV F protein, NE and MPO, respectively. NETs were magnified four times and are numbered (1, 2, 3 and 4) on the right side. Images are representative of 3 independent experiments. Images were taken in a Zeiss LSM 5 Exciter microscope. Scale bars = 5 μm. (E) Neutrophils (1 × 10 6 /mL) were stimulated with active RSV (10 4 PFU/mL) or UV-inactivated RSV (10 4 PFU/mL) for 180 min at 37 °C with 5% CO 2 . Afterwards, NETs were quantified in culture supernatants using Quant-iT <t>dsDNA</t> HS kit (Invitrogen). Data are representative of 2 independent experiments performed in triplicates and represent mean ± SEM. Data were analyzed with Mann Whitney test. *p
    Quant It Dsdna Hs Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1448 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RSV triggers the release of NETs coated with NE and MPO. ( A – D ) Neutrophils (1 × 10 5 /300 μL) were stimulated with active RSV (10 4 PFU/mL) or UV-inactivated RSV (10 4 PFU/mL) for 180 min at 37 °C with 5% CO 2 in 8-chamber culture slides. Afterwards, cells were fixed with 4% PFA and stained with (A) Hoechst 33342 (1:2000), anti-RSV fusion protein (1:1000) followed by anti-mouse PE antibody (1:500); (B) Hoechst 33342 (1:2000), anti-elastase (NE; 1:1000) followed by anti-rabbit Cy3 antibody (1:500); (C) Hoechst 33342 (1:2000), anti-myeloperoxidase PE (MPO, 1:1000) antibody; (D) Hoechst 33342 (1:2000). Overlay of the fluorescence images are shown in the penultimate panels. Arrowheads indicate the presence of extracellular <t>DNA</t> lattices co-localized with RSV F protein, NE and MPO, respectively. NETs were magnified four times and are numbered (1, 2, 3 and 4) on the right side. Images are representative of 3 independent experiments. Images were taken in a Zeiss LSM 5 Exciter microscope. Scale bars = 5 μm. (E) Neutrophils (1 × 10 6 /mL) were stimulated with active RSV (10 4 PFU/mL) or UV-inactivated RSV (10 4 PFU/mL) for 180 min at 37 °C with 5% CO 2 . Afterwards, NETs were quantified in culture supernatants using Quant-iT <t>dsDNA</t> HS kit (Invitrogen). Data are representative of 2 independent experiments performed in triplicates and represent mean ± SEM. Data were analyzed with Mann Whitney test. *p
    Dark Reader Transilluminator, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RSV triggers the release of NETs coated with NE and MPO. ( A – D ) Neutrophils (1 × 10 5 /300 μL) were stimulated with active RSV (10 4 PFU/mL) or UV-inactivated RSV (10 4 PFU/mL) for 180 min at 37 °C with 5% CO 2 in 8-chamber culture slides. Afterwards, cells were fixed with 4% PFA and stained with (A) Hoechst 33342 (1:2000), anti-RSV fusion protein (1:1000) followed by anti-mouse PE antibody (1:500); (B) Hoechst 33342 (1:2000), anti-elastase (NE; 1:1000) followed by anti-rabbit Cy3 antibody (1:500); (C) Hoechst 33342 (1:2000), anti-myeloperoxidase PE (MPO, 1:1000) antibody; (D) Hoechst 33342 (1:2000). Overlay of the fluorescence images are shown in the penultimate panels. Arrowheads indicate the presence of extracellular <t>DNA</t> lattices co-localized with RSV F protein, NE and MPO, respectively. NETs were magnified four times and are numbered (1, 2, 3 and 4) on the right side. Images are representative of 3 independent experiments. Images were taken in a Zeiss LSM 5 Exciter microscope. Scale bars = 5 μm. (E) Neutrophils (1 × 10 6 /mL) were stimulated with active RSV (10 4 PFU/mL) or UV-inactivated RSV (10 4 PFU/mL) for 180 min at 37 °C with 5% CO 2 . Afterwards, NETs were quantified in culture supernatants using Quant-iT <t>dsDNA</t> HS kit (Invitrogen). Data are representative of 2 independent experiments performed in triplicates and represent mean ± SEM. Data were analyzed with Mann Whitney test. *p
