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    Structured Review

    Thermo Fisher kbp circular dna molecules
    Schematic of the experimental ring-linear system. Fluorescently labeled tracer ring <t>DNA</t> molecules (45 <t>kbp,</t> shown in red) are uniformly dissolved in a background solution of semidilute linear DNA molecules. Dynamics are studied under a equilibrium (no flow) conditions and in b planar extensional flow. The transient molecular extension of ring polymers l circ is directly observed using SMFM
    Kbp Circular Dna Molecules, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 62699 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Effect of molecular architecture on ring polymer dynamics in semidilute linear polymer solutions"

    Article Title: Effect of molecular architecture on ring polymer dynamics in semidilute linear polymer solutions

    Journal: Nature Communications

    doi: 10.1038/s41467-019-09627-7

    Schematic of the experimental ring-linear system. Fluorescently labeled tracer ring DNA molecules (45 kbp, shown in red) are uniformly dissolved in a background solution of semidilute linear DNA molecules. Dynamics are studied under a equilibrium (no flow) conditions and in b planar extensional flow. The transient molecular extension of ring polymers l circ is directly observed using SMFM
    Figure Legend Snippet: Schematic of the experimental ring-linear system. Fluorescently labeled tracer ring DNA molecules (45 kbp, shown in red) are uniformly dissolved in a background solution of semidilute linear DNA molecules. Dynamics are studied under a equilibrium (no flow) conditions and in b planar extensional flow. The transient molecular extension of ring polymers l circ is directly observed using SMFM

    Techniques Used: Labeling, Flow Cytometry

    2) Product Images from "Susceptibility to Neurodegeneration in a Glaucoma Is Modified by Bax Gene Dosage"

    Article Title: Susceptibility to Neurodegeneration in a Glaucoma Is Modified by Bax Gene Dosage

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.0010004

    Dying RGCs Have Characteristic Features of Apoptosis (A–C) A double-labeling assay that identifies fragmented DNA using fluorescently labeled dUTP (A) and detects chromatin condensation by binding of the dye YOYO-1 (B) was used to assess the presence of these hallmarks of apoptosis in glaucomatous DBA/2J eyes at 10–11 mo of age (a time when many RGCs die). A cell in the retinal ganglion cell layer (GCL, arrowhead) has both of these features of apoptosis as indicated by double labeling (C). INL, inner nuclear layer. (D–F) Electron microscopy provided further evidence for apoptosis. (D) An example of a healthy RGC. (E) Chromatin condensation (a hallmark of apoptosis) along the inner surface of the nuclear envelope in a ganglion cell (arrows). The internal limiting membrane of the retina is indicated by arrowheads. (F) An apoptotic body in the ganglion cell layer (arrows) containing a nuclear fragment with prominent condensed chromatin (asterisk) and other cell remnants. (G) A TUNEL assay (see Materials and Methods ) was used to assess the prevalence of cell death at different ages. TUNEL labeling was not detected at 7 mo (an age prior to glaucomatous cell death) and peaked at 10–13 mo, when most RGCs die. No TUNEL-positive cells were detected in nonglaucomatous, age-matched control mice. These results support an important role of apoptosis in RGC death in spontaneous glaucoma. Scale bar, 1 μm.
    Figure Legend Snippet: Dying RGCs Have Characteristic Features of Apoptosis (A–C) A double-labeling assay that identifies fragmented DNA using fluorescently labeled dUTP (A) and detects chromatin condensation by binding of the dye YOYO-1 (B) was used to assess the presence of these hallmarks of apoptosis in glaucomatous DBA/2J eyes at 10–11 mo of age (a time when many RGCs die). A cell in the retinal ganglion cell layer (GCL, arrowhead) has both of these features of apoptosis as indicated by double labeling (C). INL, inner nuclear layer. (D–F) Electron microscopy provided further evidence for apoptosis. (D) An example of a healthy RGC. (E) Chromatin condensation (a hallmark of apoptosis) along the inner surface of the nuclear envelope in a ganglion cell (arrows). The internal limiting membrane of the retina is indicated by arrowheads. (F) An apoptotic body in the ganglion cell layer (arrows) containing a nuclear fragment with prominent condensed chromatin (asterisk) and other cell remnants. (G) A TUNEL assay (see Materials and Methods ) was used to assess the prevalence of cell death at different ages. TUNEL labeling was not detected at 7 mo (an age prior to glaucomatous cell death) and peaked at 10–13 mo, when most RGCs die. No TUNEL-positive cells were detected in nonglaucomatous, age-matched control mice. These results support an important role of apoptosis in RGC death in spontaneous glaucoma. Scale bar, 1 μm.

    Techniques Used: Labeling, Binding Assay, Electron Microscopy, TUNEL Assay, Mouse Assay

    Mechanical Axon Insult, but Not Excitotoxicity, Induces BAX-Dependent RGC Death To help distinguish between the likely roles of mechanical axon insult and excitotoxicity in cell death induction in spontaneous glaucoma, we subjected preglaucomatous DBA/2J mice of each Bax genotype to either controlled optic nerve crush or NMDA-mediated excitotoxicity. For controlled crush and NMDA, the percent RGC survival in the manipulated eye compared to the contralateral control eye is shown. For ease of comparison, the data for glaucomatous damage are the same as shown in Figure 3 . In contrast to the spontaneous glaucoma, NMDA-mediated RGC death is not dependent on BAX, as evident by the complete lack of protection from death in Bax −/− mice. As for the spontaneous glaucoma, RGC death induced by controlled optic nerve crush was completely dependent on BAX and prevented in both Bax +/− and Bax −/− mice. Overall, the effects of BAX in the face of spontaneous glaucoma and controlled crush were remarkably similar.
    Figure Legend Snippet: Mechanical Axon Insult, but Not Excitotoxicity, Induces BAX-Dependent RGC Death To help distinguish between the likely roles of mechanical axon insult and excitotoxicity in cell death induction in spontaneous glaucoma, we subjected preglaucomatous DBA/2J mice of each Bax genotype to either controlled optic nerve crush or NMDA-mediated excitotoxicity. For controlled crush and NMDA, the percent RGC survival in the manipulated eye compared to the contralateral control eye is shown. For ease of comparison, the data for glaucomatous damage are the same as shown in Figure 3 . In contrast to the spontaneous glaucoma, NMDA-mediated RGC death is not dependent on BAX, as evident by the complete lack of protection from death in Bax −/− mice. As for the spontaneous glaucoma, RGC death induced by controlled optic nerve crush was completely dependent on BAX and prevented in both Bax +/− and Bax −/− mice. Overall, the effects of BAX in the face of spontaneous glaucoma and controlled crush were remarkably similar.

    Techniques Used: Mouse Assay

    Complete Bax Deficiency Has a Protective Effect against IOP Elevation To assess the possibility that Bax deficiency may delay axon degeneration by lessening the glaucomatous insult to which RGCs are exposed, we analyzed IOP at key ages of IOP elevation. (A) The average IOP for each genotype (± SEM) and (B) the actual IOP values recorded. Bax deficiency did not prevent IOP elevation. At both 9 mo and 10.5 mo, the average IOP of both Bax +/− and Bax −/− mice was significantly elevated compared to preglaucomatous DBA/2J mice ( p
    Figure Legend Snippet: Complete Bax Deficiency Has a Protective Effect against IOP Elevation To assess the possibility that Bax deficiency may delay axon degeneration by lessening the glaucomatous insult to which RGCs are exposed, we analyzed IOP at key ages of IOP elevation. (A) The average IOP for each genotype (± SEM) and (B) the actual IOP values recorded. Bax deficiency did not prevent IOP elevation. At both 9 mo and 10.5 mo, the average IOP of both Bax +/− and Bax −/− mice was significantly elevated compared to preglaucomatous DBA/2J mice ( p

    Techniques Used: Mouse Assay

    3) Product Images from "Specific RNA binding to ordered phospholipid bilayers"

    Article Title: Specific RNA binding to ordered phospholipid bilayers

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl220

    ( A ) Fluorescence anisotropy of DPH-HPC in DMPC liposomes in the presence (gray) and in the absence (black) of RNA 10; ( B ) Dipole-field-dependent ratio of fluorescence intensities, 440/540, of RH-421 in DMPC liposomes in the presence (gray) and in the absence (black) of RNA 10. Data were collected by temperature scanning at 1°C/min. RNA concentration 1 µM, liposome concentration 75 µM.
    Figure Legend Snippet: ( A ) Fluorescence anisotropy of DPH-HPC in DMPC liposomes in the presence (gray) and in the absence (black) of RNA 10; ( B ) Dipole-field-dependent ratio of fluorescence intensities, 440/540, of RH-421 in DMPC liposomes in the presence (gray) and in the absence (black) of RNA 10. Data were collected by temperature scanning at 1°C/min. RNA concentration 1 µM, liposome concentration 75 µM.

