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  • 99
    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: 99/100, based on 246 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher picogreen dsdna reagent
    Cell seeding comparison between porous and solid ringed 2:8 PET:PU scaffolds. <t>PicoGreen</t> <t>dsDNA</t> (double stranded DNA) assay was utilized to evaluate the effect of ring structure on seeded cell density. Values were interpolated from a standard curve made from serially diluted aliquots of known cell concentrations (manual cell count) from the pre-seeding solution. One curve was generated for each experiment. (A) Comparison of average seeded cell density between porous and ringed scaffolds (n = 4 scaffolds per group, each scaffold was sampled n = 8 times for analysis). (B) Division of graft samples by ringed or inter-ring segment to evaluate the effect of ring design on homogeneity of scaffold seeding. ns: not significant, * p
    Picogreen Dsdna Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 706 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega quantifluor dsdna
    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 <t>QuantiFluor</t> <t>dsDNA</t> 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
    Quantifluor Dsdna, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Fisher Scientific dsdna
    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 <t>QuantiFluor</t> <t>dsDNA</t> 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
    Dsdna, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    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: 94/100, based on 591 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Promega 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 Promega, used in various techniques. Bioz Stars score: 96/100, based on 230 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 2088 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    InvivoGen dsdna
    TRIM29 binds and colocalizes with STING in the cytosol. a Immunoblot analysis of endogenous proteins of TRIM29 and STING precipitated with anti-STING or immunoglobulin G (IgG; control) from whole-cell lysates of BMDCs from WT mice stimulated without (No) or with <t>HSV-60</t> (2.5 μg/ml) for 8 h, and then with MG132 treatment for 3 h. Input, 20% of the BMDCs lysate. b Full-length STING and serial truncations of STING with deletion of various domains ( top ). Immunoblot analysis of purified HA-tagged TRIM29 with anti-HA ( bottom blot ), and immunoblot analysis of purified HA-tagged full-length TRIM29 with anti-HA ( middle blot ) or purified Myc-tagged full-length STING and STING truncation mutants alone with anti-Myc ( top blot ) after incubation with Myc-tagged full-length STING and STING truncation mutants and immunoprecipitation with anti-Myc ( top and middle blots ). c Full-length TRIM29 and serial truncations of TRIM29 with deletion (Δ) of various domains ( left margin ); numbers at ends indicate amino-acid positions ( top ). Below , immunoblot analysis of purified Myc-tagged STING with anti-Myc ( bottom blot ), and immunoblot analysis (with anti-HA) of purified HA-tagged full-length TRIM29 and TRIM29 truncation mutants alone ( top blot ) or after incubation with Myc-tagged STING and immunoprecipitation with anti-Myc ( middle blot ). d Colocalization of endogenous TRIM29 and STING in BEAS-2B cells. Confocal microscopy of BEAS-2B cells without stimulation, or stimulated with <t>dsDNA</t> HSV-60 for 4 h. STING was stained with Rabbit anti-STING polyclonal antibody (Cat: 19851-1-AP, Proteintech), followed by Alexa Fluor 488 goat anti-rabbit secondary antibody ( green ), while TRIM29 was stained with mouse anti-TRIM29 monoclonal antibody (sc-166707, Santa Cruz), followed by Alexa Fluor 594 goat anti-mouse secondary antibody ( red ). DAPI (4′,6-diamidino-2-phenylindole) served as the nuclei marker ( blue ). Scale bars represent 20 µm. The position of protein markers (shown in kDa) is indicated on the right . Data are representative of three independent experiments
    Dsdna, supplied by InvivoGen, used in various techniques. Bioz Stars score: 93/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bioneer Corporation dsdna
    TRIM29 binds and colocalizes with STING in the cytosol. a Immunoblot analysis of endogenous proteins of TRIM29 and STING precipitated with anti-STING or immunoglobulin G (IgG; control) from whole-cell lysates of BMDCs from WT mice stimulated without (No) or with <t>HSV-60</t> (2.5 μg/ml) for 8 h, and then with MG132 treatment for 3 h. Input, 20% of the BMDCs lysate. b Full-length STING and serial truncations of STING with deletion of various domains ( top ). Immunoblot analysis of purified HA-tagged TRIM29 with anti-HA ( bottom blot ), and immunoblot analysis of purified HA-tagged full-length TRIM29 with anti-HA ( middle blot ) or purified Myc-tagged full-length STING and STING truncation mutants alone with anti-Myc ( top blot ) after incubation with Myc-tagged full-length STING and STING truncation mutants and immunoprecipitation with anti-Myc ( top and middle blots ). c Full-length TRIM29 and serial truncations of TRIM29 with deletion (Δ) of various domains ( left margin ); numbers at ends indicate amino-acid positions ( top ). Below , immunoblot analysis of purified Myc-tagged STING with anti-Myc ( bottom blot ), and immunoblot analysis (with anti-HA) of purified HA-tagged full-length TRIM29 and TRIM29 truncation mutants alone ( top blot ) or after incubation with Myc-tagged STING and immunoprecipitation with anti-Myc ( middle blot ). d Colocalization of endogenous TRIM29 and STING in BEAS-2B cells. Confocal microscopy of BEAS-2B cells without stimulation, or stimulated with <t>dsDNA</t> HSV-60 for 4 h. STING was stained with Rabbit anti-STING polyclonal antibody (Cat: 19851-1-AP, Proteintech), followed by Alexa Fluor 488 goat anti-rabbit secondary antibody ( green ), while TRIM29 was stained with mouse anti-TRIM29 monoclonal antibody (sc-166707, Santa Cruz), followed by Alexa Fluor 594 goat anti-mouse secondary antibody ( red ). DAPI (4′,6-diamidino-2-phenylindole) served as the nuclei marker ( blue ). Scale bars represent 20 µm. The position of protein markers (shown in kDa) is indicated on the right . Data are representative of three independent experiments
    Dsdna, supplied by Bioneer Corporation, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Dynatech Laboratories dsdna
    Peptide inhibition of MAbs binding to EBNA‐1 and <t>dsDNA.</t> 1D2 (5 μg/ml) and 3D4 (0.05 μg/ml) were incubated with increasing concentrations of peptides PFM‐15 or GC‐15 and examined by ELISA for binding to EBNA‐1 (A and B, respectively). 1D2 (5 μg/ml) and 3D4 (2.5 μg/ml) were incubated with increasing concentrations of peptides PFM‐15 or GC‐15 and examined by ELISA for binding dsDNA (C and D, respectively). <t>ELISAs</t> are representative of two experiments. ELISA values were obtained in triplicate. Error bars represent standard deviations of triplicate values.
    Dsdna, supplied by Dynatech Laboratories, used in various techniques. Bioz Stars score: 94/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dsdna  (ALPCO)
    91
    ALPCO dsdna
    Peptide inhibition of MAbs binding to EBNA‐1 and <t>dsDNA.</t> 1D2 (5 μg/ml) and 3D4 (0.05 μg/ml) were incubated with increasing concentrations of peptides PFM‐15 or GC‐15 and examined by ELISA for binding to EBNA‐1 (A and B, respectively). 1D2 (5 μg/ml) and 3D4 (2.5 μg/ml) were incubated with increasing concentrations of peptides PFM‐15 or GC‐15 and examined by ELISA for binding dsDNA (C and D, respectively). <t>ELISAs</t> are representative of two experiments. ELISA values were obtained in triplicate. Error bars represent standard deviations of triplicate values.
    Dsdna, supplied by ALPCO, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dsdna  (Roche)
    96
    Roche dsdna
    Insertion of a loxP511 site. ( A ) The location of the wild-type and mutant loxP sites in the BAC backbone are indicated along with the extra mutant loxP511 site that was introduced into the BAC genomic insert via <t>galK</t> counterselection. The 95 kb region deleted by Cre-mediated recombination between the two loxP511 sites is indicated, and PCR primers used to confirm the deletion are shown as small arrows. ( B ) SpeI restriction analysis of six miniprep clones selected for the replacement of galK with a <t>dsDNA</t> oligo containing the mutant loxP511 site. Clones 3, 5 and 6 (circles) had the same restriction pattern as the unmodified BAC, indicating that DOG resistance occurred due to the intended homologous recombination event. Clones 1, 2 and 4 had large deletions and were not analyzed further. ( C ) SpeI restriction analysis of BAC miniprep DNA from clones 3, 5 and 6 after transformation into Cre-induced EL350 cells. Two clones from each parental clone were tested. The restriction pattern shows that the 95 kb region flanked by two loxP511 sites is deleted from all clones analyzed, confirming the correct insertion of loxP511 in clones 3, 5 and 6. ( D ) PCR analysis of the six clones from (C) with one primer mapping to the BAC backbone and the other to a position distal to the inserted loxP511 site.
    Dsdna, supplied by Roche, used in various techniques. Bioz Stars score: 96/100, based on 198 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Zymo Research dsdna shearase plus