    Qubit 2 0 Dsdna Hs Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RSV triggers the release of NETs coated with NE and MPO. ( A – D ) Neutrophils (1 × 10 5 /300 μL) were stimulated with active RSV (10 4 PFU/mL) or UV-inactivated RSV (10 4 PFU/mL) for 180 min at 37 °C with 5% CO 2 in 8-chamber culture slides. Afterwards, cells were fixed with 4% PFA and stained with (A) Hoechst 33342 (1:2000), anti-RSV fusion protein (1:1000) followed by anti-mouse PE antibody (1:500); (B) Hoechst 33342 (1:2000), anti-elastase (NE; 1:1000) followed by anti-rabbit Cy3 antibody (1:500); (C) Hoechst 33342 (1:2000), anti-myeloperoxidase PE (MPO, 1:1000) antibody; (D) Hoechst 33342 (1:2000). Overlay of the fluorescence images are shown in the penultimate panels. Arrowheads indicate the presence of extracellular <t>DNA</t> lattices co-localized with RSV F protein, NE and MPO, respectively. NETs were magnified four times and are numbered (1, 2, 3 and 4) on the right side. Images are representative of 3 independent experiments. Images were taken in a Zeiss LSM 5 Exciter microscope. Scale bars = 5 μm. (E) Neutrophils (1 × 10 6 /mL) were stimulated with active RSV (10 4 PFU/mL) or UV-inactivated RSV (10 4 PFU/mL) for 180 min at 37 °C with 5% CO 2 . Afterwards, NETs were quantified in culture supernatants using Quant-iT <t>dsDNA</t> HS kit (Invitrogen). Data are representative of 2 independent experiments performed in triplicates and represent mean ± SEM. Data were analyzed with Mann Whitney test. *p
    Qubit Double Stranded Dna Dsdna High Sensitivity High Sensitivity Hs Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RSV triggers the release of NETs coated with NE and MPO. ( A – D ) Neutrophils (1 × 10 5 /300 μL) were stimulated with active RSV (10 4 PFU/mL) or UV-inactivated RSV (10 4 PFU/mL) for 180 min at 37 °C with 5% CO 2 in 8-chamber culture slides. Afterwards, cells were fixed with 4% PFA and stained with (A) Hoechst 33342 (1:2000), anti-RSV fusion protein (1:1000) followed by anti-mouse PE antibody (1:500); (B) Hoechst 33342 (1:2000), anti-elastase (NE; 1:1000) followed by anti-rabbit Cy3 antibody (1:500); (C) Hoechst 33342 (1:2000), anti-myeloperoxidase PE (MPO, 1:1000) antibody; (D) Hoechst 33342 (1:2000). Overlay of the fluorescence images are shown in the penultimate panels. Arrowheads indicate the presence of extracellular <t>DNA</t> lattices co-localized with RSV F protein, NE and MPO, respectively. NETs were magnified four times and are numbered (1, 2, 3 and 4) on the right side. Images are representative of 3 independent experiments. Images were taken in a Zeiss LSM 5 Exciter microscope. Scale bars = 5 μm. (E) Neutrophils (1 × 10 6 /mL) were stimulated with active RSV (10 4 PFU/mL) or UV-inactivated RSV (10 4 PFU/mL) for 180 min at 37 °C with 5% CO 2 . Afterwards, NETs were quantified in culture supernatants using Quant-iT <t>dsDNA</t> HS kit (Invitrogen). Data are representative of 2 independent experiments performed in triplicates and represent mean ± SEM. Data were analyzed with Mann Whitney test. *p
