ethidium bromide Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher ethidium bromide
    Ethidium Bromide, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10016 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ethidium bromide/product/Thermo Fisher
    Average 99 stars, based on 10016 article reviews
    Price from $9.99 to $1999.99
    ethidium bromide - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    99
    Millipore ethidium bromide
    Ethidium Bromide, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12697 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ethidium bromide/product/Millipore
    Average 99 stars, based on 12697 article reviews
    Price from $9.99 to $1999.99
    ethidium bromide - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    99
    Bio-Rad ethidium bromide
    Ethidium Bromide, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 19284 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ethidium bromide/product/Bio-Rad
    Average 99 stars, based on 19284 article reviews
    Price from $9.99 to $1999.99
    ethidium bromide - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher ethidium homodimer 1
    Ethidium Homodimer 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2814 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ethidium homodimer 1/product/Thermo Fisher
    Average 99 stars, based on 2814 article reviews
    Price from $9.99 to $1999.99
    ethidium homodimer 1 - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    92
    Alpha Innotech ethidium bromide
    (A) Reduced fitness of Ed N-522D relative to Ed N was also illustrated by coinfecting hsp72-expressing N2a-HSP 1 and control N2a-V 2 cells with each variant at a total MOI of 1.0, using progressively increased proportions of Ed N-522D (i.e., from a 1:1 ratio with Ed N to a 100:1 ratio). The proportion of each viral variant in cell-free progeny was based upon BsmAI restriction fragment length polymorphisms of RT-PCR amplicons derived from genomic RNA, quantifying <t>ethidium</t> bromide staining intensities of products resolved on 2% agarose gels. Increasing the relative amount of Ed N-522D/N in the inoculum to 20:1 resulted in increased recovery of Ed N-522D in the progeny, although further increasing Ed N-522D in the inoculum was without effect. Results are representative of two experimental trials. Controls for the infection were individual inoculations with Ed N or Ed N-522D virus, and reactions lacking RT were used as a negative control (-) for the RT-PCR. (B) Recovery of Ed N-522D in the progeny of mixed infections was also enhanced by transfecting cells with plasmid supporting the expression of N-522D. Cells were infected with a 1:1 ratio of Ed N-522D and Ed N (combined MOI of 1.0) at 2 h posttransfection. Negative controls included nontransfected cells (-) and cells transfected with empty plasmid vector (V). Western blot analysis of uninfected cell lysates processed in parallel showed expression of the N-522D protein. Infections were performed in triplicate, and the Ed N-522D/N RT-PCR signal ratio is expressed as the mean ± SD.
    Ethidium Bromide, supplied by Alpha Innotech, used in various techniques. Bioz Stars score: 92/100, based on 4081 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ethidium bromide/product/Alpha Innotech
    Average 92 stars, based on 4081 article reviews
    Price from $9.99 to $1999.99
    ethidium bromide - by Bioz Stars, 2020-11
    92/100 stars
      Buy from Supplier

    99
    Thermo Fisher ethidium bromide staining
    (A) Reduced fitness of Ed N-522D relative to Ed N was also illustrated by coinfecting hsp72-expressing N2a-HSP 1 and control N2a-V 2 cells with each variant at a total MOI of 1.0, using progressively increased proportions of Ed N-522D (i.e., from a 1:1 ratio with Ed N to a 100:1 ratio). The proportion of each viral variant in cell-free progeny was based upon BsmAI restriction fragment length polymorphisms of RT-PCR amplicons derived from genomic RNA, quantifying <t>ethidium</t> bromide staining intensities of products resolved on 2% agarose gels. Increasing the relative amount of Ed N-522D/N in the inoculum to 20:1 resulted in increased recovery of Ed N-522D in the progeny, although further increasing Ed N-522D in the inoculum was without effect. Results are representative of two experimental trials. Controls for the infection were individual inoculations with Ed N or Ed N-522D virus, and reactions lacking RT were used as a negative control (-) for the RT-PCR. (B) Recovery of Ed N-522D in the progeny of mixed infections was also enhanced by transfecting cells with plasmid supporting the expression of N-522D. Cells were infected with a 1:1 ratio of Ed N-522D and Ed N (combined MOI of 1.0) at 2 h posttransfection. Negative controls included nontransfected cells (-) and cells transfected with empty plasmid vector (V). Western blot analysis of uninfected cell lysates processed in parallel showed expression of the N-522D protein. Infections were performed in triplicate, and the Ed N-522D/N RT-PCR signal ratio is expressed as the mean ± SD.
    Ethidium Bromide Staining, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1564 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ethidium bromide staining/product/Thermo Fisher
    Average 99 stars, based on 1564 article reviews
    Price from $9.99 to $1999.99
    ethidium bromide staining - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    94
    Syngene ethidium bromide
    Differential enrichment of H3K9/14ac and H3K4me3 histone modifications at the VPAC1 promoter A. Flow cytometric analysis of mouse CD4 + T cells and CD45R + B cells. Left Panel : Non-adherent splenocytes were stained with anti-CD4-FITC pre- (red line) and post- (blue line) CD4 positive magnetic bead separation ( Materials and Methods ) with typical purities ≥93% (n=3). Right Panel : CD4 depleted non-adherent splenocytes were subsequently stained pre- (red line) and post- (blue line) CD45R-FITC positive magnetic bead separation ( Materials and Methods ) with typical purities ≥97% (n=3). B. Bar graph for Taqman qPCR measurements for VPAC1 from indicated cell type. A hash mark indicates a shift in the y-axis. Data is represented as means +/- SEM for relative VPAC1 levels normalized to HPRT and calculated by 2 (-ΔCt) formula from three independent experiments. The asterisk (*) represents a p≤ 0.05 as compared to CD4 + T cells. C-D. ChIP analysis was performed using purified CD4 + T and CD45R + B cells followed by quantitative SYBR green PCR using VPAC1 primer set 2 or genomic negative control. C. Amplification reactions from SYBR green qPCR using primer set 2 were separated on a 3% agarose gel, stained with <t>ethidium</t> bromide and visualized by a CCD camera (Syngene). This experiment was repeated twice with similar results. D. Bar graph showing fold-enrichment over IgG control of H3K9/14ac and H3K4me3 from the indicated cell types. Cycle thresholds (Ct) were subtracted from input Ct values to obtain relative ΔCt values. Fold increases were calculated by 2 -(ΔCt) formula with non specific IgG levels normalized to input arbitrarily set to 1 (not shown). Data is presented as means +/- SEM from three biologically independent experiments (*, p≤0.05).
    Ethidium Bromide, supplied by Syngene, used in various techniques. Bioz Stars score: 94/100, based on 2933 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ethidium bromide/product/Syngene
    Average 94 stars, based on 2933 article reviews
    Price from $9.99 to $1999.99
    ethidium bromide - by Bioz Stars, 2020-11
    94/100 stars
      Buy from Supplier

    94
    Kodak Corp ethidium bromide
    Kinetic analyses of OPI and interpretation of in vitro transposition reactions. The kinetics of the transposition reaction were analyzed in standard reactions containing 6.7 nM plasmid substrate and the indicated concentrations of the transposases. Photographs of <t>ethidium</t> bromide stained agarose gels are shown. ( A ) The effects of the ASO mechanism complicates the interpretation of the gels, particularly the kinetic analyses. In the reactions with 20 nM transposase about half of the substrate is converted to the nicked intermediate in the first 2 min. However, it takes an hour or more to consume the remainder of the substrate. This biphasic behavior arises from the ASO mechanism, which operates in any reaction that contains more than one dimer of transposase. Thus, even if the transposase was 100% active and the reaction was performed with the optimum ratio of one dimer to one transposon, only half of the substrate would react initially. At the start of the reaction half of the transposons would be occupied by a dimer and would react, a quarter would be occupied by two dimers and would suffer OPI and the other quarter would be completely unoccupied. The slow phase of consumption corresponds to the redistribution of dimers from transposons which initially suffered OPI to those that were completely unbound. Even under OPI conditions, when both ends of the transposon are occupied by transposase dimers, their occasional dissociation provides a window of opportunity for synapsis. OPI can therefore be overcome by extending the incubation period so that the accumulated windows of opportunity eventually suffice to complete the reaction. Thus, the reaction with 40 nM transposase goes on to reach completion during the 8 hr incubation, despite initially suffering from OPI. With 200 nM transposase, the extended incubation is unable to overcome OPI because the windows of opportunity provided by unoccupied transposon ends are shorter at this concentration. ( B ) The nicked substrate is more sensitive to OPI and is inhibited at only 40 nM transposase. This is because any free transposon ends, made available by the dissociation of a transposase dimer, are re-bound before they can achieve synapsis. In other words, the slow synapsis of the nicked substrate requires a longer window of opportunity than is available at 40 nM transposase. Time points and transposase concentrations as in part ( A ). ( C ) The RA104 transposase mutant is resistant to OPI because the higher OFF-rate provides more windows of opportunity for synapsis (see Figure 4C and text for details). Time points and transposase concentrations as in part ( A ). DOI: http://dx.doi.org/10.7554/eLife.00668.005
    Ethidium Bromide, supplied by Kodak Corp, used in various techniques. Bioz Stars score: 94/100, based on 2079 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ethidium bromide/product/Kodak Corp
    Average 94 stars, based on 2079 article reviews
    Price from $9.99 to $1999.99
    ethidium bromide - by Bioz Stars, 2020-11
    94/100 stars
      Buy from Supplier

    99
    Millipore ethidium bromide eb
    TiO 2 NPs induced mitochondrial proapoptotic factors and subsequent apoptosis in RAW 264.7 cells. Notes: ( A ) Mechanism depicting TiO 2 NPs induced BAX activation; ( B ) RT-PCR gel images of BAX, BIM, and PUMA; ( C ) Flow cytometry analysis of apoptosis; ( D ) Quantitative analysis of apoptotic cell death. Cells were incubated with indicated concentrations of TiO 2 NPs for 24 hours. RT-PCR reactions were performed and PCR products were separated by 1% agarose gel electrophoresis and bands were visualized under UVP Biospectrum-600 (Thermo Fisher Scientific, Waltham, MA, USA) after <t>ethidium</t> bromide staining (EtBr, Sigma-Aldrich, St Louis, MO, USA). Housekeeping gene GAPDH was used as loading control. Then, apoptosis was investigated using Muse™ flowcytometric method with Muse™ Annexin V and Dead Cell Kit. Untreated cells were considered as control in the experiments. Data are presented as the mean ± standard error of mean; *** P
    Ethidium Bromide Eb, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1533 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ethidium bromide eb/product/Millipore
    Average 99 stars, based on 1533 article reviews
    Price from $9.99 to $1999.99
    ethidium bromide eb - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    92
    Promega ethidium bromide
    Agarose gel electrophoresis of one-step RT-PCR amplified products of the IBV S1 gene. Commercial IBV vaccine (IBV H120, MA5, and 4/91) and field strains (“Moroccan 30” and “Moroccan 38”) were separated by electrophoresis on a 1.5 % agarose gel stained with <t>ethidium</t> bromide. Lane 1 100 bp DNA ladder; lane 2 Moroccan 30 strain; lane 3 negative control; lane 4 Moroccan 38 strain; lanes 5, 6 negative controls; lane 7 Ma5 commercial vaccine; lane 8 H120 commercial vaccine; lane 9 4/91 commercial vaccine. Product size, 700 bp. Bp base pairs, IBV infectious bronchitis virus, RT - PCR reverse transcriptase-polymerase chain reaction
    Ethidium Bromide, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 1798 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ethidium bromide/product/Promega
    Average 92 stars, based on 1798 article reviews
    Price from $9.99 to $1999.99
    ethidium bromide - by Bioz Stars, 2020-11
    92/100 stars
      Buy from Supplier