    Insertion of a loxP511 site. ( A ) The location of the wild-type and mutant loxP sites in the BAC backbone are indicated along with the extra mutant loxP511 site that was introduced into the BAC genomic insert via <t>galK</t> counterselection. The 95 kb region deleted by Cre-mediated recombination between the two loxP511 sites is indicated, and PCR primers used to confirm the deletion are shown as small arrows. ( B ) SpeI restriction analysis of six miniprep clones selected for the replacement of galK with a <t>dsDNA</t> oligo containing the mutant loxP511 site. Clones 3, 5 and 6 (circles) had the same restriction pattern as the unmodified BAC, indicating that DOG resistance occurred due to the intended homologous recombination event. Clones 1, 2 and 4 had large deletions and were not analyzed further. ( C ) SpeI restriction analysis of BAC miniprep DNA from clones 3, 5 and 6 after transformation into Cre-induced EL350 cells. Two clones from each parental clone were tested. The restriction pattern shows that the 95 kb region flanked by two loxP511 sites is deleted from all clones analyzed, confirming the correct insertion of loxP511 in clones 3, 5 and 6. ( D ) PCR analysis of the six clones from (C) with one primer mapping to the BAC backbone and the other to a position distal to the inserted loxP511 site.
    Dsdna Shearase Plus, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dsdna kit
    Morphology, proliferation and differentiation of <t>BMSCs</t> on the MPZs and control surfaces. a) Quant-iT <t>dsDNA</t> Assay of BMSCs on the samples. The assay was repeated twice and expressed as means ± s.d.. Significant differences were determined using a one-way analysis of variance (ANOVA) followed by LSD-t test. (**) and (*) indicates a statisctic difference at p
    Dsdna Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Immco immulisa dsdna
    Morphology, proliferation and differentiation of <t>BMSCs</t> on the MPZs and control surfaces. a) Quant-iT <t>dsDNA</t> Assay of BMSCs on the samples. The assay was repeated twice and expressed as means ± s.d.. Significant differences were determined using a one-way analysis of variance (ANOVA) followed by LSD-t test. (**) and (*) indicates a statisctic difference at p
    Immulisa Dsdna, supplied by Immco, used in various techniques. Bioz Stars score: 87/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega quantifluor one dsdna system
    Morphology, proliferation and differentiation of <t>BMSCs</t> on the MPZs and control surfaces. a) Quant-iT <t>dsDNA</t> Assay of BMSCs on the samples. The assay was repeated twice and expressed as means ± s.d.. Significant differences were determined using a one-way analysis of variance (ANOVA) followed by LSD-t test. (**) and (*) indicates a statisctic difference at p
    Quantifluor One Dsdna System, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher qubit dsdna
    Morphology, proliferation and differentiation of <t>BMSCs</t> on the MPZs and control surfaces. a) Quant-iT <t>dsDNA</t> Assay of BMSCs on the samples. The assay was repeated twice and expressed as means ± s.d.. Significant differences were determined using a one-way analysis of variance (ANOVA) followed by LSD-t test. (**) and (*) indicates a statisctic difference at p
    Qubit Dsdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher 850bp dsdna
    Morphology, proliferation and differentiation of <t>BMSCs</t> on the MPZs and control surfaces. a) Quant-iT <t>dsDNA</t> Assay of BMSCs on the samples. The assay was repeated twice and expressed as means ± s.d.. Significant differences were determined using a one-way analysis of variance (ANOVA) followed by LSD-t test. (**) and (*) indicates a statisctic difference at p
    850bp Dsdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore soluble dsdna
    Reactivity of MOG-specific B cells. B cells were preincubated with 20 μ g of soluble MOG, albumin, <t>dsDNA,</t> insulin, or <t>LPS</t> before the addition of MOG-coated beads ( n = 3). (a) The frequency of MOG-BBR drops with the soluble MOG preincubation but not with albumin preincubation. (b) The recognition of MOG-coated beads decreased after the addition of soluble LPS (% of inhibition: 73%), dsDNA (11%), and insulin (35%). Competitive assay was tested in MS ( n = 3); the percentage of inhibition after soluble antigen addition was the same proportion as HI: LPS (78%), dsDNA (11%), and insulin (32%). (c) B cells were preincubated with Fab'2 anti-human IgG+IgA+IgM. The frequency of B cells recognizing MOG-coated beads was assessed on 3 HI. Fab'2 antibody modified the frequency of MOG-specific B cells (ns, P > 0.05, Wilcoxon test ).
    Soluble Dsdna, supplied by Millipore, 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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    84
    PRIMER-E bp dsdna
    Reactivity of MOG-specific B cells. B cells were preincubated with 20 μ g of soluble MOG, albumin, <t>dsDNA,</t> insulin, or <t>LPS</t> before the addition of MOG-coated beads ( n = 3). (a) The frequency of MOG-BBR drops with the soluble MOG preincubation but not with albumin preincubation. (b) The recognition of MOG-coated beads decreased after the addition of soluble LPS (% of inhibition: 73%), dsDNA (11%), and insulin (35%). Competitive assay was tested in MS ( n = 3); the percentage of inhibition after soluble antigen addition was the same proportion as HI: LPS (78%), dsDNA (11%), and insulin (32%). (c) B cells were preincubated with Fab'2 anti-human IgG+IgA+IgM. The frequency of B cells recognizing MOG-coated beads was assessed on 3 HI. Fab'2 antibody modified the frequency of MOG-specific B cells (ns, P > 0.05, Wilcoxon test ).
    Bp Dsdna, supplied by PRIMER-E, used in various techniques. Bioz Stars score: 84/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega dsdna dye
    Reactivity of MOG-specific B cells. B cells were preincubated with 20 μ g of soluble MOG, albumin, <t>dsDNA,</t> insulin, or <t>LPS</t> before the addition of MOG-coated beads ( n = 3). (a) The frequency of MOG-BBR drops with the soluble MOG preincubation but not with albumin preincubation. (b) The recognition of MOG-coated beads decreased after the addition of soluble LPS (% of inhibition: 73%), dsDNA (11%), and insulin (35%). Competitive assay was tested in MS ( n = 3); the percentage of inhibition after soluble antigen addition was the same proportion as HI: LPS (78%), dsDNA (11%), and insulin (32%). (c) B cells were preincubated with Fab'2 anti-human IgG+IgA+IgM. The frequency of B cells recognizing MOG-coated beads was assessed on 3 HI. Fab'2 antibody modified the frequency of MOG-specific B cells (ns, P > 0.05, Wilcoxon test ).
    Dsdna Dye, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Promega linear dsdna
    Reactivity of MOG-specific B cells. B cells were preincubated with 20 μ g of soluble MOG, albumin, <t>dsDNA,</t> insulin, or <t>LPS</t> before the addition of MOG-coated beads ( n = 3). (a) The frequency of MOG-BBR drops with the soluble MOG preincubation but not with albumin preincubation. (b) The recognition of MOG-coated beads decreased after the addition of soluble LPS (% of inhibition: 73%), dsDNA (11%), and insulin (35%). Competitive assay was tested in MS ( n = 3); the percentage of inhibition after soluble antigen addition was the same proportion as HI: LPS (78%), dsDNA (11%), and insulin (32%). (c) B cells were preincubated with Fab'2 anti-human IgG+IgA+IgM. The frequency of B cells recognizing MOG-coated beads was assessed on 3 HI. Fab'2 antibody modified the frequency of MOG-specific B cells (ns, P > 0.05, Wilcoxon test ).
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    Reactivity of MOG-specific B cells. B cells were preincubated with 20 μ g of soluble MOG, albumin, <t>dsDNA,</t> insulin, or <t>LPS</t> before the addition of MOG-coated beads ( n = 3). (a) The frequency of MOG-BBR drops with the soluble MOG preincubation but not with albumin preincubation. (b) The recognition of MOG-coated beads decreased after the addition of soluble LPS (% of inhibition: 73%), dsDNA (11%), and insulin (35%). Competitive assay was tested in MS ( n = 3); the percentage of inhibition after soluble antigen addition was the same proportion as HI: LPS (78%), dsDNA (11%), and insulin (32%). (c) B cells were preincubated with Fab'2 anti-human IgG+IgA+IgM. The frequency of B cells recognizing MOG-coated beads was assessed on 3 HI. Fab'2 antibody modified the frequency of MOG-specific B cells (ns, P > 0.05, Wilcoxon test ).
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    Reactivity of MOG-specific B cells. B cells were preincubated with 20 μ g of soluble MOG, albumin, <t>dsDNA,</t> insulin, or <t>LPS</t> before the addition of MOG-coated beads ( n = 3). (a) The frequency of MOG-BBR drops with the soluble MOG preincubation but not with albumin preincubation. (b) The recognition of MOG-coated beads decreased after the addition of soluble LPS (% of inhibition: 73%), dsDNA (11%), and insulin (35%). Competitive assay was tested in MS ( n = 3); the percentage of inhibition after soluble antigen addition was the same proportion as HI: LPS (78%), dsDNA (11%), and insulin (32%). (c) B cells were preincubated with Fab'2 anti-human IgG+IgA+IgM. The frequency of B cells recognizing MOG-coated beads was assessed on 3 HI. Fab'2 antibody modified the frequency of MOG-specific B cells (ns, P > 0.05, Wilcoxon test ).
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    Image Search Results