    Qubit Dsdna High Sensitivity Hs Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher qubit 3 0 fluorometer dsdna hs assay kit
    RSV triggers the release of NETs coated with NE and MPO. ( A – D ) Neutrophils (1 × 10 5 /300 μL) were stimulated with active RSV (10 4 PFU/mL) or UV-inactivated RSV (10 4 PFU/mL) for 180 min at 37 °C with 5% CO 2 in 8-chamber culture slides. Afterwards, cells were fixed with 4% PFA and stained with (A) Hoechst 33342 (1:2000), anti-RSV fusion protein (1:1000) followed by anti-mouse PE antibody (1:500); (B) Hoechst 33342 (1:2000), anti-elastase (NE; 1:1000) followed by anti-rabbit Cy3 antibody (1:500); (C) Hoechst 33342 (1:2000), anti-myeloperoxidase PE (MPO, 1:1000) antibody; (D) Hoechst 33342 (1:2000). Overlay of the fluorescence images are shown in the penultimate panels. Arrowheads indicate the presence of extracellular <t>DNA</t> lattices co-localized with RSV F protein, NE and MPO, respectively. NETs were magnified four times and are numbered (1, 2, 3 and 4) on the right side. Images are representative of 3 independent experiments. Images were taken in a Zeiss LSM 5 Exciter microscope. Scale bars = 5 μm. (E) Neutrophils (1 × 10 6 /mL) were stimulated with active RSV (10 4 PFU/mL) or UV-inactivated RSV (10 4 PFU/mL) for 180 min at 37 °C with 5% CO 2 . Afterwards, NETs were quantified in culture supernatants using Quant-iT <t>dsDNA</t> HS kit (Invitrogen). Data are representative of 2 independent experiments performed in triplicates and represent mean ± SEM. Data were analyzed with Mann Whitney test. *p
    Qubit 3 0 Fluorometer Dsdna Hs Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Extracellular <t>DNA</t> is enclosed mostly in L-EVs in plasma. ( a) 50 or 100 μg of PC3 EVs was spiked in 1 ml of normal plasma. Plasma EVs were isolated, EV DNA was extracted and (b) quantified using HS dsDNA <t>Qubit</t> Assay. (c) 50 or 100 μg of PC3 EVs was spiked in 1 ml of normal plasma. Plasma cell-free (cf)DNA was extracted and (d) quantified using HS dsDNA Qubit Assay. (e) Representative bioluminescent images showing progressive bone and visceral metastasis following intracardial injection of 1 × 10 7 luciferase-labelled PC3 cells in NOD/SCID mice (left). The bioluminescent signal was quantified weekly and was measured as radiance in p/sec/cm 2 /sr (right). (f) SCNV of MYC, AKT1, PTEN, PTK2 and KLF10 in plasma-derived L-EVs was assessed by dPCR, demonstrating that L-EVs in the plasma of a mouse model of bone metastases report tumour-specific SCNV. Copy number of each target gene was normalized to gene reference RNAse P. N = 3 for each data point.
    Dna High Sensitivity Qubit Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Storage condition of purified ChIP DNA is important. Purified ChIP DNA was adjusted to a concentration of 1 ng/μL ( a ) or 0.1 ng/μL ( b ), aliquoted into 4 different types of microcentrifuge tubes in 15 μL volume, and stored at −20 °C. DNA was quantified using Qubit dsDNA High Sensitivity assay at the indicated time points and expressed as a percentage of the amount measured at day 0. Three independent DNA samples were used in the experiment and DNA concentration from five tubes were measured at each time point. MaxyClear, Axygen® 1.7 mL MaxyClear Snaplock Microcentrifuge Tube; LoBind, Eppendorf DNA LoBind Snap Cap PCR Tube; Siliconized, Fisherbrand™ Siliconized Low-Retention Microcentrifuge Tube; Premium, Fisherbrand™ Premium Microcentrifuge Tube