    93
    Vilber Lourmat ethidium bromide
    Generation of seamless vectors for λ-Int-mediated genomic recombination. ( A ) A schematic diagram showing λ-Int-mediated recombination between genomic att H4X (located in LINE-1 elements) and att P4X (present on a plasmid target vector), leading to site-specific integration of the entire plasmid into the human LINE-1 elements, then flanked by att L4X (left junction) and att R4X (right junction). ( B ) An illustration depicting in vitro intra-molecular att H4X x att P4X recombination of a substrate plasmid, resulting in the formation of a seamless vector carrying the att L4X sequence after releasing the undesired bacterial DNA catenane ring by endonucleolytic digest. The seamless vector is subsequently targeted at genomic att H4X, leading to the insertion of payload through a second λ-Int-mediated recombination event. ( C ) SDS PAGE gel image depicting different desalted fractions of partially purified C-terminally histidine-tagged Int-h/218 (41 kDa). L, Prestained protein ladder (10–250 kDa); E, first eluted fraction from desalting column; 1, 2, 3, 4, different desalted fractions of partially purified C-terminal histidine-tagged Int-h/218. ( D ) Representative analysis of an in vitro intra-molecular att H4X x att P4X recombination with reaction buffers containing increasing KCl concentrations as indicated (left). Supercoiled seamless vector (SC-SV) and open circular seamless vector (OC-SV) obtained after λ-Int-mediated recombination were resolved via agarose gel electrophoresis in the presence of <t>ethidium</t> bromide. This resulted in DNA bands for SC-SV and OC-SV, which migrated at ∼1.2 and ∼2.2 kb, respectively, whereas the linearized second catenane DNA (linear bacterial DNA) and the unrecombined substrate plasmid migrated at ∼3 and ∼5.0 kb, respectively, as indicated. Purified seamless vector is shown at the right.
    Ethidium Bromide, supplied by Vilber Lourmat, used in various techniques. Bioz Stars score: 93/100, based on 960 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ethidium bromide/product/Vilber Lourmat
    Average 93 stars, based on 960 article reviews
    Price from $9.99 to $1999.99
    ethidium bromide - by Bioz Stars, 2020-11
    93/100 stars
      Buy from Supplier

    93
    Stratagene ethidium bromide
    Cytokine expression in Marek's disease herpesvirus (MDV)-infected chicken splenocytes. Total RNAs were prepared from spleens prepared from 3-week-old control chickens (lane 1) and from chickens infected with JM16 (lane 2), SB-1 (lane 3) and HVT (lane 4), respectively. The RNAs were reverse transcribed using oligo dT(16) as primers and the cDNAs were amplified by the polymerase chain reaction (PCR) using specific primers for interferon-γ (IFN-γ), inducible nitric oxide synthase (iNOS), interleukin (IL)-2, IL-6 and IL-8. β-actin was used as a control. The amplified DNA fragments were analysed in a 1·5% gel, stained with <t>ethidium</t> bromide and visualized on the Eagle Eye II Still Video System (Stratagene). All images were photographed as negative images except for IL-8. The sizes of the PCR fragments or expected fragments (IL-6) are indicated.
    Ethidium Bromide, supplied by Stratagene, used in various techniques. Bioz Stars score: 93/100, based on 859 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ethidium bromide/product/Stratagene
    Average 93 stars, based on 859 article reviews
    Price from $9.99 to $1999.99
    ethidium bromide - by Bioz Stars, 2020-11
    93/100 stars
      Buy from Supplier

    94
    FUJIFILM ethidium bromide
    Double-strands breaks do not accumulate in rad52 cells treated with MMS. Chromosomes were visualized by <t>ethidium</t> bromide or Southern hybridization. ( A ) After exposing rad52 cells to 0.05% MMS for 30 min heating to 55°C, which causes massive chromosome fragmentation. ( B ) No chromosome fragmentation was observed in rad52 cells treated with either 0.05 or 0.1% MMS for 30–90 min.
    Ethidium Bromide, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 94/100, based on 1046 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ethidium bromide/product/FUJIFILM
    Average 94 stars, based on 1046 article reviews
    Price from $9.99 to $1999.99
    ethidium bromide - by Bioz Stars, 2020-11
    94/100 stars
      Buy from Supplier

    96
    Millipore ethidium homodimer
    Double-strands breaks do not accumulate in rad52 cells treated with MMS. Chromosomes were visualized by <t>ethidium</t> bromide or Southern hybridization. ( A ) After exposing rad52 cells to 0.05% MMS for 30 min heating to 55°C, which causes massive chromosome fragmentation. ( B ) No chromosome fragmentation was observed in rad52 cells treated with either 0.05 or 0.1% MMS for 30–90 min.
    Ethidium Homodimer, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 135 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ethidium homodimer/product/Millipore
    Average 96 stars, based on 135 article reviews
    Price from $9.99 to $1999.99
    ethidium homodimer - by Bioz Stars, 2020-11
    96/100 stars
      Buy from Supplier

    92
    Amresco ethidium bromide
    CAY10593 impairs ATP-induced <t>ethidium</t> + uptake into RPMI 8226 cells in a non-competitive-like manner. RPMI 8226 cells in NaCl medium were pre-incubated at 37 °C for 15 min in the presence of DMSO, or 2 μM or 10 μM
    Ethidium Bromide, supplied by Amresco, used in various techniques. Bioz Stars score: 92/100, based on 426 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ethidium bromide/product/Amresco
    Average 92 stars, based on 426 article reviews
    Price from $9.99 to $1999.99
    ethidium bromide - by Bioz Stars, 2020-11
    92/100 stars
      Buy from Supplier

    93
    Fisher Scientific ethidium bromide
    Platelets are not associated with dying parasites during nonresolving, eCM-eliciting Pb A in C57BL/6 mice. The analyses were performed in thin blood films fluorescence labeled with anti-CD41 (blue: platelets) and <t>ethidium</t> bromide (red: parasites) and TUNEL (green). (A) Platelet effector-to-target (iRBC) ratios (filled blue square), platelet:uRBC (open green square) ratios, and parasitemia (filled red circle) on left axis and platelet counts/mL (open blue circle) on right axis. (B) Percent PbA parasitemia for all RBCs (red bars) ([# iRBC /# RBC ]%) and considering only RBCs with bound platelets (blue bars) ([# platelet:iRBC /# plateletRBC ]%). Platelet:RBC indicates a platelet RBC conjugate; the RBC maybe uninfected (uRBC) or infected (iRBC). (C) Percentage of RBCs with bound platelets with uninfected (red bars) ([# platelet:uRBC /# plateletRBC ]%) or infected (blue bars) RBCs: ([# platelet:iRBC /# platelet:RBC ]%). (D) The percentage of iRBCs with an adherent platelet ([# platelet:iRBC /# iRBCs ]%). (E) Representative thin blood film made on day 6PI and fluorescently labeled; blue arrows indicate platelets, red arrows indicate parasites without TUNEL, and yellow arrows indicate parasites with TUNEL (green+red). (F) Percentage on days 4 and 6PI of iRBCs exhibiting TUNEL + labeling (red bar, x-axis) ([(# TUNEL + iRBCs )/# iRBCs ]%) and TUNEL – (green bar, x-axis) ([(# TUNEL − iRBCs )/# iRBCs ]%) with the percentage of each iRBC + , TUNEL + or iRBC + , TUNEL – ([(# platelet:TUNEL − iRBCs )/# iRBCs ]%) exhibiting an adherent platelet shown as an inset (blue bar). This experiment was repeated twice (n = 5) and verified by flow cytometry. Values are average ± SEM.
    Ethidium Bromide, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 93/100, based on 492 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ethidium bromide/product/Fisher Scientific
    Average 93 stars, based on 492 article reviews
    Price from $9.99 to $1999.99
    ethidium bromide - by Bioz Stars, 2020-11
    93/100 stars
      Buy from Supplier

    99
    Thermo Fisher ultrapure ethidium bromide
    Platelets are not associated with dying parasites during nonresolving, eCM-eliciting Pb A in C57BL/6 mice. The analyses were performed in thin blood films fluorescence labeled with anti-CD41 (blue: platelets) and <t>ethidium</t> bromide (red: parasites) and TUNEL (green). (A) Platelet effector-to-target (iRBC) ratios (filled blue square), platelet:uRBC (open green square) ratios, and parasitemia (filled red circle) on left axis and platelet counts/mL (open blue circle) on right axis. (B) Percent PbA parasitemia for all RBCs (red bars) ([# iRBC /# RBC ]%) and considering only RBCs with bound platelets (blue bars) ([# platelet:iRBC /# plateletRBC ]%). Platelet:RBC indicates a platelet RBC conjugate; the RBC maybe uninfected (uRBC) or infected (iRBC). (C) Percentage of RBCs with bound platelets with uninfected (red bars) ([# platelet:uRBC /# plateletRBC ]%) or infected (blue bars) RBCs: ([# platelet:iRBC /# platelet:RBC ]%). (D) The percentage of iRBCs with an adherent platelet ([# platelet:iRBC /# iRBCs ]%). (E) Representative thin blood film made on day 6PI and fluorescently labeled; blue arrows indicate platelets, red arrows indicate parasites without TUNEL, and yellow arrows indicate parasites with TUNEL (green+red). (F) Percentage on days 4 and 6PI of iRBCs exhibiting TUNEL + labeling (red bar, x-axis) ([(# TUNEL + iRBCs )/# iRBCs ]%) and TUNEL – (green bar, x-axis) ([(# TUNEL − iRBCs )/# iRBCs ]%) with the percentage of each iRBC + , TUNEL + or iRBC + , TUNEL – ([(# platelet:TUNEL − iRBCs )/# iRBCs ]%) exhibiting an adherent platelet shown as an inset (blue bar). This experiment was repeated twice (n = 5) and verified by flow cytometry. Values are average ± SEM.
    Ultrapure Ethidium Bromide, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ultrapure ethidium bromide/product/Thermo Fisher
    Average 99 stars, based on 142 article reviews
    Price from $9.99 to $1999.99
    ultrapure ethidium bromide - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    Image Search Results


    (A) Reduced fitness of Ed N-522D relative to Ed N was also illustrated by coinfecting hsp72-expressing N2a-HSP 1 and control N2a-V 2 cells with each variant at a total MOI of 1.0, using progressively increased proportions of Ed N-522D (i.e., from a 1:1 ratio with Ed N to a 100:1 ratio). The proportion of each viral variant in cell-free progeny was based upon BsmAI restriction fragment length polymorphisms of RT-PCR amplicons derived from genomic RNA, quantifying ethidium bromide staining intensities of products resolved on 2% agarose gels. Increasing the relative amount of Ed N-522D/N in the inoculum to 20:1 resulted in increased recovery of Ed N-522D in the progeny, although further increasing Ed N-522D in the inoculum was without effect. Results are representative of two experimental trials. Controls for the infection were individual inoculations with Ed N or Ed N-522D virus, and reactions lacking RT were used as a negative control (-) for the RT-PCR. (B) Recovery of Ed N-522D in the progeny of mixed infections was also enhanced by transfecting cells with plasmid supporting the expression of N-522D. Cells were infected with a 1:1 ratio of Ed N-522D and Ed N (combined MOI of 1.0) at 2 h posttransfection. Negative controls included nontransfected cells (-) and cells transfected with empty plasmid vector (V). Western blot analysis of uninfected cell lysates processed in parallel showed expression of the N-522D protein. Infections were performed in triplicate, and the Ed N-522D/N RT-PCR signal ratio is expressed as the mean ± SD.

    Journal: Journal of Virology

    Article Title: A Single Codon in the Nucleocapsid Protein C Terminus Contributes to In Vitro and In Vivo Fitness of Edmonston Measles Virus

    doi: 10.1128/JVI.80.6.2904-2912.2006

    Figure Lengend Snippet: (A) Reduced fitness of Ed N-522D relative to Ed N was also illustrated by coinfecting hsp72-expressing N2a-HSP 1 and control N2a-V 2 cells with each variant at a total MOI of 1.0, using progressively increased proportions of Ed N-522D (i.e., from a 1:1 ratio with Ed N to a 100:1 ratio). The proportion of each viral variant in cell-free progeny was based upon BsmAI restriction fragment length polymorphisms of RT-PCR amplicons derived from genomic RNA, quantifying ethidium bromide staining intensities of products resolved on 2% agarose gels. Increasing the relative amount of Ed N-522D/N in the inoculum to 20:1 resulted in increased recovery of Ed N-522D in the progeny, although further increasing Ed N-522D in the inoculum was without effect. Results are representative of two experimental trials. Controls for the infection were individual inoculations with Ed N or Ed N-522D virus, and reactions lacking RT were used as a negative control (-) for the RT-PCR. (B) Recovery of Ed N-522D in the progeny of mixed infections was also enhanced by transfecting cells with plasmid supporting the expression of N-522D. Cells were infected with a 1:1 ratio of Ed N-522D and Ed N (combined MOI of 1.0) at 2 h posttransfection. Negative controls included nontransfected cells (-) and cells transfected with empty plasmid vector (V). Western blot analysis of uninfected cell lysates processed in parallel showed expression of the N-522D protein. Infections were performed in triplicate, and the Ed N-522D/N RT-PCR signal ratio is expressed as the mean ± SD.