    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

    DSB s formation at suboptimal pH e. (A) Left panel: PFGE in neutral conditions to resolve DSB s. Arrows show maximal density (apex) for each track. Right panel: corresponding densitometric scan of the PFGE tracks shown in A. Arrows indicate the corresponding position of apex shown on the gel. (B) qTUNEL quantification of DNA double‐stranded breaks for the highest and lowest values of the suboptimal pH e range. Frag indicates fragmentase‐digested DNA from fibroblasts used as a positive control. Values are mean ± SEM of three determinations.

    Journal: FEBS Open Bio

    Article Title: Suboptimal extracellular pH values alter DNA damage response to induced double‐strand breaks

    doi: 10.1002/2211-5463.12384

    Figure Lengend Snippet: DSB s formation at suboptimal pH e. (A) Left panel: PFGE in neutral conditions to resolve DSB s. Arrows show maximal density (apex) for each track. Right panel: corresponding densitometric scan of the PFGE tracks shown in A. Arrows indicate the corresponding position of apex shown on the gel. (B) qTUNEL quantification of DNA double‐stranded breaks for the highest and lowest values of the suboptimal pH e range. Frag indicates fragmentase‐digested DNA from fibroblasts used as a positive control. Values are mean ± SEM of three determinations.

    Article Snippet: As a positive control, digestion of the DNA was performed with 2 μL of fragmentase (M0348, NEB, Ipswich, MA, USA).

    Techniques: Positive Control

    Cell seeding comparison between porous and solid ringed 2:8 PET:PU scaffolds. PicoGreen dsDNA (double stranded DNA) assay was utilized to evaluate the effect of ring structure on seeded cell density. Values were interpolated from a standard curve made from serially diluted aliquots of known cell concentrations (manual cell count) from the pre-seeding solution. One curve was generated for each experiment. (A) Comparison of average seeded cell density between porous and ringed scaffolds (n = 4 scaffolds per group, each scaffold was sampled n = 8 times for analysis). (B) Division of graft samples by ringed or inter-ring segment to evaluate the effect of ring design on homogeneity of scaffold seeding. ns: not significant, * p

    Journal: International journal of pediatric otorhinolaryngology

    Article Title: Designing a tissue-engineered tracheal scaffold for preclinical evaluation

    doi: 10.1016/j.ijporl.2017.10.036

    Figure Lengend Snippet: Cell seeding comparison between porous and solid ringed 2:8 PET:PU scaffolds. PicoGreen dsDNA (double stranded DNA) assay was utilized to evaluate the effect of ring structure on seeded cell density. Values were interpolated from a standard curve made from serially diluted aliquots of known cell concentrations (manual cell count) from the pre-seeding solution. One curve was generated for each experiment. (A) Comparison of average seeded cell density between porous and ringed scaffolds (n = 4 scaffolds per group, each scaffold was sampled n = 8 times for analysis). (B) Division of graft samples by ringed or inter-ring segment to evaluate the effect of ring design on homogeneity of scaffold seeding. ns: not significant, * p

    Article Snippet: DNA assays were performed using PicoGreen® dsDNA reagent (Invitrogen, Carlsbad, CA, USA).

    Techniques: Positron Emission Tomography, dsDNA Assay, Cell Counting, Generated

    Bar graph representing dsDNA level evaluated by fluorimetric PicoGreen ® assay of dental pulp stem cell in different passages. The results are presented in % related to the first DPSC passage

    Journal: Veterinary Research Forum

    Article Title: DNA damage in dental pulp mesenchymal stem cells: An in vitro study

    doi: 10.30466/vrf.2018.33083

    Figure Lengend Snippet: Bar graph representing dsDNA level evaluated by fluorimetric PicoGreen ® assay of dental pulp stem cell in different passages. The results are presented in % related to the first DPSC passage

    Article Snippet: The DNA fragmentation in DPSCs from each culture cell passage was performed using an ultrasensitive protocol for quantification of the level of DNA fragmentation using Quant-iT™ PicoGreen® dsDNA (Invitrogen, Carlsbad, USA) and a protocol for quantitative assessment of DNA concentration and damage (fragmentation) employing a protocol similar to that described by Batel et al . and Georgiou et al . but adjusted to fit a 96-well cell culture plate., The method is based on the ability of the specific fluorochrome dye (PicoGreen; Thermo Fisher Scientific, Massachusetts, USA) to make a very stable complex with double-stranded DNA (dsDNA) under alkaline conditions instead of single-stranded DNA (ssDNA), proteins, sodium dodecyl sulfate and urea.

    Techniques: Picogreen Assay

    Growth of E.coli K-12 in suspensions and biofilms and eDNA released in suspended cultures and biofilm matrix in 10% LB supplemented with or without dATP (400 µM). A, growth of E.coli K-12 in suspensions and biofilms; B, eDNA in cultures and biofilm matrix. eDNA was extracted using the method we developed (see the Materials and Methods section) and quantified using the PicoGreen dsDNA Quantitation Kit (Molecular Probes, Invitrogen). P values from statistical analysis of three independent experiments were shown in the graph. Student T-test was used for statistic analysis.

    Journal: PLoS ONE

    Article Title: dATP/ATP, a Multifunctional Nucleotide, Stimulates Bacterial Cell Lysis, Extracellular DNA Release and Biofilm Development

    doi: 10.1371/journal.pone.0013355

    Figure Lengend Snippet: Growth of E.coli K-12 in suspensions and biofilms and eDNA released in suspended cultures and biofilm matrix in 10% LB supplemented with or without dATP (400 µM). A, growth of E.coli K-12 in suspensions and biofilms; B, eDNA in cultures and biofilm matrix. eDNA was extracted using the method we developed (see the Materials and Methods section) and quantified using the PicoGreen dsDNA Quantitation Kit (Molecular Probes, Invitrogen). P values from statistical analysis of three independent experiments were shown in the graph. Student T-test was used for statistic analysis.

    Article Snippet: DNA was dissolved in 50 µl of sterile MilliQ water and the concentration of DNA was measured by using the PicoGreen dsDNA Quantitation Kit (Molecular Probes, Invitrogen).

    Techniques: Quantitation Assay

    Quantifying the outcomes of viral infections of HAE ( A ) Apical washes harvested on the indicated days from mock-, PIV5-, PIV3-, and RSV-infected HAE (apical, MOI of 6) were subjected to three rounds of freeze and thaw cycles to release nuclear dsDNA. The amount of dsDNA present in the apical wash as a function of cell shedding was quantified with PicoGreen dsDNA reagent (see “Materials and Methods” for details). The mean values per HAE culture (0.6 cm 2 ) (± SEM, n = 3) from 1 to 6 days PI are shown. ( B ) Trans-epithelial electrical resistances (TEER) of HAE infected with PIV5, PIV3, RSV, or mock-infected as above were measured with an EVOM meter on 0 to 8 days PI, and the mean values (± SEM, n = 3) are shown. ( C ) Basolateral media were collected from the infected HAE from 1 to 4 days PI and type I interferon concentrations were determined with a CPE inhibition bioassay (see “Materials and Methods” for details) (± SEM, n = 3).

    Journal: Virology

    Article Title: Comparison of Differing Cytopathic Effects in Human Airway Epithelium of Parainfluenza Virus 5 (W3A), Parainfluenza Virus Type 3, and Respiratory Syncytial Virus

    doi: 10.1016/j.virol.2011.08.020

    Figure Lengend Snippet: Quantifying the outcomes of viral infections of HAE ( A ) Apical washes harvested on the indicated days from mock-, PIV5-, PIV3-, and RSV-infected HAE (apical, MOI of 6) were subjected to three rounds of freeze and thaw cycles to release nuclear dsDNA. The amount of dsDNA present in the apical wash as a function of cell shedding was quantified with PicoGreen dsDNA reagent (see “Materials and Methods” for details). The mean values per HAE culture (0.6 cm 2 ) (± SEM, n = 3) from 1 to 6 days PI are shown. ( B ) Trans-epithelial electrical resistances (TEER) of HAE infected with PIV5, PIV3, RSV, or mock-infected as above were measured with an EVOM meter on 0 to 8 days PI, and the mean values (± SEM, n = 3) are shown. ( C ) Basolateral media were collected from the infected HAE from 1 to 4 days PI and type I interferon concentrations were determined with a CPE inhibition bioassay (see “Materials and Methods” for details) (± SEM, n = 3).