    Journal: BMC Genomics

    Article Title: Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application

    doi: 10.1186/s12864-017-4371-5

    Figure Lengend Snippet: Storage condition of purified ChIP DNA is important. Purified ChIP DNA was adjusted to a concentration of 1 ng/μL ( a ) or 0.1 ng/μL ( b ), aliquoted into 4 different types of microcentrifuge tubes in 15 μL volume, and stored at −20 °C. DNA was quantified using Qubit dsDNA High Sensitivity assay at the indicated time points and expressed as a percentage of the amount measured at day 0. Three independent DNA samples were used in the experiment and DNA concentration from five tubes were measured at each time point. MaxyClear, Axygen® 1.7 mL MaxyClear Snaplock Microcentrifuge Tube; LoBind, Eppendorf DNA LoBind Snap Cap PCR Tube; Siliconized, Fisherbrand™ Siliconized Low-Retention Microcentrifuge Tube; Premium, Fisherbrand™ Premium Microcentrifuge Tube

    Article Snippet: After treatment with RNase A and proteinase K, DNA was purified by Qiagen MinElute PCR Purification Kit (Cat. # 28006, Valencia, CA) and quantified using Qubit dsDNA High Sensitivity assay (Invitrogen, Q32851).

    Techniques: Purification, Chromatin Immunoprecipitation, Concentration Assay, Sensitive Assay, Polymerase Chain Reaction

    RSV triggers the release of NETs coated with NE and MPO. ( A – D ) Neutrophils (1 × 10 5 /300 μL) were stimulated with active RSV (10 4 PFU/mL) or UV-inactivated RSV (10 4 PFU/mL) for 180 min at 37 °C with 5% CO 2 in 8-chamber culture slides. Afterwards, cells were fixed with 4% PFA and stained with (A) Hoechst 33342 (1:2000), anti-RSV fusion protein (1:1000) followed by anti-mouse PE antibody (1:500); (B) Hoechst 33342 (1:2000), anti-elastase (NE; 1:1000) followed by anti-rabbit Cy3 antibody (1:500); (C) Hoechst 33342 (1:2000), anti-myeloperoxidase PE (MPO, 1:1000) antibody; (D) Hoechst 33342 (1:2000). Overlay of the fluorescence images are shown in the penultimate panels. Arrowheads indicate the presence of extracellular DNA lattices co-localized with RSV F protein, NE and MPO, respectively. NETs were magnified four times and are numbered (1, 2, 3 and 4) on the right side. Images are representative of 3 independent experiments. Images were taken in a Zeiss LSM 5 Exciter microscope. Scale bars = 5 μm. (E) Neutrophils (1 × 10 6 /mL) were stimulated with active RSV (10 4 PFU/mL) or UV-inactivated RSV (10 4 PFU/mL) for 180 min at 37 °C with 5% CO 2 . Afterwards, NETs were quantified in culture supernatants using Quant-iT dsDNA HS kit (Invitrogen). Data are representative of 2 independent experiments performed in triplicates and represent mean ± SEM. Data were analyzed with Mann Whitney test. *p

    Journal: Scientific Reports

    Article Title: Respiratory Syncytial Virus induces the classical ROS-dependent NETosis through PAD-4 and necroptosis pathways activation

    doi: 10.1038/s41598-018-32576-y

    Figure Lengend Snippet: RSV triggers the release of NETs coated with NE and MPO. ( A – D ) Neutrophils (1 × 10 5 /300 μL) were stimulated with active RSV (10 4 PFU/mL) or UV-inactivated RSV (10 4 PFU/mL) for 180 min at 37 °C with 5% CO 2 in 8-chamber culture slides. Afterwards, cells were fixed with 4% PFA and stained with (A) Hoechst 33342 (1:2000), anti-RSV fusion protein (1:1000) followed by anti-mouse PE antibody (1:500); (B) Hoechst 33342 (1:2000), anti-elastase (NE; 1:1000) followed by anti-rabbit Cy3 antibody (1:500); (C) Hoechst 33342 (1:2000), anti-myeloperoxidase PE (MPO, 1:1000) antibody; (D) Hoechst 33342 (1:2000). Overlay of the fluorescence images are shown in the penultimate panels. Arrowheads indicate the presence of extracellular DNA lattices co-localized with RSV F protein, NE and MPO, respectively. NETs were magnified four times and are numbered (1, 2, 3 and 4) on the right side. Images are representative of 3 independent experiments. Images were taken in a Zeiss LSM 5 Exciter microscope. Scale bars = 5 μm. (E) Neutrophils (1 × 10 6 /mL) were stimulated with active RSV (10 4 PFU/mL) or UV-inactivated RSV (10 4 PFU/mL) for 180 min at 37 °C with 5% CO 2 . Afterwards, NETs were quantified in culture supernatants using Quant-iT dsDNA HS kit (Invitrogen). Data are representative of 2 independent experiments performed in triplicates and represent mean ± SEM. Data were analyzed with Mann Whitney test. *p