    Article Snippet: Bands were resolved by 2.0% agarose gel electrophoresis and stained with ethidium bromide, and ethidium bromide staining intensity was quantified using an Alpha Imager (Alpha Innotech).

    Techniques: Expressing, Variant Assay, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Staining, Infection, Negative Control, Plasmid Preparation, Transfection, Western Blot

    Relative fitness of Ed N-522D compared to Ed N based upon coinfection of hsp72-expressing N2a-HSP 1 and control N2a-V 2 cells. The inocula contained a 1:1 ratio of each virus for a combined MOI of 0.1, 1.0, or 10.0. The proportion of Ed N and Ed N-522D in the viral progeny was based upon BsmAI restriction fragment length polymorphisms of amplicons derived from RT-PCR of cell-free viral genomic RNA. Yield of amplicon was based upon ethidium bromide staining intensity of BsmAI-digested products resolved by gel electrophoresis (A). The ratio of the 133-/134- and the 267-bp products was used as a correlate of the relative fitness of Ed N-522D compared to Ed N (B). Results reflect the means of three experimental trials ± SD. Differences in ratios calculated from infection of N2a-HSP 1 and N2a-V 2 cells were not statistically significant. Controls for the infection were individual inoculations with Ed N or Ed N-522D virus, and reactions lacking RT were used as a negative control (-) for the RT-PCR.

    Journal: Journal of Virology

    Article Title: A Single Codon in the Nucleocapsid Protein C Terminus Contributes to In Vitro and In Vivo Fitness of Edmonston Measles Virus

    doi: 10.1128/JVI.80.6.2904-2912.2006

    Figure Lengend Snippet: Relative fitness of Ed N-522D compared to Ed N based upon coinfection of hsp72-expressing N2a-HSP 1 and control N2a-V 2 cells. The inocula contained a 1:1 ratio of each virus for a combined MOI of 0.1, 1.0, or 10.0. The proportion of Ed N and Ed N-522D in the viral progeny was based upon BsmAI restriction fragment length polymorphisms of amplicons derived from RT-PCR of cell-free viral genomic RNA. Yield of amplicon was based upon ethidium bromide staining intensity of BsmAI-digested products resolved by gel electrophoresis (A). The ratio of the 133-/134- and the 267-bp products was used as a correlate of the relative fitness of Ed N-522D compared to Ed N (B). Results reflect the means of three experimental trials ± SD. Differences in ratios calculated from infection of N2a-HSP 1 and N2a-V 2 cells were not statistically significant. Controls for the infection were individual inoculations with Ed N or Ed N-522D virus, and reactions lacking RT were used as a negative control (-) for the RT-PCR.

    Article Snippet: Bands were resolved by 2.0% agarose gel electrophoresis and stained with ethidium bromide, and ethidium bromide staining intensity was quantified using an Alpha Imager (Alpha Innotech).

    Techniques: Expressing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Staining, Nucleic Acid Electrophoresis, Infection, Negative Control

    Nested RT-PCR amplification of viral sequence from total lung RNA of cotton rats infected with 1 × 10 5 TCID 50 of Ed N, Ed-N522D, or a 1:1 ratio of Ed N and N-522D. Samples were processed at 4 days p.i. Amplicons were digested with BsmAI to determine the proportion of the 267-bp (Ed N) versus 133/134-bp (Ed N-522D) product on 2% agarose gels stained with ethidium bromide. Negative controls included total lung RNA from uninfected rats (U) and infected rats in which the RT step was omitted (-). Ed-N is the predominant virus identified as a consequence of mixed infections.

    Journal: Journal of Virology

    Article Title: A Single Codon in the Nucleocapsid Protein C Terminus Contributes to In Vitro and In Vivo Fitness of Edmonston Measles Virus

    doi: 10.1128/JVI.80.6.2904-2912.2006

    Figure Lengend Snippet: Nested RT-PCR amplification of viral sequence from total lung RNA of cotton rats infected with 1 × 10 5 TCID 50 of Ed N, Ed-N522D, or a 1:1 ratio of Ed N and N-522D. Samples were processed at 4 days p.i. Amplicons were digested with BsmAI to determine the proportion of the 267-bp (Ed N) versus 133/134-bp (Ed N-522D) product on 2% agarose gels stained with ethidium bromide. Negative controls included total lung RNA from uninfected rats (U) and infected rats in which the RT step was omitted (-). Ed-N is the predominant virus identified as a consequence of mixed infections.

    Article Snippet: Bands were resolved by 2.0% agarose gel electrophoresis and stained with ethidium bromide, and ethidium bromide staining intensity was quantified using an Alpha Imager (Alpha Innotech).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing, Infection, Staining

    Linear relationship between the ratio of infectious viral variants Ed N and Ed N-522D and virus-specific RT-PCR amplicon yield. Individual pools of titrated Ed N and Ed N-522D virus were combined at an N-522D/N ratio of 1:1, 1:2, 1:5, or 1:10. Total RNA was extracted, and a 267-nt genomic sequence spanning the N-P junction was amplified by RT-PCR. (A) The proportion of Ed N and Ed N-522D represented in the amplicons was based upon BsmAI restriction fragment length polymorphism. The 267-bp amplicon derived from Ed N virus is not cleaved, whereas the corresponding amplicon from Ed N-522D is cleaved to yield 134- and 133-bp fragments; these fragments resolve as a single band following 2% agarose gel electrophoresis. Reactions lacking RT were used as a negative control (-) for the RT-PCR. (B) Linear regression analysis of the ratio of N-522D and N signal intensities (i.e., ethidium bromide staining intensities of virus-specific amplicons) expressed as a function of the ratio of N-522D and N virus used in the assay. Mean signal ± SD was based upon three separate analyses. The line describing the relationship between the ratio of infectious viral variants and the ratio of virus-specific RT-PCR amplicons ( y = 0.55 x + 0.42) exhibits an excellent fit to the experimental data ( R 2 = 0.99).

    Journal: Journal of Virology

    Article Title: A Single Codon in the Nucleocapsid Protein C Terminus Contributes to In Vitro and In Vivo Fitness of Edmonston Measles Virus

    doi: 10.1128/JVI.80.6.2904-2912.2006

    Figure Lengend Snippet: Linear relationship between the ratio of infectious viral variants Ed N and Ed N-522D and virus-specific RT-PCR amplicon yield. Individual pools of titrated Ed N and Ed N-522D virus were combined at an N-522D/N ratio of 1:1, 1:2, 1:5, or 1:10. Total RNA was extracted, and a 267-nt genomic sequence spanning the N-P junction was amplified by RT-PCR. (A) The proportion of Ed N and Ed N-522D represented in the amplicons was based upon BsmAI restriction fragment length polymorphism. The 267-bp amplicon derived from Ed N virus is not cleaved, whereas the corresponding amplicon from Ed N-522D is cleaved to yield 134- and 133-bp fragments; these fragments resolve as a single band following 2% agarose gel electrophoresis. Reactions lacking RT were used as a negative control (-) for the RT-PCR. (B) Linear regression analysis of the ratio of N-522D and N signal intensities (i.e., ethidium bromide staining intensities of virus-specific amplicons) expressed as a function of the ratio of N-522D and N virus used in the assay. Mean signal ± SD was based upon three separate analyses. The line describing the relationship between the ratio of infectious viral variants and the ratio of virus-specific RT-PCR amplicons ( y = 0.55 x + 0.42) exhibits an excellent fit to the experimental data ( R 2 = 0.99).

    Article Snippet: Bands were resolved by 2.0% agarose gel electrophoresis and stained with ethidium bromide, and ethidium bromide staining intensity was quantified using an Alpha Imager (Alpha Innotech).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing, Derivative Assay, Agarose Gel Electrophoresis, Negative Control, Staining

    (A) Infectious viral progeny release for parental Ed N compared to the Ed N-522D variant in control (C) and preconditioned (PC) Vero cells following infection at an MOI of 0.01. Results are an average of two separate experimental trials and expressed as the mean ± standard error of the mean (SEM). Preconditioning of Vero cells was achieved by exposing cells in the log phase of growth to 43°C for 1.5 h, a treatment elevating cytoplasmic hsp72 for 24 to 48 h posttreatment. Cells were infected at 16 h posttreatment. Peak infectious progeny release was statistically significantly elevated for Ed N but not Ed N-522D virus, representing increases of 5- and 1.5-fold, respectively. (B) Relative fitness of Ed N-522D compared to Ed N based upon coinfection of preconditioned and control Vero cells. The inocula contained a 1:1 ratio of each virus for a combined MOI of 0.01, or 1.0. The proportion of Ed N and Ed N-522D in the viral progeny was based upon BsmAI restriction fragment length polymorphisms of amplicons derived from RT-PCR of cell-free viral genomic RNA. Yield of amplicon was based upon ethidium bromide staining intensity of BsmAI-digested products resolved by gel electrophoresis.

    Journal: Journal of Virology

    Article Title: A Single Codon in the Nucleocapsid Protein C Terminus Contributes to In Vitro and In Vivo Fitness of Edmonston Measles Virus

    doi: 10.1128/JVI.80.6.2904-2912.2006

    Figure Lengend Snippet: (A) Infectious viral progeny release for parental Ed N compared to the Ed N-522D variant in control (C) and preconditioned (PC) Vero cells following infection at an MOI of 0.01. Results are an average of two separate experimental trials and expressed as the mean ± standard error of the mean (SEM). Preconditioning of Vero cells was achieved by exposing cells in the log phase of growth to 43°C for 1.5 h, a treatment elevating cytoplasmic hsp72 for 24 to 48 h posttreatment. Cells were infected at 16 h posttreatment. Peak infectious progeny release was statistically significantly elevated for Ed N but not Ed N-522D virus, representing increases of 5- and 1.5-fold, respectively. (B) Relative fitness of Ed N-522D compared to Ed N based upon coinfection of preconditioned and control Vero cells. The inocula contained a 1:1 ratio of each virus for a combined MOI of 0.01, or 1.0. The proportion of Ed N and Ed N-522D in the viral progeny was based upon BsmAI restriction fragment length polymorphisms of amplicons derived from RT-PCR of cell-free viral genomic RNA. Yield of amplicon was based upon ethidium bromide staining intensity of BsmAI-digested products resolved by gel electrophoresis.

    Article Snippet: Bands were resolved by 2.0% agarose gel electrophoresis and stained with ethidium bromide, and ethidium bromide staining intensity was quantified using an Alpha Imager (Alpha Innotech).

    Techniques: Variant Assay, Infection, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Staining, Nucleic Acid Electrophoresis

    Differential enrichment of H3K9/14ac and H3K4me3 histone modifications at the VPAC1 promoter A. Flow cytometric analysis of mouse CD4 + T cells and CD45R + B cells. Left Panel : Non-adherent splenocytes were stained with anti-CD4-FITC pre- (red line) and post- (blue line) CD4 positive magnetic bead separation ( Materials and Methods ) with typical purities ≥93% (n=3). Right Panel : CD4 depleted non-adherent splenocytes were subsequently stained pre- (red line) and post- (blue line) CD45R-FITC positive magnetic bead separation ( Materials and Methods ) with typical purities ≥97% (n=3). B. Bar graph for Taqman qPCR measurements for VPAC1 from indicated cell type. A hash mark indicates a shift in the y-axis. Data is represented as means +/- SEM for relative VPAC1 levels normalized to HPRT and calculated by 2 (-ΔCt) formula from three independent experiments. The asterisk (*) represents a p≤ 0.05 as compared to CD4 + T cells. C-D. ChIP analysis was performed using purified CD4 + T and CD45R + B cells followed by quantitative SYBR green PCR using VPAC1 primer set 2 or genomic negative control. C. Amplification reactions from SYBR green qPCR using primer set 2 were separated on a 3% agarose gel, stained with ethidium bromide and visualized by a CCD camera (Syngene). This experiment was repeated twice with similar results. D. Bar graph showing fold-enrichment over IgG control of H3K9/14ac and H3K4me3 from the indicated cell types. Cycle thresholds (Ct) were subtracted from input Ct values to obtain relative ΔCt values. Fold increases were calculated by 2 -(ΔCt) formula with non specific IgG levels normalized to input arbitrarily set to 1 (not shown). Data is presented as means +/- SEM from three biologically independent experiments (*, p≤0.05).