    Article Snippet: The total amount of dsDNA present in the apical washes of HAE were quantified in 96-well plate format using a Quant-iT PicoGreen dsDNA Kit (Invitrogen # ).

    Techniques: Infection, Inhibition

    Circulating dsDNA level comparison between WT and mast cell deficient groups. Circulating dsDNA levels were measured by Quant-iT ™ PicoGreen dsDNA kit. Values are expressed as mean ± SEM. * p

    Journal: Journal of the American College of Surgeons

    Article Title: Mast Cells Play a Critical Role in the Systemic Inflammatory Response and End Organ Injury Resulting from Trauma

    doi: 10.1016/j.jamcollsurg.2011.08.009

    Figure Lengend Snippet: Circulating dsDNA level comparison between WT and mast cell deficient groups. Circulating dsDNA levels were measured by Quant-iT ™ PicoGreen dsDNA kit. Values are expressed as mean ± SEM. * p

    Article Snippet: Quant-iT™ PicoGreen dsDNA Kits were bought from Molecular Probes, Inc. (Eugene, OR).

    Techniques:

    HuR is required for short-term proliferation of PDA cells Relative proliferation of DOX-inducible MIA PaCa-2 cell lines treated with 0 or 2 μg/ml DOX for the indicated time points, as determined by measurement of dsDNA content by PicoGreen staining. Each data point represents the mean of 5 independent experiments ± standard error of the mean (SEM). * = p

    Journal: Oncotarget

    Article Title: Targeting the mRNA-binding protein HuR impairs malignant characteristics of pancreatic ductal adenocarcinoma cells

    doi:

    Figure Lengend Snippet: HuR is required for short-term proliferation of PDA cells Relative proliferation of DOX-inducible MIA PaCa-2 cell lines treated with 0 or 2 μg/ml DOX for the indicated time points, as determined by measurement of dsDNA content by PicoGreen staining. Each data point represents the mean of 5 independent experiments ± standard error of the mean (SEM). * = p

    Article Snippet: Double-stranded DNA (dsDNA) was stained by Quant-iT PicoGreen dsDNA reagent (Life Technologies, cat. #P7581), and fluorescence intensity was measured by a microplate reader (Tecan, part #F129015) using excitation wavelength of 485 nm and emission wavelength of 535 nm.

    Techniques: Staining

    Sorting of foreign DNA into EVs require TBK1 mediated phosphorylation of MVB12b. ( a ) Top 20 induced phosphorylated peptides upon dsDNA transfection in MEFs plotted as median with range from four experiments. ( b ) MS/MS spectrum of the identified phosphorylation of serine 222 on Mvb12b. ( c ) Amino acid sequence of protein murine MVB12b from amino acid 218-226 flanking TBK1 phospho-target Serine 222. For comparison, human MVB12b is also shown. ( d ) Immunoblot for MVB12b and β actin on cell lysates from Wt and two Mvb12b -/- clones (made with independent gRNAs). ( e, f ) Induction of Ifnb mRNA in Wt MEFs stimulated with supernatants from Wt and Mvb12b -/- MEFs transfected with dsDNA (2μg/ml) (n=6) or infected with L.monocytogenes (MOI 200) (n=4). ( g ) Quantification of % FITC-positive recipient Wt MEFs after treatment with supernatants from the indicated MEF donor cells, transfected with FITC-DNA (1ug/ml) for 6h (n=4). ( h ) Quantification of DNA in EVs isolated from Wt and Mvb12b -/- MEFs infected with L.monocytogenes for 18 h using Picogreen (n=3). ( i ) Phosphorylation of MVB12b in Wt MEFs upon dsDNA stimulation compared to Sting gt/gt and Tbk1 -/- MEFs. β actin was used as loading control. ( j ) Co-localization of STING and phospho-MVB12b 6 h after infection with L.monocytogenes . Nuclei were stained with DAPI. ( k ) Induction of Ifnb mRNA in Wt recipient MEFs stimulated with supernatants from L.monocytogenes -infected Mvb12b -/- donor MEFs reconstituted with Wt or S222A mutants (n=4). The presented data are representative of at least 2 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.

    Journal: Nature microbiology

    Article Title: Intracellular bacteria engage a STING-TBK1-MVB12b pathway to enable paracrine cGAS-STING signaling

    doi: 10.1038/s41564-019-0367-z

    Figure Lengend Snippet: Sorting of foreign DNA into EVs require TBK1 mediated phosphorylation of MVB12b. ( a ) Top 20 induced phosphorylated peptides upon dsDNA transfection in MEFs plotted as median with range from four experiments. ( b ) MS/MS spectrum of the identified phosphorylation of serine 222 on Mvb12b. ( c ) Amino acid sequence of protein murine MVB12b from amino acid 218-226 flanking TBK1 phospho-target Serine 222. For comparison, human MVB12b is also shown. ( d ) Immunoblot for MVB12b and β actin on cell lysates from Wt and two Mvb12b -/- clones (made with independent gRNAs). ( e, f ) Induction of Ifnb mRNA in Wt MEFs stimulated with supernatants from Wt and Mvb12b -/- MEFs transfected with dsDNA (2μg/ml) (n=6) or infected with L.monocytogenes (MOI 200) (n=4). ( g ) Quantification of % FITC-positive recipient Wt MEFs after treatment with supernatants from the indicated MEF donor cells, transfected with FITC-DNA (1ug/ml) for 6h (n=4). ( h ) Quantification of DNA in EVs isolated from Wt and Mvb12b -/- MEFs infected with L.monocytogenes for 18 h using Picogreen (n=3). ( i ) Phosphorylation of MVB12b in Wt MEFs upon dsDNA stimulation compared to Sting gt/gt and Tbk1 -/- MEFs. β actin was used as loading control. ( j ) Co-localization of STING and phospho-MVB12b 6 h after infection with L.monocytogenes . Nuclei were stained with DAPI. ( k ) Induction of Ifnb mRNA in Wt recipient MEFs stimulated with supernatants from L.monocytogenes -infected Mvb12b -/- donor MEFs reconstituted with Wt or S222A mutants (n=4). The presented data are representative of at least 2 independent experiments. The Ifnb mRNA levels were normalized to bactin mRNA and shown as relative levels compared to mock. Data are shown as mean ± SD. P values were calculated using 2-tailed unpaired students t-test.

    Article Snippet: Isolated DNA was quantified using Quanti-it PicoGreen dsDNA reagent and kit (Invitrogen) according to manufacturer’s instructions.

    Techniques: Transfection, Mass Spectrometry, Sequencing, Clone Assay, Infection, Isolation, Staining

    Increased biofilm formation and aggregation by Δ ldhA is associated with an increase in extracellular DNA and autolysis. (A) Biofilm formation by the wild-type, Δ ldhA , and Δ cap mutant strains in the presence or absence of DNase I. Δ cap was used as a positive control. Bacteria were resuspended in GC liquid supplemented with 1% Kellogg’s with or without DNase I to OD 600 of 0.05 and grown under static conditions for 24 h. Heat-treated DNase I was used as control (hi–DNase). Washed biofilms were stained with crystal violet and dissolved with acetic acid. The absorbance was measured at 630 nm. The experiment was performed three times in triplicate. (B) Autolysis under non-growth conditions. The wild-type and the Δ ldhA strains were resuspended in GC liquid supplemented with 1% Kellogg’s, to an OD 600 of 0.05 and grown for 3 h. Bacteria were centrifuged, washed with PBS, and resuspended in 30 mM Tris-HCL buffers at pH 6 or 8 to an OD 600 of 1. Absorbance values were acquired every 10 min for the first hour and at 20 min intervals during the second hour. The values were used to calculate the percentage of initial turbidity. One representative experiment, performed in duplicate, out of four is shown. Detection of eDNA in the culture supernatants (C) and in biofilms (D) of the wild-type and Δ ldhA strains using a fluorescence-based Quant-IT PicoGreen dsDNA assay kit. Experiments were performed three times in triplicate. (E) Sedimentation of wild-type and Δ ldhA aggregates grown in the presence or absence of DNase I. Bacteria were grown for 3 h in the presence or absence of DNase I under shaking conditions at 37°C and 5% CO 2 and then moved to static conditions at room temperature. The absorbance (OD 600 ) of the top layer of the culture was measured every 20 min. Data are presented as relative values compared to the 0 min time point (set to 1). Experiments were performed three times. Unless stated, the bars represent the means, with error bars representing the standard deviations. ∗ p