    Techniques Used: Fluorescence, Concentration Assay

    4) Product Images from "Single molecule linear analysis of DNA in nano-channel labeled with sequence specific fluorescent probes"

    Article Title: Single molecule linear analysis of DNA in nano-channel labeled with sequence specific fluorescent probes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq673

    Image of the nano-channel array in a chip that has been used for the linearization of DNA. ( A ) Different regions of a nano-channel device. ( B ) Image showing the translocation of DNA through different areas (microstructure and nano-channel) of the chip. ( C ) Image of the relaxed and linearized DNA molecules inside nano-channel array. ( D ) Size distribution of BAC 3F5 DNA molecules inside nano-channel array.
    Figure Legend Snippet: Image of the nano-channel array in a chip that has been used for the linearization of DNA. ( A ) Different regions of a nano-channel device. ( B ) Image showing the translocation of DNA through different areas (microstructure and nano-channel) of the chip. ( C ) Image of the relaxed and linearized DNA molecules inside nano-channel array. ( D ) Size distribution of BAC 3F5 DNA molecules inside nano-channel array.

    Techniques Used: Chromatin Immunoprecipitation, Translocation Assay, BAC Assay

    Image of Fosmid G248P8446G6 DNA in nano-channel (60 nm × 100 nm). The DNA was nicked with Nb.BbvCI and the free 3′ end is extended by Vent (exo-) in presence of a mixture of three unlabeled nucleotides (dAGC) and Alexa-546 labeled dUTP ( Figure 3 A) or a mixture of four unlabeled nucleotides (dNTP) ( Figure 3 B). The DNA backbone was stained with intercalated dye YOYO-1 iodide. ( A ) Eight nicked sites were thus labeled with Alexa 546 (green). Two nicking sites at ∼9.3 and ∼9.5 kb were too close to resolve optically. The DNA backbone is indicated as a blue line. The positions of the labeled dyes match well with the predicted nicking positions on the backbone. ( B ) The generated single strand flaps (by nick translation in presence of dNTP mixtures) were hybridized with dye labeled probe of sequences Cy3-TGCCTGTGAGAGG-AAATCTCAACTCTCTT-Cy3. Five out of the eight single strand flaps contain the complement of the probe sequence and thus get hybridized. (B) shows five labels (red) along with the blue backbone. All these positions match well with the predicted ones ( 6 ). ( C ) Image shows several full length flap labeled Fosmid molecules inside a nano-channel array. ( D ) The prediction of labeling efficiency of one site in a DNA molecule with maximum five labeling sites available in it. The lines show the changes in the number distribution of 1, 2, 3, 4 and 5 labeled molecules with change in labeling efficiencies (30, 50, 75, 85, 90, 95 and 98%). The gray line is the distribution of number of molecules that were experimentally obtained from the flap labeled (five sites) Fosmid G248P8446G6. This line shows its labeling efficiency ∼85–90%. The imaging procedure is described in the ‘Material and Methods’ section.
    Figure Legend Snippet: Image of Fosmid G248P8446G6 DNA in nano-channel (60 nm × 100 nm). The DNA was nicked with Nb.BbvCI and the free 3′ end is extended by Vent (exo-) in presence of a mixture of three unlabeled nucleotides (dAGC) and Alexa-546 labeled dUTP ( Figure 3 A) or a mixture of four unlabeled nucleotides (dNTP) ( Figure 3 B). The DNA backbone was stained with intercalated dye YOYO-1 iodide. ( A ) Eight nicked sites were thus labeled with Alexa 546 (green). Two nicking sites at ∼9.3 and ∼9.5 kb were too close to resolve optically. The DNA backbone is indicated as a blue line. The positions of the labeled dyes match well with the predicted nicking positions on the backbone. ( B ) The generated single strand flaps (by nick translation in presence of dNTP mixtures) were hybridized with dye labeled probe of sequences Cy3-TGCCTGTGAGAGG-AAATCTCAACTCTCTT-Cy3. Five out of the eight single strand flaps contain the complement of the probe sequence and thus get hybridized. (B) shows five labels (red) along with the blue backbone. All these positions match well with the predicted ones ( 6 ). ( C ) Image shows several full length flap labeled Fosmid molecules inside a nano-channel array. ( D ) The prediction of labeling efficiency of one site in a DNA molecule with maximum five labeling sites available in it. The lines show the changes in the number distribution of 1, 2, 3, 4 and 5 labeled molecules with change in labeling efficiencies (30, 50, 75, 85, 90, 95 and 98%). The gray line is the distribution of number of molecules that were experimentally obtained from the flap labeled (five sites) Fosmid G248P8446G6. This line shows its labeling efficiency ∼85–90%. The imaging procedure is described in the ‘Material and Methods’ section.

    Techniques Used: Labeling, Staining, Generated, Nick Translation, Sequencing, Imaging

    5) Product Images from "ATP-gated P2X7 receptors require chloride channels to promote inflammation in human macrophages"

    Article Title: ATP-gated P2X7 receptors require chloride channels to promote inflammation in human macrophages

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1801101

    P2X7R-mediated activation of a Cl − channel is responsible for human macrophage permeabilization. (a) Representative images of macrophages pre-incubated for 30 min with the non-selective Cl − channel inhibitors tannic acid (TA, 20 µM), A01 (40 µM), DIDS (100 µM), and NPPB (0.5 mM). YO-PRO-1 uptake was measured after 15 min in the presence of ATP (2 mM) in normal ECS at 37°C. The independence of YO-PRO-1 uptake on Ca 2+ flux was assessed by loading the cells with 10 µM BAPTA-AM and measuring dye uptake in Ca 2+ free ECS with 1 mM EDTA (-Ca 2+ ). Scale bars: 20 μm. (b) YO-PRO-1 fluorescence in Ca 2+ free conditions (-Ca 2+ ) was significantly different from control (“a” is equal to p
    Figure Legend Snippet: P2X7R-mediated activation of a Cl − channel is responsible for human macrophage permeabilization. (a) Representative images of macrophages pre-incubated for 30 min with the non-selective Cl − channel inhibitors tannic acid (TA, 20 µM), A01 (40 µM), DIDS (100 µM), and NPPB (0.5 mM). YO-PRO-1 uptake was measured after 15 min in the presence of ATP (2 mM) in normal ECS at 37°C. The independence of YO-PRO-1 uptake on Ca 2+ flux was assessed by loading the cells with 10 µM BAPTA-AM and measuring dye uptake in Ca 2+ free ECS with 1 mM EDTA (-Ca 2+ ). Scale bars: 20 μm. (b) YO-PRO-1 fluorescence in Ca 2+ free conditions (-Ca 2+ ) was significantly different from control (“a” is equal to p

    Techniques Used: Activation Assay, Incubation, Fluorescence

    6) Product Images from "ATP-gated P2X7 receptors require chloride channels to promote inflammation in human macrophages"

    Article Title: ATP-gated P2X7 receptors require chloride channels to promote inflammation in human macrophages

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1801101

    The P2X7R permeabilization pathway is cation selective. (a) Fluorescence images of human macrophages incubated in the presence of ATP (2 mM) with the anionic dyes Lucifer yellow (LY, 0.5 mM) or carboxyfluorescein (CF, 0.5 mM) at 37°C. Scale bars: 20 μm. (b) Quantitative comparison of anionic dye uptake by macrophages. Cells were incubated for 15 min with LY or CF in the presence of ATP (2 mM) at 37°C. “n.s.” are not significantly different from ATP (n = 4). (c) Fluorescence images of human macrophages preloaded with anionic calcein-AM (0.5 µM) for 30 min and subsequently stimulated with ATP (2 mM) for 15 min at 37°C. (d) Quantification of change in intracellular calcein fluorescence after 15 min stimulation with ATP (2 mM). There is no significant difference in fluorescence dye after ATP treatment (n = 4 separate experiments). (e) Fluorescence images of human macrophages incubated in the presence of ATP (2 mM) with the cationic dye YOYO-1 (5 µM) for 15 mins at 37°C. (f) Quantification of YOYO-1 after macrophages were incubated for 15 min with ATP (2 mM) at 37°C. There is significant uptake of dye by ATP treated cells (p
    Figure Legend Snippet: The P2X7R permeabilization pathway is cation selective. (a) Fluorescence images of human macrophages incubated in the presence of ATP (2 mM) with the anionic dyes Lucifer yellow (LY, 0.5 mM) or carboxyfluorescein (CF, 0.5 mM) at 37°C. Scale bars: 20 μm. (b) Quantitative comparison of anionic dye uptake by macrophages. Cells were incubated for 15 min with LY or CF in the presence of ATP (2 mM) at 37°C. “n.s.” are not significantly different from ATP (n = 4). (c) Fluorescence images of human macrophages preloaded with anionic calcein-AM (0.5 µM) for 30 min and subsequently stimulated with ATP (2 mM) for 15 min at 37°C. (d) Quantification of change in intracellular calcein fluorescence after 15 min stimulation with ATP (2 mM). There is no significant difference in fluorescence dye after ATP treatment (n = 4 separate experiments). (e) Fluorescence images of human macrophages incubated in the presence of ATP (2 mM) with the cationic dye YOYO-1 (5 µM) for 15 mins at 37°C. (f) Quantification of YOYO-1 after macrophages were incubated for 15 min with ATP (2 mM) at 37°C. There is significant uptake of dye by ATP treated cells (p