    Journal: Regulatory peptides

    Article Title: A Transcriptionally Permissive Epigenetic Landscape at the Vasoactive Intestinal Peptide Receptor-1 Promoter Suggests an Euchromatin Nuclear Position in Murine CD4 T Cells

    doi: 10.1016/j.regpep.2009.08.010

    Figure Lengend Snippet: Differential enrichment of H3K9/14ac and H3K4me3 histone modifications at the VPAC1 promoter A. Flow cytometric analysis of mouse CD4 + T cells and CD45R + B cells. Left Panel : Non-adherent splenocytes were stained with anti-CD4-FITC pre- (red line) and post- (blue line) CD4 positive magnetic bead separation ( Materials and Methods ) with typical purities ≥93% (n=3). Right Panel : CD4 depleted non-adherent splenocytes were subsequently stained pre- (red line) and post- (blue line) CD45R-FITC positive magnetic bead separation ( Materials and Methods ) with typical purities ≥97% (n=3). B. Bar graph for Taqman qPCR measurements for VPAC1 from indicated cell type. A hash mark indicates a shift in the y-axis. Data is represented as means +/- SEM for relative VPAC1 levels normalized to HPRT and calculated by 2 (-ΔCt) formula from three independent experiments. The asterisk (*) represents a p≤ 0.05 as compared to CD4 + T cells. C-D. ChIP analysis was performed using purified CD4 + T and CD45R + B cells followed by quantitative SYBR green PCR using VPAC1 primer set 2 or genomic negative control. C. Amplification reactions from SYBR green qPCR using primer set 2 were separated on a 3% agarose gel, stained with ethidium bromide and visualized by a CCD camera (Syngene). This experiment was repeated twice with similar results. D. Bar graph showing fold-enrichment over IgG control of H3K9/14ac and H3K4me3 from the indicated cell types. Cycle thresholds (Ct) were subtracted from input Ct values to obtain relative ΔCt values. Fold increases were calculated by 2 -(ΔCt) formula with non specific IgG levels normalized to input arbitrarily set to 1 (not shown). Data is presented as means +/- SEM from three biologically independent experiments (*, p≤0.05).

    Article Snippet: PCR amplification products were separated on a 3% ethidium bromide stained, agarose gel and visualized using a Syngene digital camera (Frederick, MD).

    Techniques: Flow Cytometry, Staining, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Purification, SYBR Green Assay, Polymerase Chain Reaction, Negative Control, Amplification, Agarose Gel Electrophoresis

    Transcriptionally permissive modifications, H3K9/K14ac and H3K4me3, are not removed during T cell signaling ChIP analysis was performed using purified CD4 T cells incubated +/- plate bound α-CD3 ( Materials and Methods ) followed by quantitative SYBR green PCR using VPAC1 primer set 2. All data is from three independent biological experiments. A. Flow cytometric analysis of early activation markers, CD69 (left two panels, increase expected) and CD127 (right far panel, decrease expected) using CD4 + T cells treated +/- anti-CD3. B. Bar graph representing relative VPAC1 expression levels in CD4 + T cells treated +/- anti-CD3 as indicated. Data is presented as means +/- SEM and an * represents a p≤0.05 as compared to cells without anti-CD3. C. Semi-quantitative amplification reactions using immunoprecipitated chromatin and primer set 2 were separated on a 3% agarose gel, stained with ethidium bromide, and visualized by a CCD camera (Syngene). D. Bar graph of fold enrichment over IgG control of H3K9/14ac and H3K4me3 from the indicated cell types. Cycle thresholds (Cts) were subtracted from input Ct values to normalize to input and fold increases were calculated by 2 -(ΔCt) formula. Non-specific IgG levels were also normalized to input and arbitrarily set to 1 (not shown). Data is presented as means +/-SEM.

    Journal: Regulatory peptides

    Article Title: A Transcriptionally Permissive Epigenetic Landscape at the Vasoactive Intestinal Peptide Receptor-1 Promoter Suggests an Euchromatin Nuclear Position in Murine CD4 T Cells

    doi: 10.1016/j.regpep.2009.08.010

    Figure Lengend Snippet: Transcriptionally permissive modifications, H3K9/K14ac and H3K4me3, are not removed during T cell signaling ChIP analysis was performed using purified CD4 T cells incubated +/- plate bound α-CD3 ( Materials and Methods ) followed by quantitative SYBR green PCR using VPAC1 primer set 2. All data is from three independent biological experiments. A. Flow cytometric analysis of early activation markers, CD69 (left two panels, increase expected) and CD127 (right far panel, decrease expected) using CD4 + T cells treated +/- anti-CD3. B. Bar graph representing relative VPAC1 expression levels in CD4 + T cells treated +/- anti-CD3 as indicated. Data is presented as means +/- SEM and an * represents a p≤0.05 as compared to cells without anti-CD3. C. Semi-quantitative amplification reactions using immunoprecipitated chromatin and primer set 2 were separated on a 3% agarose gel, stained with ethidium bromide, and visualized by a CCD camera (Syngene). D. Bar graph of fold enrichment over IgG control of H3K9/14ac and H3K4me3 from the indicated cell types. Cycle thresholds (Cts) were subtracted from input Ct values to normalize to input and fold increases were calculated by 2 -(ΔCt) formula. Non-specific IgG levels were also normalized to input and arbitrarily set to 1 (not shown). Data is presented as means +/-SEM.

    Article Snippet: PCR amplification products were separated on a 3% ethidium bromide stained, agarose gel and visualized using a Syngene digital camera (Frederick, MD).

    Techniques: Chromatin Immunoprecipitation, Purification, Incubation, SYBR Green Assay, Polymerase Chain Reaction, Flow Cytometry, Activation Assay, Expressing, Amplification, Immunoprecipitation, Agarose Gel Electrophoresis, Staining

    Kinetic analyses of OPI and interpretation of in vitro transposition reactions. The kinetics of the transposition reaction were analyzed in standard reactions containing 6.7 nM plasmid substrate and the indicated concentrations of the transposases. Photographs of ethidium bromide stained agarose gels are shown. ( A ) The effects of the ASO mechanism complicates the interpretation of the gels, particularly the kinetic analyses. In the reactions with 20 nM transposase about half of the substrate is converted to the nicked intermediate in the first 2 min. However, it takes an hour or more to consume the remainder of the substrate. This biphasic behavior arises from the ASO mechanism, which operates in any reaction that contains more than one dimer of transposase. Thus, even if the transposase was 100% active and the reaction was performed with the optimum ratio of one dimer to one transposon, only half of the substrate would react initially. At the start of the reaction half of the transposons would be occupied by a dimer and would react, a quarter would be occupied by two dimers and would suffer OPI and the other quarter would be completely unoccupied. The slow phase of consumption corresponds to the redistribution of dimers from transposons which initially suffered OPI to those that were completely unbound. Even under OPI conditions, when both ends of the transposon are occupied by transposase dimers, their occasional dissociation provides a window of opportunity for synapsis. OPI can therefore be overcome by extending the incubation period so that the accumulated windows of opportunity eventually suffice to complete the reaction. Thus, the reaction with 40 nM transposase goes on to reach completion during the 8 hr incubation, despite initially suffering from OPI. With 200 nM transposase, the extended incubation is unable to overcome OPI because the windows of opportunity provided by unoccupied transposon ends are shorter at this concentration. ( B ) The nicked substrate is more sensitive to OPI and is inhibited at only 40 nM transposase. This is because any free transposon ends, made available by the dissociation of a transposase dimer, are re-bound before they can achieve synapsis. In other words, the slow synapsis of the nicked substrate requires a longer window of opportunity than is available at 40 nM transposase. Time points and transposase concentrations as in part ( A ). ( C ) The RA104 transposase mutant is resistant to OPI because the higher OFF-rate provides more windows of opportunity for synapsis (see Figure 4C and text for details). Time points and transposase concentrations as in part ( A ). DOI: http://dx.doi.org/10.7554/eLife.00668.005

    Journal: eLife

    Article Title: The autoregulation of a eukaryotic DNA transposon

    doi: 10.7554/eLife.00668

    Figure Lengend Snippet: Kinetic analyses of OPI and interpretation of in vitro transposition reactions. The kinetics of the transposition reaction were analyzed in standard reactions containing 6.7 nM plasmid substrate and the indicated concentrations of the transposases. Photographs of ethidium bromide stained agarose gels are shown. ( A ) The effects of the ASO mechanism complicates the interpretation of the gels, particularly the kinetic analyses. In the reactions with 20 nM transposase about half of the substrate is converted to the nicked intermediate in the first 2 min. However, it takes an hour or more to consume the remainder of the substrate. This biphasic behavior arises from the ASO mechanism, which operates in any reaction that contains more than one dimer of transposase. Thus, even if the transposase was 100% active and the reaction was performed with the optimum ratio of one dimer to one transposon, only half of the substrate would react initially. At the start of the reaction half of the transposons would be occupied by a dimer and would react, a quarter would be occupied by two dimers and would suffer OPI and the other quarter would be completely unoccupied. The slow phase of consumption corresponds to the redistribution of dimers from transposons which initially suffered OPI to those that were completely unbound. Even under OPI conditions, when both ends of the transposon are occupied by transposase dimers, their occasional dissociation provides a window of opportunity for synapsis. OPI can therefore be overcome by extending the incubation period so that the accumulated windows of opportunity eventually suffice to complete the reaction. Thus, the reaction with 40 nM transposase goes on to reach completion during the 8 hr incubation, despite initially suffering from OPI. With 200 nM transposase, the extended incubation is unable to overcome OPI because the windows of opportunity provided by unoccupied transposon ends are shorter at this concentration. ( B ) The nicked substrate is more sensitive to OPI and is inhibited at only 40 nM transposase. This is because any free transposon ends, made available by the dissociation of a transposase dimer, are re-bound before they can achieve synapsis. In other words, the slow synapsis of the nicked substrate requires a longer window of opportunity than is available at 40 nM transposase. Time points and transposase concentrations as in part ( A ). ( C ) The RA104 transposase mutant is resistant to OPI because the higher OFF-rate provides more windows of opportunity for synapsis (see Figure 4C and text for details). Time points and transposase concentrations as in part ( A ). DOI: http://dx.doi.org/10.7554/eLife.00668.005

    Article Snippet: After electrophoresis, the gel was stained with ethidium bromide, destained in water, and photographed on a 310 nm transilluminator using a DC290 camera (Kodak, Rochester, NY) with a 590 DF bandpass filter.

    Techniques: In Vitro, Plasmid Preparation, Staining, Allele-specific Oligonucleotide, Incubation, Concentration Assay, Mutagenesis

    Association and dissociation rate constants. ( A ) Simulation as in Figure 3E , with k 1 fivefold up or down. We have retained the effects of allostery but here and in subsequent simulations we have ignored the effects of the slow diffusion in vivo. This allows the algorithm to run more smoothly and shortens the scale on the x axis, but does not affect the conclusions, which are based on the differential responses to changing the various parameters. ( B ) Simulation as in part ( A ) (solid line), with k 1 100-fold down. ( C ) Binding reactions with the RA104 mutant transposase were set up as in Figure 3B . In the lane with 20 nM transposase the smear between SEC2 and the position of free DNA is probably due to complexes that have dissociated during electrophoresis. Autoradiogram of an EMSA is shown. ( D ) The kinetics of the transposition reaction were analyzed in standard reactions containing 6.7 nM of supercoiled plasmid substrate and the indicated concentrations of the transposases. Photographs of ethidium bromide stained agarose gels are shown. With 200 nM wild-type transposase, the windows of opportunity for synapsis, provided by periods when one transposon end is unoccupied, are too short to allow for synapsis. The RA104 transposase mutant is resistant to OPI because the higher dissociating rate provides more windows of opportunity for synapsis. ( E ) Mutant and wild type transposase were assayed in HeLa cell culture with 8 ng or 1000 ng of helper plasmid and 500 ng of neomycin resistant reporter plasmid. DOI: http://dx.doi.org/10.7554/eLife.00668.009

    Journal: eLife

    Article Title: The autoregulation of a eukaryotic DNA transposon

    doi: 10.7554/eLife.00668

    Figure Lengend Snippet: Association and dissociation rate constants. ( A ) Simulation as in Figure 3E , with k 1 fivefold up or down. We have retained the effects of allostery but here and in subsequent simulations we have ignored the effects of the slow diffusion in vivo. This allows the algorithm to run more smoothly and shortens the scale on the x axis, but does not affect the conclusions, which are based on the differential responses to changing the various parameters. ( B ) Simulation as in part ( A ) (solid line), with k 1 100-fold down. ( C ) Binding reactions with the RA104 mutant transposase were set up as in Figure 3B . In the lane with 20 nM transposase the smear between SEC2 and the position of free DNA is probably due to complexes that have dissociated during electrophoresis. Autoradiogram of an EMSA is shown. ( D ) The kinetics of the transposition reaction were analyzed in standard reactions containing 6.7 nM of supercoiled plasmid substrate and the indicated concentrations of the transposases. Photographs of ethidium bromide stained agarose gels are shown. With 200 nM wild-type transposase, the windows of opportunity for synapsis, provided by periods when one transposon end is unoccupied, are too short to allow for synapsis. The RA104 transposase mutant is resistant to OPI because the higher dissociating rate provides more windows of opportunity for synapsis. ( E ) Mutant and wild type transposase were assayed in HeLa cell culture with 8 ng or 1000 ng of helper plasmid and 500 ng of neomycin resistant reporter plasmid. DOI: http://dx.doi.org/10.7554/eLife.00668.009

    Article Snippet: After electrophoresis, the gel was stained with ethidium bromide, destained in water, and photographed on a 310 nm transilluminator using a DC290 camera (Kodak, Rochester, NY) with a 590 DF bandpass filter.