    Journal: Frontiers in Microbiology

    Article Title: Deletion of D-Lactate Dehydrogenase A in Neisseria meningitidis Promotes Biofilm Formation Through Increased Autolysis and Extracellular DNA Release

    doi: 10.3389/fmicb.2019.00422

    Figure Lengend Snippet: Increased biofilm formation and aggregation by Δ ldhA is associated with an increase in extracellular DNA and autolysis. (A) Biofilm formation by the wild-type, Δ ldhA , and Δ cap mutant strains in the presence or absence of DNase I. Δ cap was used as a positive control. Bacteria were resuspended in GC liquid supplemented with 1% Kellogg’s with or without DNase I to OD 600 of 0.05 and grown under static conditions for 24 h. Heat-treated DNase I was used as control (hi–DNase). Washed biofilms were stained with crystal violet and dissolved with acetic acid. The absorbance was measured at 630 nm. The experiment was performed three times in triplicate. (B) Autolysis under non-growth conditions. The wild-type and the Δ ldhA strains were resuspended in GC liquid supplemented with 1% Kellogg’s, to an OD 600 of 0.05 and grown for 3 h. Bacteria were centrifuged, washed with PBS, and resuspended in 30 mM Tris-HCL buffers at pH 6 or 8 to an OD 600 of 1. Absorbance values were acquired every 10 min for the first hour and at 20 min intervals during the second hour. The values were used to calculate the percentage of initial turbidity. One representative experiment, performed in duplicate, out of four is shown. Detection of eDNA in the culture supernatants (C) and in biofilms (D) of the wild-type and Δ ldhA strains using a fluorescence-based Quant-IT PicoGreen dsDNA assay kit. Experiments were performed three times in triplicate. (E) Sedimentation of wild-type and Δ ldhA aggregates grown in the presence or absence of DNase I. Bacteria were grown for 3 h in the presence or absence of DNase I under shaking conditions at 37°C and 5% CO 2 and then moved to static conditions at room temperature. The absorbance (OD 600 ) of the top layer of the culture was measured every 20 min. Data are presented as relative values compared to the 0 min time point (set to 1). Experiments were performed three times. Unless stated, the bars represent the means, with error bars representing the standard deviations. ∗ p

    Article Snippet: Briefly, bacteria were resuspended to an OD600 of 0.05 and grown for 3 h. The bacteria were centrifuged at 13,000 rpm for 3 min, and the supernatants were collected and incubated with Quant-iT PicoGreen dsDNA dye (Thermo Fisher Scientific) at a 1:1 ratio.

    Techniques: Mutagenesis, Positive Control, Staining, Fluorescence, Picogreen Assay, Sedimentation

    DNA yields and purity from 4 PBs using 4 DNA extraction methods. Note: (A) DNA quantified with PicoGreen dsDNA Kit. (B) DNA qualified by OD260/ OD280 with NanoDrop. Values presented are means (mean ± SD) of 3 independent replicates. The horizontal line shows the ratio at 1.8, which is the index of optimal DNA purity. Significant differences ( p

    Journal: Bioengineered

    Article Title: Suitability of various DNA extraction methods for a traditional Chinese paocai system

    doi: 10.1080/21655979.2017.1300736

    Figure Lengend Snippet: DNA yields and purity from 4 PBs using 4 DNA extraction methods. Note: (A) DNA quantified with PicoGreen dsDNA Kit. (B) DNA qualified by OD260/ OD280 with NanoDrop. Values presented are means (mean ± SD) of 3 independent replicates. The horizontal line shows the ratio at 1.8, which is the index of optimal DNA purity. Significant differences ( p

    Article Snippet: The gDNA yields and quality were assessed using a Quant-iTTM PicoGreen® dsDNA Kit (Molecular Probes, Inc., Eugene, OR) and measuring absorbance at 260 and 280 nm using a spectrophotometer (NanoDrop2000TM; Thermo Scientific Inc., West Palm Beach, FL).

    Techniques: DNA Extraction

    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

    Confocal Laser Scanning Microscope (CLSM) imaging. A : Gel.NPs labeled with for Rhodamine red dye excited at 633 nm. B : NS2 gene labeled with Quantifluor exited at 514 nm. C : labeled NS2 /Gel.NPs conjugate excited simultaneously at both 633 nm and 514 nm.

    Journal: PLoS ONE

    Article Title: Gelatin nanoparticles enhance delivery of hepatitis C virus recombinant NS2 gene

    doi: 10.1371/journal.pone.0181723

    Figure Lengend Snippet: Confocal Laser Scanning Microscope (CLSM) imaging. A : Gel.NPs labeled with for Rhodamine red dye excited at 633 nm. B : NS2 gene labeled with Quantifluor exited at 514 nm. C : labeled NS2 /Gel.NPs conjugate excited simultaneously at both 633 nm and 514 nm.

    Article Snippet: LB broth Miller (Luria-Bertani, Amresco,US), Ampicillin (Sigma-Aldrich, US), Kanamycin (Sigma-Aldrich, US), Isopropyl b-D-thiogalactoside (IPTG) (≥99% (TLC), ≤0.1% Dioxane, Sigma-Aldrich, US), PBS (Sigma, US), PMSF (Sigma, Ltd. Dorset, England), 8 M urea (Sigma, US), 20 mM β-mercaptoethanol (β-ME) (Sigma-Aldrich,US) Ni2+/nitrilotriacetate (NTA)–agarose (Qiagen Inc., Valencia, USA), Bovine serum albumin (Sigma, US), Tris-buffered saline (Sigma, US), Horseradish peroxidase (HRP)-labeled Protein A (Sigma, Ltd. Dorset, England), ECL Plus™ Western Blotting Reagents from GE Healthcare (formerly Amersham Biosciences), Rhodamine red dye (PowerPlex® 16 BIO System, Promega) and Quantifluor (QuantiFluor® dsDNA System, Promega), High Pure PCR Purification Kit (Biobasic Inc., Ontario, Canada), QIAexpressionest kit (Qiagen Inc., Valencia, USA), Proteins and Molecular weight standards (Sigma-Aldrich, US), Nitrocellulose membranes (Wattman, US), QIAGEN EZ Competent Cells (Qiagen Inc., Valencia, USA), QIAprep SpinMiniprep kit (Qiagen Inc., Valencia, USA), SphI and HindIII (Roche), QIAGEN® PCR Cloning kit (Qiagen Inc., Valencia, USA), GoTaq® Green Master Mix (Promega).

    Techniques: Laser-Scanning Microscopy, Confocal Laser Scanning Microscopy, Imaging, Labeling

    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

    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

    TRIM29 binds and colocalizes with STING in the cytosol. a Immunoblot analysis of endogenous proteins of TRIM29 and STING precipitated with anti-STING or immunoglobulin G (IgG; control) from whole-cell lysates of BMDCs from WT mice stimulated without (No) or with HSV-60 (2.5 μg/ml) for 8 h, and then with MG132 treatment for 3 h. Input, 20% of the BMDCs lysate. b Full-length STING and serial truncations of STING with deletion of various domains ( top ). Immunoblot analysis of purified HA-tagged TRIM29 with anti-HA ( bottom blot ), and immunoblot analysis of purified HA-tagged full-length TRIM29 with anti-HA ( middle blot ) or purified Myc-tagged full-length STING and STING truncation mutants alone with anti-Myc ( top blot ) after incubation with Myc-tagged full-length STING and STING truncation mutants and immunoprecipitation with anti-Myc ( top and middle blots ). c Full-length TRIM29 and serial truncations of TRIM29 with deletion (Δ) of various domains ( left margin ); numbers at ends indicate amino-acid positions ( top ). Below , immunoblot analysis of purified Myc-tagged STING with anti-Myc ( bottom blot ), and immunoblot analysis (with anti-HA) of purified HA-tagged full-length TRIM29 and TRIM29 truncation mutants alone ( top blot ) or after incubation with Myc-tagged STING and immunoprecipitation with anti-Myc ( middle blot ). d Colocalization of endogenous TRIM29 and STING in BEAS-2B cells. Confocal microscopy of BEAS-2B cells without stimulation, or stimulated with dsDNA HSV-60 for 4 h. STING was stained with Rabbit anti-STING polyclonal antibody (Cat: 19851-1-AP, Proteintech), followed by Alexa Fluor 488 goat anti-rabbit secondary antibody ( green ), while TRIM29 was stained with mouse anti-TRIM29 monoclonal antibody (sc-166707, Santa Cruz), followed by Alexa Fluor 594 goat anti-mouse secondary antibody ( red ). DAPI (4′,6-diamidino-2-phenylindole) served as the nuclei marker ( blue ). Scale bars represent 20 µm. The position of protein markers (shown in kDa) is indicated on the right . Data are representative of three independent experiments