    Techniques Used: Fluorescence, Incubation

    7) Product Images from "Visualization of oligonucleotide probes and point mutations in interphase nuclei and DNA fibers using rolling circle DNA amplification"

    Article Title: Visualization of oligonucleotide probes and point mutations in interphase nuclei and DNA fibers using rolling circle DNA amplification

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

    doi: 10.1073/pnas.061026198

    RCA detection of wt and mu alleles at the G542X locus of the CFTR gene. ( A ) Nuclei from wt, homozygous mu, and heterozygous cells in G 1 phase ( Upper ) or G 2 phase ( Lower ) of cell cycle. ( B ) Discrimination of wt and mu alleles of the G542X locus on stretched DNA fibers.
    Figure Legend Snippet: RCA detection of wt and mu alleles at the G542X locus of the CFTR gene. ( A ) Nuclei from wt, homozygous mu, and heterozygous cells in G 1 phase ( Upper ) or G 2 phase ( Lower ) of cell cycle. ( B ) Discrimination of wt and mu alleles of the G542X locus on stretched DNA fibers.

    Techniques Used:

    Visualization of ODN probes hybridized to 50-mer target sequences in the CFTR gene in interphase nuclei and DNA fibers. ( A ) RCA detection of probes targeted to the G542X locus (FITC), the Δ508 locus (Cy-3), and the M1101K locus (Cy5) in normal human lymphocytes. Merged images (com) show that signals from all three loci colocalize. ( B ) Hybridization of the Δ508 locus probe to nuclei of HeLa cells. ( C ) Cohybridization of two PAC clones (extended green and red signals) with Δ508 (yellow), G542X (green), and M1101K (white) oligomer probes. Eight patterns ( a – h ) of hybridization are produced (see text).
    Figure Legend Snippet: Visualization of ODN probes hybridized to 50-mer target sequences in the CFTR gene in interphase nuclei and DNA fibers. ( A ) RCA detection of probes targeted to the G542X locus (FITC), the Δ508 locus (Cy-3), and the M1101K locus (Cy5) in normal human lymphocytes. Merged images (com) show that signals from all three loci colocalize. ( B ) Hybridization of the Δ508 locus probe to nuclei of HeLa cells. ( C ) Cohybridization of two PAC clones (extended green and red signals) with Δ508 (yellow), G542X (green), and M1101K (white) oligomer probes. Eight patterns ( a – h ) of hybridization are produced (see text).

    Techniques Used: Hybridization, Clone Assay, Produced

    8) Product Images from "PLASMALEMMA PERMEABILITY AND NECROTIC CELL DEATH PHENOTYPES AFTER INTRACEREBRAL HEMORRHAGE IN MICE"

    Article Title: PLASMALEMMA PERMEABILITY AND NECROTIC CELL DEATH PHENOTYPES AFTER INTRACEREBRAL HEMORRHAGE IN MICE

    Journal: Stroke; a journal of cerebral circulation

    doi: 10.1161/STROKEAHA.111.635672

    Identification of permeable cell types after intracerebral hemorrhage (ICH). (A-C) Detection of cell types with plasmalemma permeability to YOYO-1 iodide at 6 h after ICH. (A) Representative photomicrographs of YOYO-1+ cells (a), IBA-1+ (b), and overlay (c) showing no colocalization. (B) Representative photomicrographs of YOYO-1+ cells (d), GFAP+ cells (e), and overlay (f) showing no colocalization. (C) Representative photomicrographs of NeuN+ neurons (g) that colocalized with YOYO-1 (h) at 6 h after ICH (i, overlay) suggesting that neurons are particularly sensitive to plasmalemma damage early after ICH. (D) Detection of cell types with plasmalemma permeability to YOYO-1 at 24 h after ICH. By 24 h, YOYO-1+ cells (j) colocalized with IBA-1+ cells with morphological features of microglia (k) as shown in the overlay (l). YOYO-1+ cells (m) also colocalized with IBA-1+ cells with the morphological appearance of macrophages (n) at 24 h (o, overlay). Scale bar, 10 um for each panel.
    Figure Legend Snippet: Identification of permeable cell types after intracerebral hemorrhage (ICH). (A-C) Detection of cell types with plasmalemma permeability to YOYO-1 iodide at 6 h after ICH. (A) Representative photomicrographs of YOYO-1+ cells (a), IBA-1+ (b), and overlay (c) showing no colocalization. (B) Representative photomicrographs of YOYO-1+ cells (d), GFAP+ cells (e), and overlay (f) showing no colocalization. (C) Representative photomicrographs of NeuN+ neurons (g) that colocalized with YOYO-1 (h) at 6 h after ICH (i, overlay) suggesting that neurons are particularly sensitive to plasmalemma damage early after ICH. (D) Detection of cell types with plasmalemma permeability to YOYO-1 at 24 h after ICH. By 24 h, YOYO-1+ cells (j) colocalized with IBA-1+ cells with morphological features of microglia (k) as shown in the overlay (l). YOYO-1+ cells (m) also colocalized with IBA-1+ cells with the morphological appearance of macrophages (n) at 24 h (o, overlay). Scale bar, 10 um for each panel.

    Techniques Used: Permeability

    Plasmalemma resealing after intracerebral hemorrhage. Mice were subjected to ICH and administered YOYO-1 at 24 or 48 h and PI at 48 or 72 h, respectively. (a–d) representative photomicrographs of brain sections showing YOYO-1+ and PI+ cells labeled at 24–48 h. Note that the majority of YOYO-1+ cells are also PI+, however some YOYO-1+ cells are PI-negative (arrows). Scale bars: (a, b) 60 um; (c, d), 20 um.
    Figure Legend Snippet: Plasmalemma resealing after intracerebral hemorrhage. Mice were subjected to ICH and administered YOYO-1 at 24 or 48 h and PI at 48 or 72 h, respectively. (a–d) representative photomicrographs of brain sections showing YOYO-1+ and PI+ cells labeled at 24–48 h. Note that the majority of YOYO-1+ cells are also PI+, however some YOYO-1+ cells are PI-negative (arrows). Scale bars: (a, b) 60 um; (c, d), 20 um.

    Techniques Used: Mouse Assay, Labeling

    9) Product Images from "Three-dimensional Nanowire Structures for Ultra-Fast Separation of DNA, Protein and RNA Molecules"

    Article Title: Three-dimensional Nanowire Structures for Ultra-Fast Separation of DNA, Protein and RNA Molecules

    Journal: Scientific Reports

    doi: 10.1038/srep10584

    Separation and mobility of protein molecules in the 3D nanowire structures. ( a ) Separation of (1) trypsin inhibitor (20.1 kDa), (2) protein A (45 kDa), (3) streptavidin (52.8 kDa), (4) β-galactosidase (116 kDa) and (5) fibrinogen (340 kDa). The electropherograms were obtained at 2000 μm from the entrance of the 3D nanowire structures. The applied electric field in the separation channel was 500 V/cm. ( b ) The electropherograms of each type of protein molecule to verify the migration time of each separation peak ( E = 2000 V/cm, L = 500 μm). ( c ) Semi-log plot of electrophoretic mobility as a function of molecular weight under the applied electric field of 500V/cm.
    Figure Legend Snippet: Separation and mobility of protein molecules in the 3D nanowire structures. ( a ) Separation of (1) trypsin inhibitor (20.1 kDa), (2) protein A (45 kDa), (3) streptavidin (52.8 kDa), (4) β-galactosidase (116 kDa) and (5) fibrinogen (340 kDa). The electropherograms were obtained at 2000 μm from the entrance of the 3D nanowire structures. The applied electric field in the separation channel was 500 V/cm. ( b ) The electropherograms of each type of protein molecule to verify the migration time of each separation peak ( E = 2000 V/cm, L = 500 μm). ( c ) Semi-log plot of electrophoretic mobility as a function of molecular weight under the applied electric field of 500V/cm.