    Techniques: Diffusion-based Assay, In Vivo, Binding Assay, Mutagenesis, Electrophoresis, Plasmid Preparation, Staining, Cell Culture

    EMSA analysis of transpososome assembly shows that SEC1 arises from dissociation of the PEC. Transposon ends were encoded on radiolabeled linear DNA fragments. Binding reactions were incubated at 37 °C for 2 hr, separated on a 5% native polyacrylamide gel and recorded by phosphoimaging. ( A ) SEC1 and SEC2 represent a single transposon end bound by a transposase monomer and dimer, respectively (see main text for details). SEC2 comes to dominate the reaction as the transposase concentration rises. There is a significant transition between 4 and 8 nM transposase when SEC1 largely disappears. This corresponds to the point at which the transposon ends become sub-stoichiometric to the transposase dimers. At this point no free transposon ends remain as they are all bound by transposase. According to the S-NEC mechanism (see Figure 1 for details), SEC2 is converted to the transpososome (=PEC) by recruitment of a naked transposon end. OPI occurs when the transposon ends are sub-stoichiometric to transposase dimers and there is a shortage of free transposon ends available for recruitment. Note that the various species observed in these binding reactions are identical to those observed in reactions with the related Mos1 and Himar1 transposons, which display the same behavior for example ( Dawson and Finnegan, 2003 ; Lipkow et al., 2004 ). The present data suggests that the PEC in all three of these related systems is unstable in the EMSA and dissociates into two SEC1 complexes soon after the start of electrophoresis. Thus, in agreement with the S-NEC mechanism, SEC1 disappears at the point in the titration when the transposon ends become sub-stoichiometric to transposase dimers. ( B ) A fixed amount of transposase was titrated with an increasing amount of free transposon ends. The appearance of SEC1 coincides exactly with the appearance of the free transposon ends, which are required for PEC assembly in the S-NEC model. As the amount of transposon ends is increased further, the amount of SEC1 increases. This reflects mass action, which drives PEC assembly by favoring the capture of a free transposon end (see part D below for confirmation). This supports the data in part ( A ) in suggesting that SEC1 arises from the dissociation of the PEC. ( C ) Binding reactions were with a single-chain transposase dimer, in which two monomers are concatenated by a linker peptide joining the C-terminus of one to the N-terminus of the other. Concatenation of the subunits stabilizes the PEC, which is now detected in the gel. As the transposase concentration increases, the PEC disappears at the same point as the free DNA and gives way to SEC2. This behavior is identical to SEC1 in parts ( A ) and ( B ). The single-chain dimer of transposase is fully proficient for the transposition reaction (not shown), demonstrating that SEC1 is not an obligate intermediate of the reaction. This supports the data in parts ( A ) and ( B ) further confirming that SEC1 arises from the dissociation of the PEC. ( D ) In vitro transposition reactions were performed with a plasmid substrate encoding a single transposon end. Reactions were stopped at the indicated times and deproteinated before analysis on a 1.1% agarose TBE gel stained with ethidium bromide. All three sets of transposition reactions contained the same amounts of transposase and supercoiled plasmid substrate. However, the respective reaction volumes were adjusted to 500 µl, 50 µl and 5 µl to achieve the indicated concentrations. Transpososome assembly requires bimolecular synapsis between ends located on different molecules, as illustrated below the gels. This is inefficient owing to the relatively low concentration of one transposon end with respect to another when on separate molecules. When such a transpososome performs cleavage, followed by integration into an unreacted substrate molecule, the product is a linear molecule three times the size of the substrate ( Claeys Bouuaert et al., 2011 ). There is very little reaction when the substrate concentration is low. This reflects the inefficiency of second end recruitment. At high substrate concentration, mass action drives the reaction by favoring second-end recruitment. DOI: http://dx.doi.org/10.7554/eLife.00668.008

    Journal: eLife

    Article Title: The autoregulation of a eukaryotic DNA transposon

    doi: 10.7554/eLife.00668

    Figure Lengend Snippet: EMSA analysis of transpososome assembly shows that SEC1 arises from dissociation of the PEC. Transposon ends were encoded on radiolabeled linear DNA fragments. Binding reactions were incubated at 37 °C for 2 hr, separated on a 5% native polyacrylamide gel and recorded by phosphoimaging. ( A ) SEC1 and SEC2 represent a single transposon end bound by a transposase monomer and dimer, respectively (see main text for details). SEC2 comes to dominate the reaction as the transposase concentration rises. There is a significant transition between 4 and 8 nM transposase when SEC1 largely disappears. This corresponds to the point at which the transposon ends become sub-stoichiometric to the transposase dimers. At this point no free transposon ends remain as they are all bound by transposase. According to the S-NEC mechanism (see Figure 1 for details), SEC2 is converted to the transpososome (=PEC) by recruitment of a naked transposon end. OPI occurs when the transposon ends are sub-stoichiometric to transposase dimers and there is a shortage of free transposon ends available for recruitment. Note that the various species observed in these binding reactions are identical to those observed in reactions with the related Mos1 and Himar1 transposons, which display the same behavior for example ( Dawson and Finnegan, 2003 ; Lipkow et al., 2004 ). The present data suggests that the PEC in all three of these related systems is unstable in the EMSA and dissociates into two SEC1 complexes soon after the start of electrophoresis. Thus, in agreement with the S-NEC mechanism, SEC1 disappears at the point in the titration when the transposon ends become sub-stoichiometric to transposase dimers. ( B ) A fixed amount of transposase was titrated with an increasing amount of free transposon ends. The appearance of SEC1 coincides exactly with the appearance of the free transposon ends, which are required for PEC assembly in the S-NEC model. As the amount of transposon ends is increased further, the amount of SEC1 increases. This reflects mass action, which drives PEC assembly by favoring the capture of a free transposon end (see part D below for confirmation). This supports the data in part ( A ) in suggesting that SEC1 arises from the dissociation of the PEC. ( C ) Binding reactions were with a single-chain transposase dimer, in which two monomers are concatenated by a linker peptide joining the C-terminus of one to the N-terminus of the other. Concatenation of the subunits stabilizes the PEC, which is now detected in the gel. As the transposase concentration increases, the PEC disappears at the same point as the free DNA and gives way to SEC2. This behavior is identical to SEC1 in parts ( A ) and ( B ). The single-chain dimer of transposase is fully proficient for the transposition reaction (not shown), demonstrating that SEC1 is not an obligate intermediate of the reaction. This supports the data in parts ( A ) and ( B ) further confirming that SEC1 arises from the dissociation of the PEC. ( D ) In vitro transposition reactions were performed with a plasmid substrate encoding a single transposon end. Reactions were stopped at the indicated times and deproteinated before analysis on a 1.1% agarose TBE gel stained with ethidium bromide. All three sets of transposition reactions contained the same amounts of transposase and supercoiled plasmid substrate. However, the respective reaction volumes were adjusted to 500 µl, 50 µl and 5 µl to achieve the indicated concentrations. Transpososome assembly requires bimolecular synapsis between ends located on different molecules, as illustrated below the gels. This is inefficient owing to the relatively low concentration of one transposon end with respect to another when on separate molecules. When such a transpososome performs cleavage, followed by integration into an unreacted substrate molecule, the product is a linear molecule three times the size of the substrate ( Claeys Bouuaert et al., 2011 ). There is very little reaction when the substrate concentration is low. This reflects the inefficiency of second end recruitment. At high substrate concentration, mass action drives the reaction by favoring second-end recruitment. DOI: http://dx.doi.org/10.7554/eLife.00668.008

    Article Snippet: After electrophoresis, the gel was stained with ethidium bromide, destained in water, and photographed on a 310 nm transilluminator using a DC290 camera (Kodak, Rochester, NY) with a 590 DF bandpass filter.

    Techniques: Binding Assay, Incubation, Concentration Assay, Electrophoresis, Titration, In Vitro, Plasmid Preparation, Staining

    TiO 2 NPs induced mitochondrial proapoptotic factors and subsequent apoptosis in RAW 264.7 cells. Notes: ( A ) Mechanism depicting TiO 2 NPs induced BAX activation; ( B ) RT-PCR gel images of BAX, BIM, and PUMA; ( C ) Flow cytometry analysis of apoptosis; ( D ) Quantitative analysis of apoptotic cell death. Cells were incubated with indicated concentrations of TiO 2 NPs for 24 hours. RT-PCR reactions were performed and PCR products were separated by 1% agarose gel electrophoresis and bands were visualized under UVP Biospectrum-600 (Thermo Fisher Scientific, Waltham, MA, USA) after ethidium bromide staining (EtBr, Sigma-Aldrich, St Louis, MO, USA). Housekeeping gene GAPDH was used as loading control. Then, apoptosis was investigated using Muse™ flowcytometric method with Muse™ Annexin V and Dead Cell Kit. Untreated cells were considered as control in the experiments. Data are presented as the mean ± standard error of mean; *** P

    Journal: International Journal of Nanomedicine

    Article Title: Immunotoxicity of titanium dioxide nanoparticles via simultaneous induction of apoptosis and multiple toll-like receptors signaling through ROS-dependent SAPK/JNK and p38 MAPK activation

    doi: 10.2147/IJN.S176087

    Figure Lengend Snippet: TiO 2 NPs induced mitochondrial proapoptotic factors and subsequent apoptosis in RAW 264.7 cells. Notes: ( A ) Mechanism depicting TiO 2 NPs induced BAX activation; ( B ) RT-PCR gel images of BAX, BIM, and PUMA; ( C ) Flow cytometry analysis of apoptosis; ( D ) Quantitative analysis of apoptotic cell death. Cells were incubated with indicated concentrations of TiO 2 NPs for 24 hours. RT-PCR reactions were performed and PCR products were separated by 1% agarose gel electrophoresis and bands were visualized under UVP Biospectrum-600 (Thermo Fisher Scientific, Waltham, MA, USA) after ethidium bromide staining (EtBr, Sigma-Aldrich, St Louis, MO, USA). Housekeeping gene GAPDH was used as loading control. Then, apoptosis was investigated using Muse™ flowcytometric method with Muse™ Annexin V and Dead Cell Kit. Untreated cells were considered as control in the experiments. Data are presented as the mean ± standard error of mean; *** P

    Article Snippet: PCR products were separated by 1% agarose gel electrophoresis and bands were visualized under UVP Biospectrum-600 imaging system (Thermo Fisher Scientific) after ethidium bromide staining (EtBr; Sigma-Aldrich).