    Journal: Nature Communications

    Article Title: TRIM29 promotes DNA virus infections by inhibiting innate immune response

    doi: 10.1038/s41467-017-00101-w

    Figure Lengend Snippet: TRIM29 binds and colocalizes with STING in the cytosol. a Immunoblot analysis of endogenous proteins of TRIM29 and STING precipitated with anti-STING or immunoglobulin G (IgG; control) from whole-cell lysates of BMDCs from WT mice stimulated without (No) or with HSV-60 (2.5 μg/ml) for 8 h, and then with MG132 treatment for 3 h. Input, 20% of the BMDCs lysate. b Full-length STING and serial truncations of STING with deletion of various domains ( top ). Immunoblot analysis of purified HA-tagged TRIM29 with anti-HA ( bottom blot ), and immunoblot analysis of purified HA-tagged full-length TRIM29 with anti-HA ( middle blot ) or purified Myc-tagged full-length STING and STING truncation mutants alone with anti-Myc ( top blot ) after incubation with Myc-tagged full-length STING and STING truncation mutants and immunoprecipitation with anti-Myc ( top and middle blots ). c Full-length TRIM29 and serial truncations of TRIM29 with deletion (Δ) of various domains ( left margin ); numbers at ends indicate amino-acid positions ( top ). Below , immunoblot analysis of purified Myc-tagged STING with anti-Myc ( bottom blot ), and immunoblot analysis (with anti-HA) of purified HA-tagged full-length TRIM29 and TRIM29 truncation mutants alone ( top blot ) or after incubation with Myc-tagged STING and immunoprecipitation with anti-Myc ( middle blot ). d Colocalization of endogenous TRIM29 and STING in BEAS-2B cells. Confocal microscopy of BEAS-2B cells without stimulation, or stimulated with dsDNA HSV-60 for 4 h. STING was stained with Rabbit anti-STING polyclonal antibody (Cat: 19851-1-AP, Proteintech), followed by Alexa Fluor 488 goat anti-rabbit secondary antibody ( green ), while TRIM29 was stained with mouse anti-TRIM29 monoclonal antibody (sc-166707, Santa Cruz), followed by Alexa Fluor 594 goat anti-mouse secondary antibody ( red ). DAPI (4′,6-diamidino-2-phenylindole) served as the nuclei marker ( blue ). Scale bars represent 20 µm. The position of protein markers (shown in kDa) is indicated on the right . Data are representative of three independent experiments

    Article Snippet: Reagents The dsDNA from vaccinia virus (VACV-70, Catalog: tlrl-vav70n), dsDNA from herpes simplex virus type 1 (HSV-60, Catalog: tlrl-hsv60n), poly(dG:dC) (Catalog: tlrl-pgcc), c-di-GMP (Catalog: tlrl-nacdg23), cGAMP (Catalog: tlrl-nacga23), R848 (TLR7/8 agonist, Catalog: tlrl-r848), CpG A (Catalog: tlrl-2216), CpG B (Catalog: tlrl-2006), and zymosan (Catalog: tlrl-zyn) were from Invivogen.

    Techniques: Mouse Assay, Purification, Incubation, Immunoprecipitation, Confocal Microscopy, Staining, Marker

    TRIM29 plays an important role in regulating IFN-I production in dendritic cells. a Immunoblot analysis of TRIM29 in human monocyte-derived dendritic cells (MonoDC) treated with control shRNA with scrambled sequence (sh-Ctrl), shRNA targeting mRNA encoding TRIM29 (two shRNAs: T29-a and T29-b). GAPDH serves as a loading control throughout. b ELISA of IFN-α and IFN-β in MonoDCs treated with scrambled shRNA (sh-Ctrl) and left unstimulated (Mock) or treated with shRNA as above and then stimulated for 16 h with dsDNA derived from herpes simplex virus type 1 (HSV-60, 2.5 μg/ml) or cGAMP (1.0 μg/ml) delivered by Lipofectamine 3000. c Immunoblot analysis of TRIM29 in mouse D2SC cells treated with control shRNA with scrambled sequence (sh-Ctrl), shRNA targeting mRNA encoding TRIM29 (two shRNAs: T29-#1 and T29-#2). GAPDH serves as a loading control throughout. d ELISA of IFN-α and IFN-β in D2SC cells treated with scrambled shRNA (sh-Ctrl) and left unstimulated (Mock) or treated with shRNA as above and then stimulated for 16 h with dsDNA from vaccinia virus (VACV-70, 2.5 μg/ml) or cGAMP (1.0 μg/ml) delivered by Lipofectamine 3000. e ELISA of IFN-α, IFN-β, and IL-6 in BMDCs from WT and Trim29 -KO mice after 16 h of stimulation with dsDNA from vaccinia virus (VACV-70, 2.5 μg/ml), dsDNA from HSV-1 virus (HSV-60, 2.5 μg/ml), c-di-GMP (2.5 μg/ml), or cGAMP (1.0 μg/ml) delivered by Lipofectamine 3000. f ELISA of IFN-β in BMDCs from WT and Trim29 -KO mice mock infected or infected with HSV-1 or adenovirus (adeno) at multiplicity of infection (MOI) of 5 for 6, 12, or 20. Each symbol represents the value from independent experimental cells in each well; small horizontal lines indicate the average of triplicates. Mock, cells without stimulation or infection. * P

    Journal: Nature Communications

    Article Title: TRIM29 promotes DNA virus infections by inhibiting innate immune response

    doi: 10.1038/s41467-017-00101-w

    Figure Lengend Snippet: TRIM29 plays an important role in regulating IFN-I production in dendritic cells. a Immunoblot analysis of TRIM29 in human monocyte-derived dendritic cells (MonoDC) treated with control shRNA with scrambled sequence (sh-Ctrl), shRNA targeting mRNA encoding TRIM29 (two shRNAs: T29-a and T29-b). GAPDH serves as a loading control throughout. b ELISA of IFN-α and IFN-β in MonoDCs treated with scrambled shRNA (sh-Ctrl) and left unstimulated (Mock) or treated with shRNA as above and then stimulated for 16 h with dsDNA derived from herpes simplex virus type 1 (HSV-60, 2.5 μg/ml) or cGAMP (1.0 μg/ml) delivered by Lipofectamine 3000. c Immunoblot analysis of TRIM29 in mouse D2SC cells treated with control shRNA with scrambled sequence (sh-Ctrl), shRNA targeting mRNA encoding TRIM29 (two shRNAs: T29-#1 and T29-#2). GAPDH serves as a loading control throughout. d ELISA of IFN-α and IFN-β in D2SC cells treated with scrambled shRNA (sh-Ctrl) and left unstimulated (Mock) or treated with shRNA as above and then stimulated for 16 h with dsDNA from vaccinia virus (VACV-70, 2.5 μg/ml) or cGAMP (1.0 μg/ml) delivered by Lipofectamine 3000. e ELISA of IFN-α, IFN-β, and IL-6 in BMDCs from WT and Trim29 -KO mice after 16 h of stimulation with dsDNA from vaccinia virus (VACV-70, 2.5 μg/ml), dsDNA from HSV-1 virus (HSV-60, 2.5 μg/ml), c-di-GMP (2.5 μg/ml), or cGAMP (1.0 μg/ml) delivered by Lipofectamine 3000. f ELISA of IFN-β in BMDCs from WT and Trim29 -KO mice mock infected or infected with HSV-1 or adenovirus (adeno) at multiplicity of infection (MOI) of 5 for 6, 12, or 20. Each symbol represents the value from independent experimental cells in each well; small horizontal lines indicate the average of triplicates. Mock, cells without stimulation or infection. * P

    Article Snippet: Reagents The dsDNA from vaccinia virus (VACV-70, Catalog: tlrl-vav70n), dsDNA from herpes simplex virus type 1 (HSV-60, Catalog: tlrl-hsv60n), poly(dG:dC) (Catalog: tlrl-pgcc), c-di-GMP (Catalog: tlrl-nacdg23), cGAMP (Catalog: tlrl-nacga23), R848 (TLR7/8 agonist, Catalog: tlrl-r848), CpG A (Catalog: tlrl-2216), CpG B (Catalog: tlrl-2006), and zymosan (Catalog: tlrl-zyn) were from Invivogen.