    Techniques Used: Migration, Molecular Weight

    10) Product Images from "Reducible DNA nanoparticles enhance in vitro gene transfer via an extracellular mechanism"

    Article Title: Reducible DNA nanoparticles enhance in vitro gene transfer via an extracellular mechanism

    Journal: Journal of controlled release : official journal of the Controlled Release Society

    doi: 10.1016/j.jconrel.2010.04.031

    SS-DNA NPs show higher cellular uptake compared to CS-DNA NPs by flow cytometry. Flow cytometric analysis of uptake of YOYO-1 labeled CS-DNA NPs (A) or SS-DNA NPs (B) by HeLa cells at dosage (μg pDNA/well in 6-well plates) indicated in the figure.
    Figure Legend Snippet: SS-DNA NPs show higher cellular uptake compared to CS-DNA NPs by flow cytometry. Flow cytometric analysis of uptake of YOYO-1 labeled CS-DNA NPs (A) or SS-DNA NPs (B) by HeLa cells at dosage (μg pDNA/well in 6-well plates) indicated in the figure.

    Techniques Used: Flow Cytometry, Cytometry, Labeling

    11) Product Images from "Convex lens-induced nanoscale templating"

    Article Title: Convex lens-induced nanoscale templating

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

    doi: 10.1073/pnas.1321089111

    Denaturation mapping of λ-DNA in 50-nm channels. ( A ) Kymograph showing initial loading of DNA into a nanochannels and release of YOYO-1 dye to reveal the characteristic λ-DNA melting profile. ( B ) Kymograph taken ∼1 min after loading
    Figure Legend Snippet: Denaturation mapping of λ-DNA in 50-nm channels. ( A ) Kymograph showing initial loading of DNA into a nanochannels and release of YOYO-1 dye to reveal the characteristic λ-DNA melting profile. ( B ) Kymograph taken ∼1 min after loading

    Techniques Used: Denaturation Mapping

    12) Product Images from "Reversible Positioning of Single Molecules inside Zero-Mode Waveguides"

    Article Title: Reversible Positioning of Single Molecules inside Zero-Mode Waveguides

    Journal: Nano Letters

    doi: 10.1021/nl503134x

    DNA focusing into a NZMW. Fluorescence time traces from a single NZMW that contains a 3 nm diameter pore in an array of ZMWs is monitored for the fluorescence from 6000 bp DNA labeled with YOYO-1 (9 pixel region of interest for each NZMW, 10.8 ms exposure time, signal-averaged to 100 ms). Inset illustrates DNA entering the illumination volume of a NZMW as it migrates toward the pore, resulting in increased ZMW fluorescence. ZMW arrays are shown in fluorescence images (i)–(iii) with (i) being an averaged image of all frames in the experiment, and (ii) and (iii) being the membrane under respective −850 and 850 mV. Colored arrows identify ZMWs with corresponding colored fluorescence traces in bottom plot. The red arrow identifies a NZMW. Green and red backgrounds in the fluorescence traces correspond to periods of positive and negative voltage, respectively (see Supporting Information for electrical trace).
    Figure Legend Snippet: DNA focusing into a NZMW. Fluorescence time traces from a single NZMW that contains a 3 nm diameter pore in an array of ZMWs is monitored for the fluorescence from 6000 bp DNA labeled with YOYO-1 (9 pixel region of interest for each NZMW, 10.8 ms exposure time, signal-averaged to 100 ms). Inset illustrates DNA entering the illumination volume of a NZMW as it migrates toward the pore, resulting in increased ZMW fluorescence. ZMW arrays are shown in fluorescence images (i)–(iii) with (i) being an averaged image of all frames in the experiment, and (ii) and (iii) being the membrane under respective −850 and 850 mV. Colored arrows identify ZMWs with corresponding colored fluorescence traces in bottom plot. The red arrow identifies a NZMW. Green and red backgrounds in the fluorescence traces correspond to periods of positive and negative voltage, respectively (see Supporting Information for electrical trace).

    Techniques Used: Fluorescence, Labeling, Mass Spectrometry

    13) Product Images from "VCE‐004.3, a cannabidiol aminoquinone derivative, prevents bleomycin‐induced skin fibrosis and inflammation through PPARγ‐ and CB2 receptor‐dependent pathways"

    Article Title: VCE‐004.3, a cannabidiol aminoquinone derivative, prevents bleomycin‐induced skin fibrosis and inflammation through PPARγ‐ and CB2 receptor‐dependent pathways

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.14450

    VCE‐004.3 is a functional CB 1 antagonist and CB 2 agonist. (A) VCE‐004.3 binding affinity to CB 1 receptor membranes. Results are expressed as mean ± SEM ( n = 5). (B) CB 1 receptor antagonism. HEK293‐T‐CB 1 cells transfected with pCRE‐luc plasmid were pretreated for 30 min with VCE‐004.3, stimulated with WIN55,212 (1 μM) for 6 h and lysed for luciferase activity. Results are expressed as mean ± SD ( n = 5). (C) Binding affinity of VCE‐004.3 to CB 2 receptor membranes. Results are presented as mean ± SD ( n = 5). (D) VCE‐004.3 is a functional CB 2 receptor agonist. HEK293‐T cells stably expressing CB 2 receptors were transfected with pCRE‐luc plasmid and pretreated for 30 min with VCE‐004.3 or WIN55,212 as positive control. Then, cells were stimulated with forskolin (FSK; 10 μM) for 6 h and lysed for luciferase activity. Results are shown as mean ± SD ( n = 5). * P
    Figure Legend Snippet: VCE‐004.3 is a functional CB 1 antagonist and CB 2 agonist. (A) VCE‐004.3 binding affinity to CB 1 receptor membranes. Results are expressed as mean ± SEM ( n = 5). (B) CB 1 receptor antagonism. HEK293‐T‐CB 1 cells transfected with pCRE‐luc plasmid were pretreated for 30 min with VCE‐004.3, stimulated with WIN55,212 (1 μM) for 6 h and lysed for luciferase activity. Results are expressed as mean ± SD ( n = 5). (C) Binding affinity of VCE‐004.3 to CB 2 receptor membranes. Results are presented as mean ± SD ( n = 5). (D) VCE‐004.3 is a functional CB 2 receptor agonist. HEK293‐T cells stably expressing CB 2 receptors were transfected with pCRE‐luc plasmid and pretreated for 30 min with VCE‐004.3 or WIN55,212 as positive control. Then, cells were stimulated with forskolin (FSK; 10 μM) for 6 h and lysed for luciferase activity. Results are shown as mean ± SD ( n = 5). * P

    Techniques Used: Functional Assay, Binding Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Stable Transfection, Expressing, Positive Control

    VCE‐004.3 competed with RGZ for the canonical pocket and binds to an alternate site in PPARγ. NIH‐3T3 cells were transfected with PPARγ‐GAL4 plus GAL4‐luc. (A) Cells were pretreated with VCE‐004.3 for 1 h and then incubated for 6 h in the presence of RGZ ( n = 5). (B) Cells were pretreated with VCE‐004.3 for 1 h and then washed with PBS and stimulated with RGZ for 6 h ( n = 5). (C) PPARγ LBD structures 3B0R and 5U5L bound to VCE‐004.3 (blue) with and without GW9662 (orange). (D and E) NIH‐3T3 cells were transfected with PPARγ‐GAL4 plus GAL4‐luc, pretreated with T0070907 (5 μM) for 30 min and stimulated with RGZ (D) or VCE‐004.3 (E) for 6 h ( n = 5). Cells were lysed and tested for luciferase activity. Results are shown as mean ± SD. * P
    Figure Legend Snippet: VCE‐004.3 competed with RGZ for the canonical pocket and binds to an alternate site in PPARγ. NIH‐3T3 cells were transfected with PPARγ‐GAL4 plus GAL4‐luc. (A) Cells were pretreated with VCE‐004.3 for 1 h and then incubated for 6 h in the presence of RGZ ( n = 5). (B) Cells were pretreated with VCE‐004.3 for 1 h and then washed with PBS and stimulated with RGZ for 6 h ( n = 5). (C) PPARγ LBD structures 3B0R and 5U5L bound to VCE‐004.3 (blue) with and without GW9662 (orange). (D and E) NIH‐3T3 cells were transfected with PPARγ‐GAL4 plus GAL4‐luc, pretreated with T0070907 (5 μM) for 30 min and stimulated with RGZ (D) or VCE‐004.3 (E) for 6 h ( n = 5). Cells were lysed and tested for luciferase activity. Results are shown as mean ± SD. * P