    Techniques: Activation Assay, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Cytometry, Incubation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    Agarose gel electrophoresis of one-step RT-PCR amplified products of the IBV S1 gene. Commercial IBV vaccine (IBV H120, MA5, and 4/91) and field strains (“Moroccan 30” and “Moroccan 38”) were separated by electrophoresis on a 1.5 % agarose gel stained with ethidium bromide. Lane 1 100 bp DNA ladder; lane 2 Moroccan 30 strain; lane 3 negative control; lane 4 Moroccan 38 strain; lanes 5, 6 negative controls; lane 7 Ma5 commercial vaccine; lane 8 H120 commercial vaccine; lane 9 4/91 commercial vaccine. Product size, 700 bp. Bp base pairs, IBV infectious bronchitis virus, RT - PCR reverse transcriptase-polymerase chain reaction

    Journal: BMC Research Notes

    Article Title: Comparison of SYBR green I real-time RT-PCR with conventional agarose gel-based RT-PCR for the diagnosis of infectious bronchitis virus infection in chickens in Morocco

    doi: 10.1186/s13104-016-2037-z

    Figure Lengend Snippet: Agarose gel electrophoresis of one-step RT-PCR amplified products of the IBV S1 gene. Commercial IBV vaccine (IBV H120, MA5, and 4/91) and field strains (“Moroccan 30” and “Moroccan 38”) were separated by electrophoresis on a 1.5 % agarose gel stained with ethidium bromide. Lane 1 100 bp DNA ladder; lane 2 Moroccan 30 strain; lane 3 negative control; lane 4 Moroccan 38 strain; lanes 5, 6 negative controls; lane 7 Ma5 commercial vaccine; lane 8 H120 commercial vaccine; lane 9 4/91 commercial vaccine. Product size, 700 bp. Bp base pairs, IBV infectious bronchitis virus, RT - PCR reverse transcriptase-polymerase chain reaction

    Article Snippet: The amplified PCR products of the N gene were evaluated by 1.5 % agarose gel electrophoresis in Tris–borate–EDTA buffer stained with ethidium bromide (Promega).

    Techniques: Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Amplification, Electrophoresis, Staining, Negative Control, Polymerase Chain Reaction

    Agarose gel electrophoresis of conventional RT-PCR products from the N gene of IBV H120 strain. Amplified products were separated by electrophoresis on a 1.5 % agarose gel and stained with ethidium bromide. Lane 1 100 bp DNA ladder; lane 2 10 7 copies/μl; lane 3 10 6 copies/μl; lane 4 10 5 copies/μl; lane 5 10 4 copies/μl; lane 6 10 3 copies/μl; lane 7 10 2 copies/μl; lane 8 10 copies/μl. IBV infectious bronchitis virus, RT - PCR reverse transcriptase-polymerase chain reaction

    Journal: BMC Research Notes

    Article Title: Comparison of SYBR green I real-time RT-PCR with conventional agarose gel-based RT-PCR for the diagnosis of infectious bronchitis virus infection in chickens in Morocco

    doi: 10.1186/s13104-016-2037-z

    Figure Lengend Snippet: Agarose gel electrophoresis of conventional RT-PCR products from the N gene of IBV H120 strain. Amplified products were separated by electrophoresis on a 1.5 % agarose gel and stained with ethidium bromide. Lane 1 100 bp DNA ladder; lane 2 10 7 copies/μl; lane 3 10 6 copies/μl; lane 4 10 5 copies/μl; lane 5 10 4 copies/μl; lane 6 10 3 copies/μl; lane 7 10 2 copies/μl; lane 8 10 copies/μl. IBV infectious bronchitis virus, RT - PCR reverse transcriptase-polymerase chain reaction

    Article Snippet: The amplified PCR products of the N gene were evaluated by 1.5 % agarose gel electrophoresis in Tris–borate–EDTA buffer stained with ethidium bromide (Promega).

    Techniques: Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Amplification, Electrophoresis, Staining, Polymerase Chain Reaction

    Agarose gel electrophoresis of conventional RT-PCR amplified products from infectious bronchitis virus N gene. Electrophoresis of PCR amplification after serial dilutions of the IBV Beaudette strain in a 1.5 % agarose gel stained with ethidium bromide. Lanes 1 and 10 50-bp DNA ladder; lane 2 negative control; lane 3 10 8 copies/μl; lane 4 10 7 copies/μl; lane 5 10 6 copies/μl; lane 6 10 5 copies/μl; lane 7 10 4 copies/μl; lane 8 10 3 copies/μl; lane 9 10 2 copies/μl. bp base pairs, IBV infectious bronchitis virus, RT - PCR reverse transcriptase-polymerase chain reaction

    Journal: BMC Research Notes

    Article Title: Comparison of SYBR green I real-time RT-PCR with conventional agarose gel-based RT-PCR for the diagnosis of infectious bronchitis virus infection in chickens in Morocco

    doi: 10.1186/s13104-016-2037-z

    Figure Lengend Snippet: Agarose gel electrophoresis of conventional RT-PCR amplified products from infectious bronchitis virus N gene. Electrophoresis of PCR amplification after serial dilutions of the IBV Beaudette strain in a 1.5 % agarose gel stained with ethidium bromide. Lanes 1 and 10 50-bp DNA ladder; lane 2 negative control; lane 3 10 8 copies/μl; lane 4 10 7 copies/μl; lane 5 10 6 copies/μl; lane 6 10 5 copies/μl; lane 7 10 4 copies/μl; lane 8 10 3 copies/μl; lane 9 10 2 copies/μl. bp base pairs, IBV infectious bronchitis virus, RT - PCR reverse transcriptase-polymerase chain reaction

    Article Snippet: The amplified PCR products of the N gene were evaluated by 1.5 % agarose gel electrophoresis in Tris–borate–EDTA buffer stained with ethidium bromide (Promega).

    Techniques: Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Amplification, Electrophoresis, Polymerase Chain Reaction, Staining, Negative Control

    Generation of seamless vectors for λ-Int-mediated genomic recombination. ( A ) A schematic diagram showing λ-Int-mediated recombination between genomic att H4X (located in LINE-1 elements) and att P4X (present on a plasmid target vector), leading to site-specific integration of the entire plasmid into the human LINE-1 elements, then flanked by att L4X (left junction) and att R4X (right junction). ( B ) An illustration depicting in vitro intra-molecular att H4X x att P4X recombination of a substrate plasmid, resulting in the formation of a seamless vector carrying the att L4X sequence after releasing the undesired bacterial DNA catenane ring by endonucleolytic digest. The seamless vector is subsequently targeted at genomic att H4X, leading to the insertion of payload through a second λ-Int-mediated recombination event. ( C ) SDS PAGE gel image depicting different desalted fractions of partially purified C-terminally histidine-tagged Int-h/218 (41 kDa). L, Prestained protein ladder (10–250 kDa); E, first eluted fraction from desalting column; 1, 2, 3, 4, different desalted fractions of partially purified C-terminal histidine-tagged Int-h/218. ( D ) Representative analysis of an in vitro intra-molecular att H4X x att P4X recombination with reaction buffers containing increasing KCl concentrations as indicated (left). Supercoiled seamless vector (SC-SV) and open circular seamless vector (OC-SV) obtained after λ-Int-mediated recombination were resolved via agarose gel electrophoresis in the presence of ethidium bromide. This resulted in DNA bands for SC-SV and OC-SV, which migrated at ∼1.2 and ∼2.2 kb, respectively, whereas the linearized second catenane DNA (linear bacterial DNA) and the unrecombined substrate plasmid migrated at ∼3 and ∼5.0 kb, respectively, as indicated. Purified seamless vector is shown at the right.

    Journal: Nucleic Acids Research

    Article Title: A novel λ integrase-mediated seamless vector transgenesis platform for therapeutic protein expression

    doi: 10.1093/nar/gky500

    Figure Lengend Snippet: Generation of seamless vectors for λ-Int-mediated genomic recombination. ( A ) A schematic diagram showing λ-Int-mediated recombination between genomic att H4X (located in LINE-1 elements) and att P4X (present on a plasmid target vector), leading to site-specific integration of the entire plasmid into the human LINE-1 elements, then flanked by att L4X (left junction) and att R4X (right junction). ( B ) An illustration depicting in vitro intra-molecular att H4X x att P4X recombination of a substrate plasmid, resulting in the formation of a seamless vector carrying the att L4X sequence after releasing the undesired bacterial DNA catenane ring by endonucleolytic digest. The seamless vector is subsequently targeted at genomic att H4X, leading to the insertion of payload through a second λ-Int-mediated recombination event. ( C ) SDS PAGE gel image depicting different desalted fractions of partially purified C-terminally histidine-tagged Int-h/218 (41 kDa). L, Prestained protein ladder (10–250 kDa); E, first eluted fraction from desalting column; 1, 2, 3, 4, different desalted fractions of partially purified C-terminal histidine-tagged Int-h/218. ( D ) Representative analysis of an in vitro intra-molecular att H4X x att P4X recombination with reaction buffers containing increasing KCl concentrations as indicated (left). Supercoiled seamless vector (SC-SV) and open circular seamless vector (OC-SV) obtained after λ-Int-mediated recombination were resolved via agarose gel electrophoresis in the presence of ethidium bromide. This resulted in DNA bands for SC-SV and OC-SV, which migrated at ∼1.2 and ∼2.2 kb, respectively, whereas the linearized second catenane DNA (linear bacterial DNA) and the unrecombined substrate plasmid migrated at ∼3 and ∼5.0 kb, respectively, as indicated. Purified seamless vector is shown at the right.

    Article Snippet: The PCR samples were analyzed by electrophoresis in 1% agarose (Seakem Agarose, Lonza, USA) gels in 1× TBE (Tris-boric acid-EDTA buffer) containing 0.5 μg/ml ethidium bromide and PCR-amplified products were compared with DNA standard markers and digitally documented under UV illumination (Quantum Vilber Lourmat, Germany).

    Techniques: Plasmid Preparation, In Vitro, Sequencing, SDS Page, Purification, Agarose Gel Electrophoresis

    Cytokine expression in Marek's disease herpesvirus (MDV)-infected chicken splenocytes. Total RNAs were prepared from spleens prepared from 3-week-old control chickens (lane 1) and from chickens infected with JM16 (lane 2), SB-1 (lane 3) and HVT (lane 4), respectively. The RNAs were reverse transcribed using oligo dT(16) as primers and the cDNAs were amplified by the polymerase chain reaction (PCR) using specific primers for interferon-γ (IFN-γ), inducible nitric oxide synthase (iNOS), interleukin (IL)-2, IL-6 and IL-8. β-actin was used as a control. The amplified DNA fragments were analysed in a 1·5% gel, stained with ethidium bromide and visualized on the Eagle Eye II Still Video System (Stratagene). All images were photographed as negative images except for IL-8. The sizes of the PCR fragments or expected fragments (IL-6) are indicated.

    Journal: Immunology

    Article Title: Expression of cytokine genes in Marek's disease virus-infected chickens and chicken embryo fibroblast cultures

    doi: 10.1046/j.1365-2567.2000.00008.x

    Figure Lengend Snippet: Cytokine expression in Marek's disease herpesvirus (MDV)-infected chicken splenocytes. Total RNAs were prepared from spleens prepared from 3-week-old control chickens (lane 1) and from chickens infected with JM16 (lane 2), SB-1 (lane 3) and HVT (lane 4), respectively. The RNAs were reverse transcribed using oligo dT(16) as primers and the cDNAs were amplified by the polymerase chain reaction (PCR) using specific primers for interferon-γ (IFN-γ), inducible nitric oxide synthase (iNOS), interleukin (IL)-2, IL-6 and IL-8. β-actin was used as a control. The amplified DNA fragments were analysed in a 1·5% gel, stained with ethidium bromide and visualized on the Eagle Eye II Still Video System (Stratagene). All images were photographed as negative images except for IL-8. The sizes of the PCR fragments or expected fragments (IL-6) are indicated.

    Article Snippet: The amplified fragments were analysed by electrophoreses in 1·5% agarose gels, stained with ethidium bromide and visualized on the Eagle Eye II Still Video System (Stratagene) using positive or negative images.

    Techniques: Expressing, Infection, Amplification, Polymerase Chain Reaction, Staining

    Expression of selected cytokine genes in chick embryo fibroblasts (CEF) stimulated with interferon-γ (IFN-γ) and lipopolysaccharide (LPS). Total RNAs were prepared from 5 × 10 6 CEF cells 18 hr post-treatment with 50 units/ml of recombinant chicken IFN-γ (rChIFN-γ) (lane 1), 25 ng/ml of LPS (lane 2), 50 units/ml of rChIFN-γ+ 25 ng/ml of LPS (lane 3) or control (lane 4). Total RNAs were reverse transcribed using oligo-dT(16) as primers. cDNAs were amplified using primers specific for interleukin (IL)-2, IL-6, IL-8, IFN-α, IFN-γ, inducible nitric oxide synthase (iNOS) and β-actin, and a ‘touch-down’ polymerase chain reaction (PCR) procedure. PCR products were separated in 1·5% agarose gels, stained with ethidium bromide, visualized on the Eagle Eye II Still Video System (Stratagene) and photographed as negative images. The sizes of the respective PCR fragments are indicated by arrows.