    Article Snippet: After the stimulation period, culture supernatant was collected and extracellular DNA was measured using the dsDNA Picogreen kit or Quant-iT dsDNA HS kit (both from Invitrogen), following manufacturer’s instructions and obtaining similar results.

    Techniques: Staining, Fluorescence, Microscopy, MANN-WHITNEY

    Extracellular DNA is enclosed mostly in L-EVs in plasma. ( a) 50 or 100 μg of PC3 EVs was spiked in 1 ml of normal plasma. Plasma EVs were isolated, EV DNA was extracted and (b) quantified using HS dsDNA Qubit Assay. (c) 50 or 100 μg of PC3 EVs was spiked in 1 ml of normal plasma. Plasma cell-free (cf)DNA was extracted and (d) quantified using HS dsDNA Qubit Assay. (e) Representative bioluminescent images showing progressive bone and visceral metastasis following intracardial injection of 1 × 10 7 luciferase-labelled PC3 cells in NOD/SCID mice (left). The bioluminescent signal was quantified weekly and was measured as radiance in p/sec/cm 2 /sr (right). (f) SCNV of MYC, AKT1, PTEN, PTK2 and KLF10 in plasma-derived L-EVs was assessed by dPCR, demonstrating that L-EVs in the plasma of a mouse model of bone metastases report tumour-specific SCNV. Copy number of each target gene was normalized to gene reference RNAse P. N = 3 for each data point.

    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: Extracellular DNA is enclosed mostly in L-EVs in plasma. ( a) 50 or 100 μg of PC3 EVs was spiked in 1 ml of normal plasma. Plasma EVs were isolated, EV DNA was extracted and (b) quantified using HS dsDNA Qubit Assay. (c) 50 or 100 μg of PC3 EVs was spiked in 1 ml of normal plasma. Plasma cell-free (cf)DNA was extracted and (d) quantified using HS dsDNA Qubit Assay. (e) Representative bioluminescent images showing progressive bone and visceral metastasis following intracardial injection of 1 × 10 7 luciferase-labelled PC3 cells in NOD/SCID mice (left). The bioluminescent signal was quantified weekly and was measured as radiance in p/sec/cm 2 /sr (right). (f) SCNV of MYC, AKT1, PTEN, PTK2 and KLF10 in plasma-derived L-EVs was assessed by dPCR, demonstrating that L-EVs in the plasma of a mouse model of bone metastases report tumour-specific SCNV. Copy number of each target gene was normalized to gene reference RNAse P. N = 3 for each data point.

    Article Snippet: Final library quality control was performed using the DNA High Sensitivity Qubit Kit (Invitrogen), the Bioanalyzer High Sensitivity Chip Kit (Agilent) and the 7900HT Fast qPCR machine (Applied Biosystems). qPCR was performed using the Illumina Universal Library Quantification Kit from KAPA Biosystems.

    Techniques: Isolation, HS DSDNA Qubit Assay, Injection, Luciferase, Mouse Assay, Size-exclusion Chromatography, Derivative Assay, Digital PCR

    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: Final library quality control was performed using the DNA High Sensitivity Qubit Kit (Invitrogen), the Bioanalyzer High Sensitivity Chip Kit (Agilent) and the 7900HT Fast qPCR machine (Applied Biosystems). qPCR was performed using the Illumina Universal Library Quantification Kit from KAPA Biosystems.

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