    Techniques Used: Transfection, Incubation, Luciferase, Activity Assay

    VCE‐004.3 prevented BLM‐induced myofibroblast accumulation and fibroblast to myofibroblast differentiation. (A) Mice were injected with BLM for 3 weeks and treated in parallel with i.p. injections of RGZ, VCE‐004.3 or vehicle. Representative images of α‐SMA + cells in skin (green) detected by immunostaining and their corresponding quantification are shown ( n = 6 animals per group). (B and C) NIH‐3T3 differentiation into myofibroblasts. Cells were incubated in low serum conditions (1% FBS) for 24 h. Then, cells were pretreated with VCE‐004.3 for 1 h and stimulated with TGFβ1 for 24 h (B, upper panel). Cells were immunostained for α‐SMA (green) and their nuclei stained with DAPI (blue) (B, bottom panel). (C) α‐SMA protein expression was determined by Western blot. Values under the gel indicate α‐SMA protein signal intensities after normalization to tubulin signal intensities ( n = 5).
    Figure Legend Snippet: VCE‐004.3 prevented BLM‐induced myofibroblast accumulation and fibroblast to myofibroblast differentiation. (A) Mice were injected with BLM for 3 weeks and treated in parallel with i.p. injections of RGZ, VCE‐004.3 or vehicle. Representative images of α‐SMA + cells in skin (green) detected by immunostaining and their corresponding quantification are shown ( n = 6 animals per group). (B and C) NIH‐3T3 differentiation into myofibroblasts. Cells were incubated in low serum conditions (1% FBS) for 24 h. Then, cells were pretreated with VCE‐004.3 for 1 h and stimulated with TGFβ1 for 24 h (B, upper panel). Cells were immunostained for α‐SMA (green) and their nuclei stained with DAPI (blue) (B, bottom panel). (C) α‐SMA protein expression was determined by Western blot. Values under the gel indicate α‐SMA protein signal intensities after normalization to tubulin signal intensities ( n = 5).

    Techniques Used: Mouse Assay, Injection, Immunostaining, Incubation, Staining, Expressing, Western Blot

    Effect of VCE‐004.3 on collagen gene transcription and synthesis in vitro . (A) NIH‐3T3‐Col1A2‐luc cells were pretreated with VCE‐004.3 for 1 h and stimulated with TGFβ1 for the following 24 h ( n = 5). (B) NIH‐3T3 cells were transfected with CAGA‐luc plasmid, pretreated with VCE‐004.3 for 1 h and stimulated with TGFβ1 for 6 h ( n = 5). Cells were tested for luciferase activity. (C) Serum‐starved NIH‐3T3 cells were preincubated with the compound for 1 h and stimulated with TGFβ1 for 2 h. Protein expression was studied by Western blot, and values under images represent mean fold induction of signal intensities after β‐actin normalization ( n = 5). (D and E) NHDFs were serum starved (1% FBS) for 24 h. Then, cells were pretreated with VCE‐004.3 for 1 h and stimulated with TGFβ1 for 24 h. Soluble collagen in the culture medium was measured using the Sircol Assay (D), and collagen deposits were studied by the Sirius Red method (E). Results are presented as mean percentage of inhibition ± SD taking TGFβ1 alone as 100%. * P
    Figure Legend Snippet: Effect of VCE‐004.3 on collagen gene transcription and synthesis in vitro . (A) NIH‐3T3‐Col1A2‐luc cells were pretreated with VCE‐004.3 for 1 h and stimulated with TGFβ1 for the following 24 h ( n = 5). (B) NIH‐3T3 cells were transfected with CAGA‐luc plasmid, pretreated with VCE‐004.3 for 1 h and stimulated with TGFβ1 for 6 h ( n = 5). Cells were tested for luciferase activity. (C) Serum‐starved NIH‐3T3 cells were preincubated with the compound for 1 h and stimulated with TGFβ1 for 2 h. Protein expression was studied by Western blot, and values under images represent mean fold induction of signal intensities after β‐actin normalization ( n = 5). (D and E) NHDFs were serum starved (1% FBS) for 24 h. Then, cells were pretreated with VCE‐004.3 for 1 h and stimulated with TGFβ1 for 24 h. Soluble collagen in the culture medium was measured using the Sircol Assay (D), and collagen deposits were studied by the Sirius Red method (E). Results are presented as mean percentage of inhibition ± SD taking TGFβ1 alone as 100%. * P

    Techniques Used: In Vitro, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Expressing, Western Blot, Inhibition

    VCE‐004.3 is a selective PPARγ agonist. (A) Schematic for synthesis of VCE‐004.3. (B) The binding affinity of VCE‐004.3 and RGZ to PPARγ. Results are presented as a logarithmic scale, and IC 50 values were calculated as the 50% inhibition of percentage of fluorescence polarization ( n = 5). (C) PPARγ transcriptional activity induced by VCE‐004.3 its cytotoxicity in NIH‐3T3 fibroblasts. Cells were co‐transfected with PPARγ‐GAL4 and GAL4‐luc constructs, stimulated with VCE‐004.3 or RGZ as a positive control for 6 h and lysed for luciferase activity. Results are presented in a bar chart and shown as fold induction compared to control ( n = 10). To test cytotoxicity, cells were treated with the compound for 6 h in the presence of YOYO‐1, and fluorescence intensity was measured and presented in a dot chart ( n = 5). (D and E) HEK‐293T cells were co‐transfected with PPARα‐GAL4 (D) or PPARδ‐GAL4 (E) and GAL4‐luc, treated with VCE‐004.3 for 6 h and lysed for luciferase activity ( n = 5). Results are shown as mean ± SD. * P
    Figure Legend Snippet: VCE‐004.3 is a selective PPARγ agonist. (A) Schematic for synthesis of VCE‐004.3. (B) The binding affinity of VCE‐004.3 and RGZ to PPARγ. Results are presented as a logarithmic scale, and IC 50 values were calculated as the 50% inhibition of percentage of fluorescence polarization ( n = 5). (C) PPARγ transcriptional activity induced by VCE‐004.3 its cytotoxicity in NIH‐3T3 fibroblasts. Cells were co‐transfected with PPARγ‐GAL4 and GAL4‐luc constructs, stimulated with VCE‐004.3 or RGZ as a positive control for 6 h and lysed for luciferase activity. Results are presented in a bar chart and shown as fold induction compared to control ( n = 10). To test cytotoxicity, cells were treated with the compound for 6 h in the presence of YOYO‐1, and fluorescence intensity was measured and presented in a dot chart ( n = 5). (D and E) HEK‐293T cells were co‐transfected with PPARα‐GAL4 (D) or PPARδ‐GAL4 (E) and GAL4‐luc, treated with VCE‐004.3 for 6 h and lysed for luciferase activity ( n = 5). Results are shown as mean ± SD. * P

    Techniques Used: Binding Assay, Inhibition, Fluorescence, Activity Assay, Transfection, Construct, Positive Control, Luciferase

    Systemic and topical treatment with VCE‐004.3 reversed fibrosis in a BLM‐induced model of established skin fibrosis. Mice were injected with BLM for 6 weeks and treated with the compound during the last 3 weeks of BLM challenge. (A) Representative images of Masson's trichrome staining of skin sections and their respective measurements of the thickness of dermal and subcutaneous adipose layers. (B) Representative images of collagen staining by picrosirius red dye and their quantification. (C) Expression of fibrosis‐related genes in the skin. Values are presented as mean ± SEM ( n = 9 animals per group). * P
    Figure Legend Snippet: Systemic and topical treatment with VCE‐004.3 reversed fibrosis in a BLM‐induced model of established skin fibrosis. Mice were injected with BLM for 6 weeks and treated with the compound during the last 3 weeks of BLM challenge. (A) Representative images of Masson's trichrome staining of skin sections and their respective measurements of the thickness of dermal and subcutaneous adipose layers. (B) Representative images of collagen staining by picrosirius red dye and their quantification. (C) Expression of fibrosis‐related genes in the skin. Values are presented as mean ± SEM ( n = 9 animals per group). * P

    Techniques Used: Mouse Assay, Injection, Staining, Expressing

    VCE‐004.3 alleviated skin inflammation and collagen accumulation induced by BLM. Mice were injected with BLM for 3 weeks and treated in parallel with i.p. injections of RGZ, VCE‐004.3 or vehicle. (A) Representative images of Masson's trichrome staining of skin sections and their respective measurement of the thickness of the layer of dermal and subcutaneous adipose tissue. (B) Representative images of collagen staining by picrosirius red dye and their quantification. (C) Gene expression of profibrotic and pro‐inflammatory genes including Il‐6 , Tgfβ , Il‐4 , Ccl2 , Il‐1β and Il‐13 were measured by q‐RT‐PCR. Results are presented as mean ± SEM referred to control group ( n = 9 animals per group). * P
    Figure Legend Snippet: VCE‐004.3 alleviated skin inflammation and collagen accumulation induced by BLM. Mice were injected with BLM for 3 weeks and treated in parallel with i.p. injections of RGZ, VCE‐004.3 or vehicle. (A) Representative images of Masson's trichrome staining of skin sections and their respective measurement of the thickness of the layer of dermal and subcutaneous adipose tissue. (B) Representative images of collagen staining by picrosirius red dye and their quantification. (C) Gene expression of profibrotic and pro‐inflammatory genes including Il‐6 , Tgfβ , Il‐4 , Ccl2 , Il‐1β and Il‐13 were measured by q‐RT‐PCR. Results are presented as mean ± SEM referred to control group ( n = 9 animals per group). * P