    Journal: Immunology

    Article Title: Expression of cytokine genes in Marek's disease virus-infected chickens and chicken embryo fibroblast cultures

    doi: 10.1046/j.1365-2567.2000.00008.x

    Figure Lengend Snippet: Expression of selected cytokine genes in chick embryo fibroblasts (CEF) stimulated with interferon-γ (IFN-γ) and lipopolysaccharide (LPS). Total RNAs were prepared from 5 × 10 6 CEF cells 18 hr post-treatment with 50 units/ml of recombinant chicken IFN-γ (rChIFN-γ) (lane 1), 25 ng/ml of LPS (lane 2), 50 units/ml of rChIFN-γ+ 25 ng/ml of LPS (lane 3) or control (lane 4). Total RNAs were reverse transcribed using oligo-dT(16) as primers. cDNAs were amplified using primers specific for interleukin (IL)-2, IL-6, IL-8, IFN-α, IFN-γ, inducible nitric oxide synthase (iNOS) and β-actin, and a ‘touch-down’ polymerase chain reaction (PCR) procedure. PCR products were separated in 1·5% agarose gels, stained with ethidium bromide, visualized on the Eagle Eye II Still Video System (Stratagene) and photographed as negative images. The sizes of the respective PCR fragments are indicated by arrows.

    Article Snippet: The amplified fragments were analysed by electrophoreses in 1·5% agarose gels, stained with ethidium bromide and visualized on the Eagle Eye II Still Video System (Stratagene) using positive or negative images.

    Techniques: Expressing, Recombinant, Amplification, Polymerase Chain Reaction, Staining

    Temporal expression of cytokines in N2a chickens infected at 1 day of age with Marek's disease herpesvirus (MDV) strain JM16/19. Total RNA was prepared from pools of three spleens obtained from non-infected (N) and JM16/19-infected (I) chickens on 3, 6, 9, 12 and 15 days post-infection (p.i.) and used for reverse transcription–polymerase chain reaction (RT–PCR) analysis. Each lane represents a pool of three spleens obtained from different chickens. Primers specific for chicken interleukin (IL)-1β, IL-2, IL-6, IL-8, interferon-γ (IFN-γ), inducible nitric oxide synthase (iNOS) and β-actin were used. PCR products were electrophoresed on 1·5% agarose, stained with ethidium bromide and visualized on the Eagle Eye II Still Video System (Stratagene), and photographed as positive images. DNA markers (123-bp ladder) were used at both sides of the gel and the sizes of the respective cytokine gene fragments are indicated.

    Journal: Immunology

    Article Title: Expression of cytokine genes in Marek's disease virus-infected chickens and chicken embryo fibroblast cultures

    doi: 10.1046/j.1365-2567.2000.00008.x

    Figure Lengend Snippet: Temporal expression of cytokines in N2a chickens infected at 1 day of age with Marek's disease herpesvirus (MDV) strain JM16/19. Total RNA was prepared from pools of three spleens obtained from non-infected (N) and JM16/19-infected (I) chickens on 3, 6, 9, 12 and 15 days post-infection (p.i.) and used for reverse transcription–polymerase chain reaction (RT–PCR) analysis. Each lane represents a pool of three spleens obtained from different chickens. Primers specific for chicken interleukin (IL)-1β, IL-2, IL-6, IL-8, interferon-γ (IFN-γ), inducible nitric oxide synthase (iNOS) and β-actin were used. PCR products were electrophoresed on 1·5% agarose, stained with ethidium bromide and visualized on the Eagle Eye II Still Video System (Stratagene), and photographed as positive images. DNA markers (123-bp ladder) were used at both sides of the gel and the sizes of the respective cytokine gene fragments are indicated.

    Article Snippet: The amplified fragments were analysed by electrophoreses in 1·5% agarose gels, stained with ethidium bromide and visualized on the Eagle Eye II Still Video System (Stratagene) using positive or negative images.

    Techniques: Expressing, Infection, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Staining

    Expression of interleukin-1β (IL-1β) in chick embryo fibroblasts (CEF) stimulated with interferon-γ (IFN-γ) and lipopolysaccharide (LPS). Total RNAs were prepared from 5 × 10 6 CEF cells 18 hr post-treatment with 50 units/ml of recombinant chicken IFN-γ (rChIFN-γ) (lane 1), 25 ng/ml of LPS (lane 2), 50 units/ml of rChIFN-γ+ 25 ng/ml of LPS (lane 3) or control (lane 4). Total RNA was reverse transcribed with oligo dT(16) as primers. cDNA was amplified using primers specific for IL-1β and β-actin, using a ‘touch-down’ polymerase chain reaction (PCR) procedure. PCR products were separated in 1·5% agarose, stained with ethidium bromide, visualized on the Eagle Eye II Still Video System (Stratagene) and photographed as positive images. M, molecular weight ladder.

    Journal: Immunology

    Article Title: Expression of cytokine genes in Marek's disease virus-infected chickens and chicken embryo fibroblast cultures

    doi: 10.1046/j.1365-2567.2000.00008.x

    Figure Lengend Snippet: Expression of interleukin-1β (IL-1β) in chick embryo fibroblasts (CEF) stimulated with interferon-γ (IFN-γ) and lipopolysaccharide (LPS). Total RNAs were prepared from 5 × 10 6 CEF cells 18 hr post-treatment with 50 units/ml of recombinant chicken IFN-γ (rChIFN-γ) (lane 1), 25 ng/ml of LPS (lane 2), 50 units/ml of rChIFN-γ+ 25 ng/ml of LPS (lane 3) or control (lane 4). Total RNA was reverse transcribed with oligo dT(16) as primers. cDNA was amplified using primers specific for IL-1β and β-actin, using a ‘touch-down’ polymerase chain reaction (PCR) procedure. PCR products were separated in 1·5% agarose, stained with ethidium bromide, visualized on the Eagle Eye II Still Video System (Stratagene) and photographed as positive images. M, molecular weight ladder.

    Article Snippet: The amplified fragments were analysed by electrophoreses in 1·5% agarose gels, stained with ethidium bromide and visualized on the Eagle Eye II Still Video System (Stratagene) using positive or negative images.

    Techniques: Expressing, Recombinant, Amplification, Polymerase Chain Reaction, Staining, Molecular Weight

    Double-strands breaks do not accumulate in rad52 cells treated with MMS. Chromosomes were visualized by ethidium bromide or Southern hybridization. ( A ) After exposing rad52 cells to 0.05% MMS for 30 min heating to 55°C, which causes massive chromosome fragmentation. ( B ) No chromosome fragmentation was observed in rad52 cells treated with either 0.05 or 0.1% MMS for 30–90 min.

    Journal: Nucleic Acids Research

    Article Title: Methyl methanesulfonate (MMS) produces heat-labile DNA damage but no detectable in vivo DNA double-strand breaks

    doi: 10.1093/nar/gki681

    Figure Lengend Snippet: Double-strands breaks do not accumulate in rad52 cells treated with MMS. Chromosomes were visualized by ethidium bromide or Southern hybridization. ( A ) After exposing rad52 cells to 0.05% MMS for 30 min heating to 55°C, which causes massive chromosome fragmentation. ( B ) No chromosome fragmentation was observed in rad52 cells treated with either 0.05 or 0.1% MMS for 30–90 min.

    Article Snippet: Separation was performed on a CHEF DR III equipment (BioRad; 120° field angle, 240 s switch time, 4 V/cm, 14°C) for 18 h. The gel was stained with ethidium bromide overnight and subsequently analysed by a scanning fluorescence reader (FLA-3000, Fujifilm) using Image Gauge software.

    Techniques: Hybridization

    MMS produces heat-labile DNA damage in S.cerevisiae . PFGE of yeast chromosomes after a 0.5 h MMS treatment (0.05%) and treatment with proteinase K at 50°C ( A ) or at 30°C ( B ) for 24 h. Chromosomes were visualized by ethidium bromide or Southern hybridization to highlight chromosome VIII directly after MMS treatments or following repair as indicated.

    Journal: Nucleic Acids Research

    Article Title: Methyl methanesulfonate (MMS) produces heat-labile DNA damage but no detectable in vivo DNA double-strand breaks

    doi: 10.1093/nar/gki681

    Figure Lengend Snippet: MMS produces heat-labile DNA damage in S.cerevisiae . PFGE of yeast chromosomes after a 0.5 h MMS treatment (0.05%) and treatment with proteinase K at 50°C ( A ) or at 30°C ( B ) for 24 h. Chromosomes were visualized by ethidium bromide or Southern hybridization to highlight chromosome VIII directly after MMS treatments or following repair as indicated.

    Article Snippet: Separation was performed on a CHEF DR III equipment (BioRad; 120° field angle, 240 s switch time, 4 V/cm, 14°C) for 18 h. The gel was stained with ethidium bromide overnight and subsequently analysed by a scanning fluorescence reader (FLA-3000, Fujifilm) using Image Gauge software.

    Techniques: Hybridization

    Alkylating agents induce DSBs randomly in DNA. The DNA of AA8 cells was labelled with 14 C-thymidine ( 14 C-TdR), either homogeneously for 24 h or specifically at sites of replication for 30 min, prior to exposure to 3 mM MMS, 10 μM MNNG, 2 mM HU or γ-rays (50 Gy). DNA was separated utilizing PFGE and visualized by ethidium bromide staining ( A ) and autoradiography ( B ).

    Journal: Nucleic Acids Research

    Article Title: Methyl methanesulfonate (MMS) produces heat-labile DNA damage but no detectable in vivo DNA double-strand breaks

    doi: 10.1093/nar/gki681

    Figure Lengend Snippet: Alkylating agents induce DSBs randomly in DNA. The DNA of AA8 cells was labelled with 14 C-thymidine ( 14 C-TdR), either homogeneously for 24 h or specifically at sites of replication for 30 min, prior to exposure to 3 mM MMS, 10 μM MNNG, 2 mM HU or γ-rays (50 Gy). DNA was separated utilizing PFGE and visualized by ethidium bromide staining ( A ) and autoradiography ( B ).

    Article Snippet: Separation was performed on a CHEF DR III equipment (BioRad; 120° field angle, 240 s switch time, 4 V/cm, 14°C) for 18 h. The gel was stained with ethidium bromide overnight and subsequently analysed by a scanning fluorescence reader (FLA-3000, Fujifilm) using Image Gauge software.

    Techniques: Staining, Autoradiography

    CAY10593 impairs ATP-induced ethidium + uptake into RPMI 8226 cells in a non-competitive-like manner. RPMI 8226 cells in NaCl medium were pre-incubated at 37 °C for 15 min in the presence of DMSO, or 2 μM or 10 μM

    Journal: Purinergic Signalling

    Article Title: CAY10593 inhibits the human P2X7 receptor independently of phospholipase D1 stimulation

    doi: 10.1007/s11302-013-9371-6

    Figure Lengend Snippet: CAY10593 impairs ATP-induced ethidium + uptake into RPMI 8226 cells in a non-competitive-like manner. RPMI 8226 cells in NaCl medium were pre-incubated at 37 °C for 15 min in the presence of DMSO, or 2 μM or 10 μM

    Article Snippet: Dimethyl sulphoxide (DMSO) and ethidium bromide were from Amresco (Solon, OH).

    Techniques: Incubation

    CAY10593 impairs ATP-induced ethidium + uptake in primary human peripheral blood mononuclear cells (PBMCs). PBMCs in NaCl medium were pre-incubated for 15 min in the presence of DMSO or 10 μM CAY10593. Cells were then incubated

    Journal: Purinergic Signalling

    Article Title: CAY10593 inhibits the human P2X7 receptor independently of phospholipase D1 stimulation

    doi: 10.1007/s11302-013-9371-6

    Figure Lengend Snippet: CAY10593 impairs ATP-induced ethidium + uptake in primary human peripheral blood mononuclear cells (PBMCs). PBMCs in NaCl medium were pre-incubated for 15 min in the presence of DMSO or 10 μM CAY10593. Cells were then incubated

    Article Snippet: Dimethyl sulphoxide (DMSO) and ethidium bromide were from Amresco (Solon, OH).