    Techniques: Derivative Assay, shRNA, Sequencing, Enzyme-linked Immunosorbent Assay, Mouse Assay, Infection

    Peptide inhibition of MAbs binding to EBNA‐1 and dsDNA. 1D2 (5 μg/ml) and 3D4 (0.05 μg/ml) were incubated with increasing concentrations of peptides PFM‐15 or GC‐15 and examined by ELISA for binding to EBNA‐1 (A and B, respectively). 1D2 (5 μg/ml) and 3D4 (2.5 μg/ml) were incubated with increasing concentrations of peptides PFM‐15 or GC‐15 and examined by ELISA for binding dsDNA (C and D, respectively). ELISAs are representative of two experiments. ELISA values were obtained in triplicate. Error bars represent standard deviations of triplicate values.

    Journal: Immunity, Inflammation and Disease

    Article Title: Mapping an epitope in EBNA‐1 that is recognized by monoclonal antibodies to EBNA‐1 that cross‐react with dsDNA

    doi: 10.1002/iid3.119

    Figure Lengend Snippet: Peptide inhibition of MAbs binding to EBNA‐1 and dsDNA. 1D2 (5 μg/ml) and 3D4 (0.05 μg/ml) were incubated with increasing concentrations of peptides PFM‐15 or GC‐15 and examined by ELISA for binding to EBNA‐1 (A and B, respectively). 1D2 (5 μg/ml) and 3D4 (2.5 μg/ml) were incubated with increasing concentrations of peptides PFM‐15 or GC‐15 and examined by ELISA for binding dsDNA (C and D, respectively). ELISAs are representative of two experiments. ELISA values were obtained in triplicate. Error bars represent standard deviations of triplicate values.

    Article Snippet: Peptide inhibition ELISAs For peptide inhibition of MAb binding to dsDNA, 3D4 (2.5 μg/ml) and 16D2 (5 μg/ml) were pre‐incubated for 1 h at 37°C with increasing concentrations of peptide PFM‐15 or GC‐15 (concentrations of peptide ranging from 0.1 μg/ml to 100 μg/ml) and then transferred to pre‐blocked dsDNA coated Immulon‐2 plates (Dynatech Laboratories Inc., Chantilly, VA).

    Techniques: Inhibition, Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay

    IgG antibody to EBNA‐1 cross‐reacts with dsDNA. IgG MAb, 3D4 binds strongly to EBNA‐1 (A) and cross‐reacts with dsDNA (B) as determined by ELISA. ELISAs are representative of three experiments. ELISA values were obtained in triplicate. Error bars indicate standard deviations of triplicates.

    Journal: Immunity, Inflammation and Disease

    Article Title: Mapping an epitope in EBNA‐1 that is recognized by monoclonal antibodies to EBNA‐1 that cross‐react with dsDNA

    doi: 10.1002/iid3.119

    Figure Lengend Snippet: IgG antibody to EBNA‐1 cross‐reacts with dsDNA. IgG MAb, 3D4 binds strongly to EBNA‐1 (A) and cross‐reacts with dsDNA (B) as determined by ELISA. ELISAs are representative of three experiments. ELISA values were obtained in triplicate. Error bars indicate standard deviations of triplicates.

    Article Snippet: Peptide inhibition ELISAs For peptide inhibition of MAb binding to dsDNA, 3D4 (2.5 μg/ml) and 16D2 (5 μg/ml) were pre‐incubated for 1 h at 37°C with increasing concentrations of peptide PFM‐15 or GC‐15 (concentrations of peptide ranging from 0.1 μg/ml to 100 μg/ml) and then transferred to pre‐blocked dsDNA coated Immulon‐2 plates (Dynatech Laboratories Inc., Chantilly, VA).

    Techniques: Enzyme-linked Immunosorbent Assay

    16D2 cross‐reacts with dsDNA. 16D2 was tested by ELISA for binding to EBNA‐1 (A) and dsDNA (B). Binding to dsDNA was confirmed by immunostaining of the kinetoplast of Crithidia luciliae (C). Immunostaining of an isotype control MAb is shown in D (D). 16D2 was tested by ELISA for reactivity with a panel of antigens; Sm, LPS, BSA, and proteinase 3 (PR3) (E). Results are representative of three experiments. ELISA values were obtained in triplicate. Error bars indicate standard deviations of triplicate values.

    Journal: Immunity, Inflammation and Disease

    Article Title: Mapping an epitope in EBNA‐1 that is recognized by monoclonal antibodies to EBNA‐1 that cross‐react with dsDNA

    doi: 10.1002/iid3.119

    Figure Lengend Snippet: 16D2 cross‐reacts with dsDNA. 16D2 was tested by ELISA for binding to EBNA‐1 (A) and dsDNA (B). Binding to dsDNA was confirmed by immunostaining of the kinetoplast of Crithidia luciliae (C). Immunostaining of an isotype control MAb is shown in D (D). 16D2 was tested by ELISA for reactivity with a panel of antigens; Sm, LPS, BSA, and proteinase 3 (PR3) (E). Results are representative of three experiments. ELISA values were obtained in triplicate. Error bars indicate standard deviations of triplicate values.

    Article Snippet: Peptide inhibition ELISAs For peptide inhibition of MAb binding to dsDNA, 3D4 (2.5 μg/ml) and 16D2 (5 μg/ml) were pre‐incubated for 1 h at 37°C with increasing concentrations of peptide PFM‐15 or GC‐15 (concentrations of peptide ranging from 0.1 μg/ml to 100 μg/ml) and then transferred to pre‐blocked dsDNA coated Immulon‐2 plates (Dynatech Laboratories Inc., Chantilly, VA).

    Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Immunostaining

    MAbs that bind to the carboxyl region of EBNA‐1 but do not cross‐react with dsDNA, recognize a different epitope than the cross‐reactive antibodies. (A) The IgG isotype of the non‐cross‐reactive MAbs 2F8, 14E10, 4F6, and 4B8 were determined by ELISA. (B) The non cross‐reactive MAbs, were tested by ELISA at a range of concentrations, for binding to EBNA‐1. 3D4 was used as the reference standard. MAbs were tested by ELISA for binding to dsDNA (C), to the amino and carboxyl regions of EBNA‐1 (D) and to two truncated carboxyl fragments; aa 452–641 and aa 459–607 (E). MAb concentrations used were 12.5 μg/ml in (C) and 1.25 μg/ml in (D) and (E). ELISAs are representative of two experiments. ELISA values were obtained in triplicate. Error bars represent the standard deviations of triplicates.

    Journal: Immunity, Inflammation and Disease

    Article Title: Mapping an epitope in EBNA‐1 that is recognized by monoclonal antibodies to EBNA‐1 that cross‐react with dsDNA

    doi: 10.1002/iid3.119

    Figure Lengend Snippet: MAbs that bind to the carboxyl region of EBNA‐1 but do not cross‐react with dsDNA, recognize a different epitope than the cross‐reactive antibodies. (A) The IgG isotype of the non‐cross‐reactive MAbs 2F8, 14E10, 4F6, and 4B8 were determined by ELISA. (B) The non cross‐reactive MAbs, were tested by ELISA at a range of concentrations, for binding to EBNA‐1. 3D4 was used as the reference standard. MAbs were tested by ELISA for binding to dsDNA (C), to the amino and carboxyl regions of EBNA‐1 (D) and to two truncated carboxyl fragments; aa 452–641 and aa 459–607 (E). MAb concentrations used were 12.5 μg/ml in (C) and 1.25 μg/ml in (D) and (E). ELISAs are representative of two experiments. ELISA values were obtained in triplicate. Error bars represent the standard deviations of triplicates.

    Article Snippet: Peptide inhibition ELISAs For peptide inhibition of MAb binding to dsDNA, 3D4 (2.5 μg/ml) and 16D2 (5 μg/ml) were pre‐incubated for 1 h at 37°C with increasing concentrations of peptide PFM‐15 or GC‐15 (concentrations of peptide ranging from 0.1 μg/ml to 100 μg/ml) and then transferred to pre‐blocked dsDNA coated Immulon‐2 plates (Dynatech Laboratories Inc., Chantilly, VA).

    Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay

    Insertion of a loxP511 site. ( A ) The location of the wild-type and mutant loxP sites in the BAC backbone are indicated along with the extra mutant loxP511 site that was introduced into the BAC genomic insert via galK counterselection. The 95 kb region deleted by Cre-mediated recombination between the two loxP511 sites is indicated, and PCR primers used to confirm the deletion are shown as small arrows. ( B ) SpeI restriction analysis of six miniprep clones selected for the replacement of galK with a dsDNA oligo containing the mutant loxP511 site. Clones 3, 5 and 6 (circles) had the same restriction pattern as the unmodified BAC, indicating that DOG resistance occurred due to the intended homologous recombination event. Clones 1, 2 and 4 had large deletions and were not analyzed further. ( C ) SpeI restriction analysis of BAC miniprep DNA from clones 3, 5 and 6 after transformation into Cre-induced EL350 cells. Two clones from each parental clone were tested. The restriction pattern shows that the 95 kb region flanked by two loxP511 sites is deleted from all clones analyzed, confirming the correct insertion of loxP511 in clones 3, 5 and 6. ( D ) PCR analysis of the six clones from (C) with one primer mapping to the BAC backbone and the other to a position distal to the inserted loxP511 site.

    Journal: Nucleic Acids Research

    Article Title: Simple and highly efficient BAC recombineering using galK selection

    doi: 10.1093/nar/gni035

    Figure Lengend Snippet: Insertion of a loxP511 site. ( A ) The location of the wild-type and mutant loxP sites in the BAC backbone are indicated along with the extra mutant loxP511 site that was introduced into the BAC genomic insert via galK counterselection. The 95 kb region deleted by Cre-mediated recombination between the two loxP511 sites is indicated, and PCR primers used to confirm the deletion are shown as small arrows. ( B ) SpeI restriction analysis of six miniprep clones selected for the replacement of galK with a dsDNA oligo containing the mutant loxP511 site. Clones 3, 5 and 6 (circles) had the same restriction pattern as the unmodified BAC, indicating that DOG resistance occurred due to the intended homologous recombination event. Clones 1, 2 and 4 had large deletions and were not analyzed further. ( C ) SpeI restriction analysis of BAC miniprep DNA from clones 3, 5 and 6 after transformation into Cre-induced EL350 cells. Two clones from each parental clone were tested. The restriction pattern shows that the 95 kb region flanked by two loxP511 sites is deleted from all clones analyzed, confirming the correct insertion of loxP511 in clones 3, 5 and 6. ( D ) PCR analysis of the six clones from (C) with one primer mapping to the BAC backbone and the other to a position distal to the inserted loxP511 site.

    Article Snippet: Oligos for recombineering Oligos used to replace the galK -targeting cassette were obtained from Invitrogen. dsDNA was used and the oligos (sense and antisense) annealed in vitro : 10 μg of each oligo (sense and antisense) was mixed in an eppendorf tube in a total volume of 100 μl of 1× PCR buffer (Expand High Fidelity PCR kit, Roche Applied Science) and boiled for 5 min, allowed to cool to room temperature for 30 min, ethanol precipitated and resuspended in 100 μl ddH2 O to a final concentration of 200 ng/μl double-stranded oligo.

    Techniques: Mutagenesis, BAC Assay, Polymerase Chain Reaction, Clone Assay, Homologous Recombination, Transformation Assay

    Overview of the galK selection scheme. The result of the first targeting event is the insertion of constitutively active galK into a defined position on the BAC by selection on minimal medium containing galactose and chloramphenicol to select for the maintenance of the BAC. The bacteria are now phenotypically Gal + . Next, the galK cassette is replaced by a dsDNA oligo, a PCR product, or a cloned dsDNA fragment carrying a desired mutation (indicated by a star) and flanked by the same homology arms used in the first selection step. This is achieved by negative selection using minimal medium containing 2-deoxy-galactose (DOG) with glycerol as the sole carbon source. The bacteria become phenotypically Gal − . H1 and H2, homology arms 1 and 2, respectively; cat , chloramphenicol acetyl transferase gene; ori2, BAC origin of replication; galK , E.coli galactokinase gene driven by a minimal promoter.

    Journal: Nucleic Acids Research

    Article Title: Simple and highly efficient BAC recombineering using galK selection

    doi: 10.1093/nar/gni035

    Figure Lengend Snippet: Overview of the galK selection scheme. The result of the first targeting event is the insertion of constitutively active galK into a defined position on the BAC by selection on minimal medium containing galactose and chloramphenicol to select for the maintenance of the BAC. The bacteria are now phenotypically Gal + . Next, the galK cassette is replaced by a dsDNA oligo, a PCR product, or a cloned dsDNA fragment carrying a desired mutation (indicated by a star) and flanked by the same homology arms used in the first selection step. This is achieved by negative selection using minimal medium containing 2-deoxy-galactose (DOG) with glycerol as the sole carbon source. The bacteria become phenotypically Gal − . H1 and H2, homology arms 1 and 2, respectively; cat , chloramphenicol acetyl transferase gene; ori2, BAC origin of replication; galK , E.coli galactokinase gene driven by a minimal promoter.

    Article Snippet: Oligos for recombineering Oligos used to replace the galK -targeting cassette were obtained from Invitrogen. dsDNA was used and the oligos (sense and antisense) annealed in vitro : 10 μg of each oligo (sense and antisense) was mixed in an eppendorf tube in a total volume of 100 μl of 1× PCR buffer (Expand High Fidelity PCR kit, Roche Applied Science) and boiled for 5 min, allowed to cool to room temperature for 30 min, ethanol precipitated and resuspended in 100 μl ddH2 O to a final concentration of 200 ng/μl double-stranded oligo.

    Techniques: Selection, BAC Assay, Polymerase Chain Reaction, Clone Assay, Mutagenesis, Chloramphenicol Acetyltransferase Assay

    Morphology, proliferation and differentiation of BMSCs on the MPZs and control surfaces. a) Quant-iT dsDNA Assay of BMSCs on the samples. The assay was repeated twice and expressed as means ± s.d.. Significant differences were determined using a one-way analysis of variance (ANOVA) followed by LSD-t test. (**) and (*) indicates a statisctic difference at p

    Journal: Theranostics

    Article Title: Bone-Inspired Spatially Specific Piezoelectricity Induces Bone Regeneration

    doi: 10.7150/thno.19748

    Figure Lengend Snippet: Morphology, proliferation and differentiation of BMSCs on the MPZs and control surfaces. a) Quant-iT dsDNA Assay of BMSCs on the samples. The assay was repeated twice and expressed as means ± s.d.. Significant differences were determined using a one-way analysis of variance (ANOVA) followed by LSD-t test. (**) and (*) indicates a statisctic difference at p

    Article Snippet: Proliferation of BMSCs was determined using Quant-iT dsDNA Assay (PicoGreen, Invitrogen, USA).

    Techniques: dsDNA 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 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

    Reactivity of MOG-specific B cells. B cells were preincubated with 20 μ g of soluble MOG, albumin, dsDNA, insulin, or LPS before the addition of MOG-coated beads ( n = 3). (a) The frequency of MOG-BBR drops with the soluble MOG preincubation but not with albumin preincubation. (b) The recognition of MOG-coated beads decreased after the addition of soluble LPS (% of inhibition: 73%), dsDNA (11%), and insulin (35%). Competitive assay was tested in MS ( n = 3); the percentage of inhibition after soluble antigen addition was the same proportion as HI: LPS (78%), dsDNA (11%), and insulin (32%). (c) B cells were preincubated with Fab'2 anti-human IgG+IgA+IgM. The frequency of B cells recognizing MOG-coated beads was assessed on 3 HI. Fab'2 antibody modified the frequency of MOG-specific B cells (ns, P > 0.05, Wilcoxon test ).

    Journal: Journal of Immunology Research

    Article Title: Decreased Frequency of Circulating Myelin Oligodendrocyte Glycoprotein B Lymphocytes in Patients with Relapsing-Remitting Multiple Sclerosis

    doi: 10.1155/2015/673503

    Figure Lengend Snippet: Reactivity of MOG-specific B cells. B cells were preincubated with 20 μ g of soluble MOG, albumin, dsDNA, insulin, or LPS before the addition of MOG-coated beads ( n = 3). (a) The frequency of MOG-BBR drops with the soluble MOG preincubation but not with albumin preincubation. (b) The recognition of MOG-coated beads decreased after the addition of soluble LPS (% of inhibition: 73%), dsDNA (11%), and insulin (35%). Competitive assay was tested in MS ( n = 3); the percentage of inhibition after soluble antigen addition was the same proportion as HI: LPS (78%), dsDNA (11%), and insulin (32%). (c) B cells were preincubated with Fab'2 anti-human IgG+IgA+IgM. The frequency of B cells recognizing MOG-coated beads was assessed on 3 HI. Fab'2 antibody modified the frequency of MOG-specific B cells (ns, P > 0.05, Wilcoxon test ).

    Article Snippet: Briefly, soluble dsDNA (Sigma, France), LPS (Sigma), and insulin (Sigma) were added during 30 minutes on B cells prior to the incubation with MOG-coated beads.

    Techniques: Inhibition, Mass Spectrometry, Modification