    Techniques Used: Mouse Assay, Injection, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction

    VCE‐004.3 inhibited BLM‐induced ERK1/2 phosphorylation in vivo . Mice were injected with BLM for 3 weeks and treated in parallel with i.p. injections of RGZ, VCE‐004.3 or vehicle. Representative images of p‐ERK + cells detected by immunostaining and their respective quantification. Values are presented as mean ± SEM ( n = 9 animals per group). * P
    Figure Legend Snippet: VCE‐004.3 inhibited BLM‐induced ERK1/2 phosphorylation in vivo . Mice were injected with BLM for 3 weeks and treated in parallel with i.p. injections of RGZ, VCE‐004.3 or vehicle. Representative images of p‐ERK + cells detected by immunostaining and their respective quantification. Values are presented as mean ± SEM ( n = 9 animals per group). * P

    Techniques Used: In Vivo, Mouse Assay, Injection, Immunostaining

    VCE‐004.3 prevented stimulation of ERK signalling by SSc autoantibodies and fibroblast migration in vitro . NHDFs were serum‐starved (1% FBS) for 24 h and then preincubated with VCE‐004.3 for 1 h and stimulated with lSSc (A) or dSSc (B) IgG for 15 min. (C) NIH‐3T3 cells were serum‐starved (1% FBS) for 24 h and then preincubated with VCE‐004.3 for 15 min and stimulated with PDGF‐BB for 5 min. Protein expression was determined by Western blot, and values under the gel indicate mean fold induction of signal intensities. (D) NHDF monolayers were scratched and treated with VCE‐004.3 at the indicated doses in the presence of TGFβ1. Results are presented as percentage of wound closure (confluence) ± SD ( n = 5). * P
    Figure Legend Snippet: VCE‐004.3 prevented stimulation of ERK signalling by SSc autoantibodies and fibroblast migration in vitro . NHDFs were serum‐starved (1% FBS) for 24 h and then preincubated with VCE‐004.3 for 1 h and stimulated with lSSc (A) or dSSc (B) IgG for 15 min. (C) NIH‐3T3 cells were serum‐starved (1% FBS) for 24 h and then preincubated with VCE‐004.3 for 15 min and stimulated with PDGF‐BB for 5 min. Protein expression was determined by Western blot, and values under the gel indicate mean fold induction of signal intensities. (D) NHDF monolayers were scratched and treated with VCE‐004.3 at the indicated doses in the presence of TGFβ1. Results are presented as percentage of wound closure (confluence) ± SD ( n = 5). * P

    Techniques Used: Migration, In Vitro, Expressing, Western Blot

    VCE‐004.3 reduced inflammatory cell infiltration in the skin. Mice were injected with BLM for 3 weeks and treated in parallel with i.p. injections of RGZ, VCE‐004.3 or vehicle. Representative images of the mast cell degranulation process are shown, as detected by toluidine blue staining and F4/80 + macrophages and CD3 + T‐lymphocyte infiltration in the skin detected by immunostaining and their corresponding quantification ( n = 9 animals per group). Results represent the mean ± SEM. * P
    Figure Legend Snippet: VCE‐004.3 reduced inflammatory cell infiltration in the skin. Mice were injected with BLM for 3 weeks and treated in parallel with i.p. injections of RGZ, VCE‐004.3 or vehicle. Representative images of the mast cell degranulation process are shown, as detected by toluidine blue staining and F4/80 + macrophages and CD3 + T‐lymphocyte infiltration in the skin detected by immunostaining and their corresponding quantification ( n = 9 animals per group). Results represent the mean ± SEM. * P

    Techniques Used: Mouse Assay, Injection, Staining, Immunostaining

    14) Product Images from "The histidine-rich peptide LAH4-L1 strongly promotes PAMAM-mediated transfection at low nitrogen to phosphorus ratios in the presence of serum"

    Article Title: The histidine-rich peptide LAH4-L1 strongly promotes PAMAM-mediated transfection at low nitrogen to phosphorus ratios in the presence of serum

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-10049-y

    DNA release kinetics of PS and PSL complexes in HeLa cells under fluorescence microscope at a magnification of 400-fold. Plasmid DNA (SB transposase) was labeled with YOYO®-1(green). Arrows show the release of plasmid DNA into the cytosol.
    Figure Legend Snippet: DNA release kinetics of PS and PSL complexes in HeLa cells under fluorescence microscope at a magnification of 400-fold. Plasmid DNA (SB transposase) was labeled with YOYO®-1(green). Arrows show the release of plasmid DNA into the cytosol.

    Techniques Used: Fluorescence, Microscopy, Plasmid Preparation, Labeling

    Cellular distribution of PS and PSL complexes (YOYO®-1 labeled, green) in HeLa cells at 4 h post transfection under fluorescence microscope at a magnification of 630-fold. Late endosome/lysosome was stained with Lysotracker Red DND-99 (red). The colocalization of PS or PSL complexes with lysosomes is represented in yellow in merged images.
    Figure Legend Snippet: Cellular distribution of PS and PSL complexes (YOYO®-1 labeled, green) in HeLa cells at 4 h post transfection under fluorescence microscope at a magnification of 630-fold. Late endosome/lysosome was stained with Lysotracker Red DND-99 (red). The colocalization of PS or PSL complexes with lysosomes is represented in yellow in merged images.

    Techniques Used: Labeling, Transfection, Fluorescence, Microscopy, Staining

    15) Product Images from "A Modified Aggregate Culture for Chondrogenesis of Human Adipose-Derived Stem Cells Genetically Modified with Growth and Differentiation Factor 5"

    Article Title: A Modified Aggregate Culture for Chondrogenesis of Human Adipose-Derived Stem Cells Genetically Modified with Growth and Differentiation Factor 5

    Journal: BioResearch Open Access

    doi: 10.1089/biores.2013.0014

    Safranin O staining of aggregates (A) and immunostaining of type II collagen in aggregates (B, top ) of human adipose stem cells with (GDF5) or without (non-GDF5) infection of ad-GDF5 and cultured in BM or CM for 3 weeks. Counterstaining was performed with a DNA fluorescent dye YOYO-1 (B, bottom ) . The negative control group was the non-GDF5/CM group without incubation of the primary antibody. Bar=100 μm.
    Figure Legend Snippet: Safranin O staining of aggregates (A) and immunostaining of type II collagen in aggregates (B, top ) of human adipose stem cells with (GDF5) or without (non-GDF5) infection of ad-GDF5 and cultured in BM or CM for 3 weeks. Counterstaining was performed with a DNA fluorescent dye YOYO-1 (B, bottom ) . The negative control group was the non-GDF5/CM group without incubation of the primary antibody. Bar=100 μm.

    Techniques Used: Staining, Immunostaining, Infection, Cell Culture, Negative Control, Incubation

    16) Product Images from "Microfluidic long DNA sample preparation from cells"

    Article Title: Microfluidic long DNA sample preparation from cells

    Journal: Lab on a chip

    doi: 10.1039/c8lc01163j

    Fluorescent image of YOYO-stained, device-extracted DNA, stretched on silanized glass in 100 µm wide and 5 µm deep PDMS channel. The image is stitched to cover 8 ROIs of an Andor Zyla camera at 100x magnification. The 32 µm scale bar corresponds to 100 kbp.
    Figure Legend Snippet: Fluorescent image of YOYO-stained, device-extracted DNA, stretched on silanized glass in 100 µm wide and 5 µm deep PDMS channel. The image is stitched to cover 8 ROIs of an Andor Zyla camera at 100x magnification. The 32 µm scale bar corresponds to 100 kbp.

    Techniques Used: Staining

    17) Product Images from "Layered Structure and Complex Mechanochemistry Underlie Strength and Versatility in a Bacterial Adhesive"

    Article Title: Layered Structure and Complex Mechanochemistry Underlie Strength and Versatility in a Bacterial Adhesive

    Journal: mBio

    doi: 10.1128/mBio.02359-17

    Caulobacter crescentus. (A and B) TEM (A) and AFM (B) images of a C. crescentus CB15 wild-type cell. (C) Schematic of Caulobacter attached to a surface under fluid flow. The holdfast size is in the order of tens of nanometer in diameter. The holdfast is circled in red. (D) AFM image of purified holdfasts attached to a mica surface in dH 2 O.
    Figure Legend Snippet: Caulobacter crescentus. (A and B) TEM (A) and AFM (B) images of a C. crescentus CB15 wild-type cell. (C) Schematic of Caulobacter attached to a surface under fluid flow. The holdfast size is in the order of tens of nanometer in diameter. The holdfast is circled in red. (D) AFM image of purified holdfasts attached to a mica surface in dH 2 O.