    Techniques: Incubation

    PLD1 is not required for ATP-induced ethidium + uptake into RPMI 8226 cells. a RNA was isolated from A431 (positive control) and RPMI 8226 cells, and then analysed by RT-PCR using primers for PLD1 and PLD2. RNA substituted with H 2 O was used as a negative

    Journal: Purinergic Signalling

    Article Title: CAY10593 inhibits the human P2X7 receptor independently of phospholipase D1 stimulation

    doi: 10.1007/s11302-013-9371-6

    Figure Lengend Snippet: PLD1 is not required for ATP-induced ethidium + uptake into RPMI 8226 cells. a RNA was isolated from A431 (positive control) and RPMI 8226 cells, and then analysed by RT-PCR using primers for PLD1 and PLD2. RNA substituted with H 2 O was used as a negative

    Article Snippet: Dimethyl sulphoxide (DMSO) and ethidium bromide were from Amresco (Solon, OH).

    Techniques: Isolation, Positive Control, Reverse Transcription Polymerase Chain Reaction

    P2X7 antagonists impair ATP-induced ethidium + uptake into RPMI 8226 cells. RPMI 8226 cells in NaCl medium were pre-incubated at 37 °C for 15 min in the absence or presence of varying concentrations of antagonist (as indicated).

    Journal: Purinergic Signalling

    Article Title: CAY10593 inhibits the human P2X7 receptor independently of phospholipase D1 stimulation

    doi: 10.1007/s11302-013-9371-6

    Figure Lengend Snippet: P2X7 antagonists impair ATP-induced ethidium + uptake into RPMI 8226 cells. RPMI 8226 cells in NaCl medium were pre-incubated at 37 °C for 15 min in the absence or presence of varying concentrations of antagonist (as indicated).

    Article Snippet: Dimethyl sulphoxide (DMSO) and ethidium bromide were from Amresco (Solon, OH).

    Techniques: Incubation

    CAY10593, CAY10594 and halopemide impair ATP-induced ethidium + uptake into RPMI 8226 cells in a concentration-dependent manner. Cells in NaCl medium were pre-incubated at 37 °C for 15 min in the presence of DMSO or varying concentrations

    Journal: Purinergic Signalling

    Article Title: CAY10593 inhibits the human P2X7 receptor independently of phospholipase D1 stimulation

    doi: 10.1007/s11302-013-9371-6

    Figure Lengend Snippet: CAY10593, CAY10594 and halopemide impair ATP-induced ethidium + uptake into RPMI 8226 cells in a concentration-dependent manner. Cells in NaCl medium were pre-incubated at 37 °C for 15 min in the presence of DMSO or varying concentrations

    Article Snippet: Dimethyl sulphoxide (DMSO) and ethidium bromide were from Amresco (Solon, OH).

    Techniques: Concentration Assay, Incubation

    PLD antagonists impair ATP-induced CD23 shedding and ethidium + uptake in RPMI 8226 cells. a , b Cells in NaCl medium were pre-incubated at 37 °C for 15 min in the presence of DMSO, or 10 μM CAY10593, CAY10594 or

    Journal: Purinergic Signalling

    Article Title: CAY10593 inhibits the human P2X7 receptor independently of phospholipase D1 stimulation

    doi: 10.1007/s11302-013-9371-6

    Figure Lengend Snippet: PLD antagonists impair ATP-induced CD23 shedding and ethidium + uptake in RPMI 8226 cells. a , b Cells in NaCl medium were pre-incubated at 37 °C for 15 min in the presence of DMSO, or 10 μM CAY10593, CAY10594 or

    Article Snippet: Dimethyl sulphoxide (DMSO) and ethidium bromide were from Amresco (Solon, OH).

    Techniques: Incubation

    Platelets are not associated with dying parasites during nonresolving, eCM-eliciting Pb A in C57BL/6 mice. The analyses were performed in thin blood films fluorescence labeled with anti-CD41 (blue: platelets) and ethidium bromide (red: parasites) and TUNEL (green). (A) Platelet effector-to-target (iRBC) ratios (filled blue square), platelet:uRBC (open green square) ratios, and parasitemia (filled red circle) on left axis and platelet counts/mL (open blue circle) on right axis. (B) Percent PbA parasitemia for all RBCs (red bars) ([# iRBC /# RBC ]%) and considering only RBCs with bound platelets (blue bars) ([# platelet:iRBC /# plateletRBC ]%). Platelet:RBC indicates a platelet RBC conjugate; the RBC maybe uninfected (uRBC) or infected (iRBC). (C) Percentage of RBCs with bound platelets with uninfected (red bars) ([# platelet:uRBC /# plateletRBC ]%) or infected (blue bars) RBCs: ([# platelet:iRBC /# platelet:RBC ]%). (D) The percentage of iRBCs with an adherent platelet ([# platelet:iRBC /# iRBCs ]%). (E) Representative thin blood film made on day 6PI and fluorescently labeled; blue arrows indicate platelets, red arrows indicate parasites without TUNEL, and yellow arrows indicate parasites with TUNEL (green+red). (F) Percentage on days 4 and 6PI of iRBCs exhibiting TUNEL + labeling (red bar, x-axis) ([(# TUNEL + iRBCs )/# iRBCs ]%) and TUNEL – (green bar, x-axis) ([(# TUNEL − iRBCs )/# iRBCs ]%) with the percentage of each iRBC + , TUNEL + or iRBC + , TUNEL – ([(# platelet:TUNEL − iRBCs )/# iRBCs ]%) exhibiting an adherent platelet shown as an inset (blue bar). This experiment was repeated twice (n = 5) and verified by flow cytometry. Values are average ± SEM.

    Journal: Blood

    Article Title: Platelets activate a pathogenic response to blood-stage Plasmodium infection but not a protective immune response

    doi: 10.1182/blood-2016-08-733519

    Figure Lengend Snippet: Platelets are not associated with dying parasites during nonresolving, eCM-eliciting Pb A in C57BL/6 mice. The analyses were performed in thin blood films fluorescence labeled with anti-CD41 (blue: platelets) and ethidium bromide (red: parasites) and TUNEL (green). (A) Platelet effector-to-target (iRBC) ratios (filled blue square), platelet:uRBC (open green square) ratios, and parasitemia (filled red circle) on left axis and platelet counts/mL (open blue circle) on right axis. (B) Percent PbA parasitemia for all RBCs (red bars) ([# iRBC /# RBC ]%) and considering only RBCs with bound platelets (blue bars) ([# platelet:iRBC /# plateletRBC ]%). Platelet:RBC indicates a platelet RBC conjugate; the RBC maybe uninfected (uRBC) or infected (iRBC). (C) Percentage of RBCs with bound platelets with uninfected (red bars) ([# platelet:uRBC /# plateletRBC ]%) or infected (blue bars) RBCs: ([# platelet:iRBC /# platelet:RBC ]%). (D) The percentage of iRBCs with an adherent platelet ([# platelet:iRBC /# iRBCs ]%). (E) Representative thin blood film made on day 6PI and fluorescently labeled; blue arrows indicate platelets, red arrows indicate parasites without TUNEL, and yellow arrows indicate parasites with TUNEL (green+red). (F) Percentage on days 4 and 6PI of iRBCs exhibiting TUNEL + labeling (red bar, x-axis) ([(# TUNEL + iRBCs )/# iRBCs ]%) and TUNEL – (green bar, x-axis) ([(# TUNEL − iRBCs )/# iRBCs ]%) with the percentage of each iRBC + , TUNEL + or iRBC + , TUNEL – ([(# platelet:TUNEL − iRBCs )/# iRBCs ]%) exhibiting an adherent platelet shown as an inset (blue bar). This experiment was repeated twice (n = 5) and verified by flow cytometry. Values are average ± SEM.

    Article Snippet: Parasite DNA and RNA were fluorescence labeled with 12 µM ethidium bromide (Fisher Scientific, Fair Lawn, NJ) in PBS for 15 minutes.

    Techniques: Mouse Assay, Fluorescence, Labeling, TUNEL Assay, Infection, Flow Cytometry, Cytometry

    Platelets are not associated with dying parasites during resolving non-eCM Pc a infection in thin blood films. The analyses were performed in thin blood films fluorescence labeled with anti-CD41 (blue; platelets), ethidium bromide (red: parasites), and TUNEL (green). (A) Platelet effector-to-target (iRBC) ratios (filled blue square), platelet:uRBC ratios (open green square), and parasitemia (filled red circle). Platelet:RBC indicates a platelet RBC conjugate; the RBC maybe uninfected (uRBC) or infected (iRBC). (B) Percent PbA parasitemia for all RBCs (red square) ([# iRBC /# RBC ]%) and considering only RBCs with bound platelets (blue square) ([# platelet:iRBC/ # platelet:RBC ]%). (C) Percentage of RBCs with bound platelets with uninfected (red) ([# platelet:uRBC /# platelet:RBC ]%) or infected (blue bars) RBCs: ([# platelet:iRBC /# platelet:RBC ]%). (D) The percentage of iRBCs with an adherent platelet ([# platelet:iRBC /# iRBC ]%). (E) Representative thin blood film made on day 6PI and fluorescently labeled; blue arrows indicate platelets, red arrows indicate parasites without TUNEL, and yellow arrows indicate parasites with TUNEL (green+red). (F) Percentage on days 6, 8, and 12PI of iRBCs exhibiting TUNEL + labeling (green bar, x-axis) ([(# TUNEL + iRBCs )/# iRBCs ]%) and TUNEL – (red bar, x-axis) ([(# TUNEL − iRBCs )/# iRBCs ]%) with the percentage of each iRBC+, TUNEL + or iRBC + , TUNEL – ([(# platelet:TUNEL − iRBCs )/# iRBCs ]%) exhibiting an adherent platelet shown as an inset (blue bar). This experiment was repeated twice (n = 5) and verified by flow cytometry. Values are average ± SEM.

    Journal: Blood

    Article Title: Platelets activate a pathogenic response to blood-stage Plasmodium infection but not a protective immune response

    doi: 10.1182/blood-2016-08-733519

    Figure Lengend Snippet: Platelets are not associated with dying parasites during resolving non-eCM Pc a infection in thin blood films. The analyses were performed in thin blood films fluorescence labeled with anti-CD41 (blue; platelets), ethidium bromide (red: parasites), and TUNEL (green). (A) Platelet effector-to-target (iRBC) ratios (filled blue square), platelet:uRBC ratios (open green square), and parasitemia (filled red circle). Platelet:RBC indicates a platelet RBC conjugate; the RBC maybe uninfected (uRBC) or infected (iRBC). (B) Percent PbA parasitemia for all RBCs (red square) ([# iRBC /# RBC ]%) and considering only RBCs with bound platelets (blue square) ([# platelet:iRBC/ # platelet:RBC ]%). (C) Percentage of RBCs with bound platelets with uninfected (red) ([# platelet:uRBC /# platelet:RBC ]%) or infected (blue bars) RBCs: ([# platelet:iRBC /# platelet:RBC ]%). (D) The percentage of iRBCs with an adherent platelet ([# platelet:iRBC /# iRBC ]%). (E) Representative thin blood film made on day 6PI and fluorescently labeled; blue arrows indicate platelets, red arrows indicate parasites without TUNEL, and yellow arrows indicate parasites with TUNEL (green+red). (F) Percentage on days 6, 8, and 12PI of iRBCs exhibiting TUNEL + labeling (green bar, x-axis) ([(# TUNEL + iRBCs )/# iRBCs ]%) and TUNEL – (red bar, x-axis) ([(# TUNEL − iRBCs )/# iRBCs ]%) with the percentage of each iRBC+, TUNEL + or iRBC + , TUNEL – ([(# platelet:TUNEL − iRBCs )/# iRBCs ]%) exhibiting an adherent platelet shown as an inset (blue bar). This experiment was repeated twice (n = 5) and verified by flow cytometry. Values are average ± SEM.

    Article Snippet: Parasite DNA and RNA were fluorescence labeled with 12 µM ethidium bromide (Fisher Scientific, Fair Lawn, NJ) in PBS for 15 minutes.

    Techniques: Infection, Fluorescence, Labeling, TUNEL Assay, Flow Cytometry, Cytometry