    Techniques Used: Transmission Electron Microscopy, Flow Cytometry, Purification

    18) Product Images from "Importin-7 Mediates Nuclear Trafficking of DNA in Mammalian Cells"

    Article Title: Importin-7 Mediates Nuclear Trafficking of DNA in Mammalian Cells

    Journal: Traffic (Copenhagen, Denmark)

    doi: 10.1111/tra.12021

    Imp7 induces nuclear import of human mtDNA A) mtDNA was obtained from purified HeLa cells mitochondria after ultracentrifugation through a 4.5 m CsCl gradient and run on a 0.8% agarose gel. The band migrating with an apparent molecular weight of > 23 kb was excised and eluted. MW, molecular weight markers; lanes 1 and 2, two different mtDNA preparations. B) Gel-fractionated mtDNA was analyzed by Southern blot with a COX I specific probe. Left panel, agarose gel after ethidium bromide staining; right panel, Southern blot. Lane 1, undigested mtDNA; lane 2, mtDNA cut with BamHI; lane 3, mtDNA cut with ClaI; lane 4, mtDNA cut with PvuII, lane 5, mtDNA cut with XhoI. These enzymes cut once in the mtDNA sequence 33 . C) Purified mtDNA analyzed by PCR with primers specific for 28S rDNA or for COX I. MW, molecular weight markers, lanes 1–5, serial dilutions of genomic DNA from 30 to 0.3 ng amplified using 28S rDNA specific primers; lanes 6, no genomic DNA; lanes 7–10, serial dilutions of purified mtDNA from 30 to 1 ng; lane 11, mtDNA (1.25 ng) amplified with COX I specific primers; lane 12, no mtDNA. D) Nuclear import assay into permeabilized HeLa cells in the presence of 20 ng labelled mtDNA and: buffer (−E); 1× energy mix (E); 1× energy mix + 1× Ran mix (E + Ran); 1× energy mix + 1× Ran mix + 1 µ m imp7 (imp7). Results are representative of two independent experiments. Scale bar = 10 µm.
    Figure Legend Snippet: Imp7 induces nuclear import of human mtDNA A) mtDNA was obtained from purified HeLa cells mitochondria after ultracentrifugation through a 4.5 m CsCl gradient and run on a 0.8% agarose gel. The band migrating with an apparent molecular weight of > 23 kb was excised and eluted. MW, molecular weight markers; lanes 1 and 2, two different mtDNA preparations. B) Gel-fractionated mtDNA was analyzed by Southern blot with a COX I specific probe. Left panel, agarose gel after ethidium bromide staining; right panel, Southern blot. Lane 1, undigested mtDNA; lane 2, mtDNA cut with BamHI; lane 3, mtDNA cut with ClaI; lane 4, mtDNA cut with PvuII, lane 5, mtDNA cut with XhoI. These enzymes cut once in the mtDNA sequence 33 . C) Purified mtDNA analyzed by PCR with primers specific for 28S rDNA or for COX I. MW, molecular weight markers, lanes 1–5, serial dilutions of genomic DNA from 30 to 0.3 ng amplified using 28S rDNA specific primers; lanes 6, no genomic DNA; lanes 7–10, serial dilutions of purified mtDNA from 30 to 1 ng; lane 11, mtDNA (1.25 ng) amplified with COX I specific primers; lane 12, no mtDNA. D) Nuclear import assay into permeabilized HeLa cells in the presence of 20 ng labelled mtDNA and: buffer (−E); 1× energy mix (E); 1× energy mix + 1× Ran mix (E + Ran); 1× energy mix + 1× Ran mix + 1 µ m imp7 (imp7). Results are representative of two independent experiments. Scale bar = 10 µm.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Molecular Weight, Southern Blot, Staining, Sequencing, Polymerase Chain Reaction, Amplification

    19) Product Images from "Ultrafast Transient Absorption Spectra of Photoexcited YOYO-1 molecules call for additional investigations of their fluorescence quenching mechanism"

    Article Title: Ultrafast Transient Absorption Spectra of Photoexcited YOYO-1 molecules call for additional investigations of their fluorescence quenching mechanism

    Journal: Journal of photochemistry and photobiology. A, Chemistry

    doi: 10.1016/j.jphotochem.2018.09.012

    (A) YOYO-1 absorption and emission spectra in energy scale. The blue solid line is the absorption of YOYO-1 in water, the blue dashed line is the YOYO-1 absorption in DNA, and the red dashed line is the YOYO-1 emission in DNA. (B) Scheme of band assignment in the TA spectra. The YOYO-1 molecular orbitals are coupled with its atomic nucleus vibrations (Franck-Condon principle) to represent a distribution of molecules with the same electronic structure but different vibrational states in the solution at a given moment. S n indicates one excited electron on the n th electronic orbital (often delocalized) and another electron in the ground state leaving a vacancy on the HOMO of the original host atom of the excited electron right after the excitation (delocalized over time). Arrows show the absorptions (blue arrows), the vibrational relaxation (twisted red arrows), and the emission (straight red arrow) of YOYO-1.
    Figure Legend Snippet: (A) YOYO-1 absorption and emission spectra in energy scale. The blue solid line is the absorption of YOYO-1 in water, the blue dashed line is the YOYO-1 absorption in DNA, and the red dashed line is the YOYO-1 emission in DNA. (B) Scheme of band assignment in the TA spectra. The YOYO-1 molecular orbitals are coupled with its atomic nucleus vibrations (Franck-Condon principle) to represent a distribution of molecules with the same electronic structure but different vibrational states in the solution at a given moment. S n indicates one excited electron on the n th electronic orbital (often delocalized) and another electron in the ground state leaving a vacancy on the HOMO of the original host atom of the excited electron right after the excitation (delocalized over time). Arrows show the absorptions (blue arrows), the vibrational relaxation (twisted red arrows), and the emission (straight red arrow) of YOYO-1.

    Techniques Used:

    The probe TA spectra change (Δ A ) after the pump excitation of 10 μM YOYO-1 (A) in water, (B) in DMSO, and (C) 460 μM DNA basepair (300 μg/mL phage λ-DNA) upon different pump-probe delay times. The lower row is the zoom-in of the first 100 ps in the spectra above. Experiments were repeated with two sets of different samples on different days. Consistency has been observed. Pump pulse is 100 fs, 486 nm (2.55 eV) laser; and probe pulse is 100 fs, white light laser. (D-F) Sample spectra in (A-C) over delay time 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, and 1500 ps
    Figure Legend Snippet: The probe TA spectra change (Δ A ) after the pump excitation of 10 μM YOYO-1 (A) in water, (B) in DMSO, and (C) 460 μM DNA basepair (300 μg/mL phage λ-DNA) upon different pump-probe delay times. The lower row is the zoom-in of the first 100 ps in the spectra above. Experiments were repeated with two sets of different samples on different days. Consistency has been observed. Pump pulse is 100 fs, 486 nm (2.55 eV) laser; and probe pulse is 100 fs, white light laser. (D-F) Sample spectra in (A-C) over delay time 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, and 1500 ps

    Techniques Used:

    20) Product Images from "Development of switchable polymers to address the dilemma of stability and cargo release in polycationic nucleic acid carriers"

    Article Title: Development of switchable polymers to address the dilemma of stability and cargo release in polycationic nucleic acid carriers

    Journal: Biomaterials

    doi: 10.1016/j.biomaterials.2017.02.036

    (A). In vitro cellular uptake of YOYO-1 labeled DNA delivered by IP and SP. Data are shown as mean ± SD (n=3). (B). Confocal images of HeLa cells treated with polyplexes containing YOYO-1 labeled DNA for 4 h and stained with LysoTracker Red. DAPI (4′,6-diamidino-2-phenylindole, blue) was used to stain cell nuclei. Scale bar: 10 μm. (C). Colocalization ratio of YOYO-1-DNA with LysoTracker Red stained endosomes. Results were presented as the mean of 15 individual cells. Data are shown as mean ± SD (n=15; student's t test, *p
    Figure Legend Snippet: (A). In vitro cellular uptake of YOYO-1 labeled DNA delivered by IP and SP. Data are shown as mean ± SD (n=3). (B). Confocal images of HeLa cells treated with polyplexes containing YOYO-1 labeled DNA for 4 h and stained with LysoTracker Red. DAPI (4′,6-diamidino-2-phenylindole, blue) was used to stain cell nuclei. Scale bar: 10 μm. (C). Colocalization ratio of YOYO-1-DNA with LysoTracker Red stained endosomes. Results were presented as the mean of 15 individual cells. Data are shown as mean ± SD (n=15; student's t test, *p

    Techniques Used: In Vitro, Labeling, Staining

    21) Product Images from "DNA interrogation by the CRISPR RNA-guided endonuclease Cas9"

    Article Title: DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

    Journal: Nature

    doi: 10.1038/nature13011

    DNA curtains assay for target binding by Cas9:RNA a, Schematic of a single-tethered DNA curtain 26 , 27 . b , Wild-type Cas9 or dCas9 was programmed with crRNA:tracrRNA targeting one of six sites. c , YOYO1-stained DNA (green) bound by QD-tagged dCas9 (magenta) programmed with λ2 guide RNA. d, dCas9:RNA binding distributions; error bars represent 95% confidence intervals obtained through bootstrap analysis 28 . e , Image of apo-Cas9 bound to DNA curtains bound to apo-Cas9. f, Binding distribution of apo-Cas9; error bars represent 95% confidence intervals. g, Lifetimes of DNA-bound apo-Cas9 and Cas9:RNA after injection of λ2 crRNA:tracrRNA (100 nM) or heparin (10 μg mL −1 ).
    Figure Legend Snippet: DNA curtains assay for target binding by Cas9:RNA a, Schematic of a single-tethered DNA curtain 26 , 27 . b , Wild-type Cas9 or dCas9 was programmed with crRNA:tracrRNA targeting one of six sites. c , YOYO1-stained DNA (green) bound by QD-tagged dCas9 (magenta) programmed with λ2 guide RNA. d, dCas9:RNA binding distributions; error bars represent 95% confidence intervals obtained through bootstrap analysis 28 . e , Image of apo-Cas9 bound to DNA curtains bound to apo-Cas9. f, Binding distribution of apo-Cas9; error bars represent 95% confidence intervals. g, Lifetimes of DNA-bound apo-Cas9 and Cas9:RNA after injection of λ2 crRNA:tracrRNA (100 nM) or heparin (10 μg mL −1 ).

    Techniques Used: Binding Assay, Staining, RNA Binding Assay, Injection

    22) Product Images from "Phage Mu Gam protein promotes NHEJ in concert with Escherichia coli ligase"

    Article Title: Phage Mu Gam protein promotes NHEJ in concert with Escherichia coli ligase

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

    doi: 10.1073/pnas.1816606115

    MuGam is located predominantly at DNA ends. ( A ) EMSA assay. Linear dsDNA (100 bp) was incubated with increasing amounts of tagless Gam, electrophoresed on a 5% native acrylamide gel, and visualized by ethidium bromide (EtBr) staining. C, DNA alone control. Position of size markers is indicated on the right. ( B ) Fluorescent FLAG-Gam (magenta) binds λ−DNA organized at microfabricated barriers (green, labeled with YOYO1 dye). Turning off buffer flow retracts both Gam and DNA to the barriers (black arrow), indicating that Gam is on the DNA. ( C ) A binding distribution of Gam along the DNA shows a strong preference for DNA ends. Gray region indicates the experimental uncertainty in defining the DNA end. Error bars were determined by bootstrap analysis. Red line denotes the Gaussian fit. ( D for experimental details.
    Figure Legend Snippet: MuGam is located predominantly at DNA ends. ( A ) EMSA assay. Linear dsDNA (100 bp) was incubated with increasing amounts of tagless Gam, electrophoresed on a 5% native acrylamide gel, and visualized by ethidium bromide (EtBr) staining. C, DNA alone control. Position of size markers is indicated on the right. ( B ) Fluorescent FLAG-Gam (magenta) binds λ−DNA organized at microfabricated barriers (green, labeled with YOYO1 dye). Turning off buffer flow retracts both Gam and DNA to the barriers (black arrow), indicating that Gam is on the DNA. ( C ) A binding distribution of Gam along the DNA shows a strong preference for DNA ends. Gray region indicates the experimental uncertainty in defining the DNA end. Error bars were determined by bootstrap analysis. Red line denotes the Gaussian fit. ( D for experimental details.

    Techniques Used: Incubation, Acrylamide Gel Assay, Staining, Labeling, Flow Cytometry, Binding Assay

    23) Product Images from "Three-dimensional Nanowire Structures for Ultra-Fast Separation of DNA, Protein and RNA Molecules"

    Article Title: Three-dimensional Nanowire Structures for Ultra-Fast Separation of DNA, Protein and RNA Molecules

    Journal: Scientific Reports

    doi: 10.1038/srep10584

    Separation and mobility of DNA molecules in the 3D nanowire structures. ( a ) Separation of 50 bp (40 ng/μL), 100 bp (30 ng/μL), 200 bp (30 ng/μL), 300 bp (30 ng/μL), 500 bp (30 ng/μL) and 1000 bp (30 ng/μL) molecules in the 3D nanowire structures. The electropherograms were obtained at 500 μm from the entrance of the 3D nanowire structures. The applied electric field in the separation channel was 100 V/cm. ( b ) The electropherogram of each type of DNA molecule to verify the migration time of each separation peak ( E = 100 V/cm, L = 500 μm). ( c ) Semi-log plot of electrophoretic mobility as a function of DNA size under the applied electric field of 100V/cm.
    Figure Legend Snippet: Separation and mobility of DNA molecules in the 3D nanowire structures. ( a ) Separation of 50 bp (40 ng/μL), 100 bp (30 ng/μL), 200 bp (30 ng/μL), 300 bp (30 ng/μL), 500 bp (30 ng/μL) and 1000 bp (30 ng/μL) molecules in the 3D nanowire structures. The electropherograms were obtained at 500 μm from the entrance of the 3D nanowire structures. The applied electric field in the separation channel was 100 V/cm. ( b ) The electropherogram of each type of DNA molecule to verify the migration time of each separation peak ( E = 100 V/cm, L = 500 μm). ( c ) Semi-log plot of electrophoretic mobility as a function of DNA size under the applied electric field of 100V/cm.

    Techniques Used: Migration

    3D nanowire structures. ( a ) Photograph of a device for the 3D nanowire structures; scale bar 5 mm. ( b ) SEM image of SnO 2 nanowires embedded in a microchannel; scale bar 1 μm. ( c ) Schematic of Au catalyst assisted VLS 3D nanowire growth; nanowire backbone were growth in [100] direction (1), then Au catalyst decorate along nanowire backbone (2), after that, the first nanowire branches growth in [001] direction (3), Au catalysts were deposited on the first nanowire branches and the second nanowire branches were growth by VLS technique as a cycle (4). ( d ) SEM image of the 3D nanowire structures; scale bar 100 nm. (e) Pore size distribution in the 3D nanowire structures.
    Figure Legend Snippet: 3D nanowire structures. ( a ) Photograph of a device for the 3D nanowire structures; scale bar 5 mm. ( b ) SEM image of SnO 2 nanowires embedded in a microchannel; scale bar 1 μm. ( c ) Schematic of Au catalyst assisted VLS 3D nanowire growth; nanowire backbone were growth in [100] direction (1), then Au catalyst decorate along nanowire backbone (2), after that, the first nanowire branches growth in [001] direction (3), Au catalysts were deposited on the first nanowire branches and the second nanowire branches were growth by VLS technique as a cycle (4). ( d ) SEM image of the 3D nanowire structures; scale bar 100 nm. (e) Pore size distribution in the 3D nanowire structures.

    Techniques Used:

    Separation and mobility of protein molecules in the 3D nanowire structures. ( a ) Separation of (1) trypsin inhibitor (20.1 kDa), (2) protein A (45 kDa), (3) streptavidin (52.8 kDa), (4) β-galactosidase (116 kDa) and (5) fibrinogen (340 kDa). The electropherograms were obtained at 2000 μm from the entrance of the 3D nanowire structures. The applied electric field in the separation channel was 500 V/cm. ( b ) The electropherograms of each type of protein molecule to verify the migration time of each separation peak ( E = 2000 V/cm, L = 500 μm). ( c ) Semi-log plot of electrophoretic mobility as a function of molecular weight under the applied electric field of 500V/cm.
    Figure Legend Snippet: Separation and mobility of protein molecules in the 3D nanowire structures. ( a ) Separation of (1) trypsin inhibitor (20.1 kDa), (2) protein A (45 kDa), (3) streptavidin (52.8 kDa), (4) β-galactosidase (116 kDa) and (5) fibrinogen (340 kDa). The electropherograms were obtained at 2000 μm from the entrance of the 3D nanowire structures. The applied electric field in the separation channel was 500 V/cm. ( b ) The electropherograms of each type of protein molecule to verify the migration time of each separation peak ( E = 2000 V/cm, L = 500 μm). ( c ) Semi-log plot of electrophoretic mobility as a function of molecular weight under the applied electric field of 500V/cm.

    Techniques Used: Migration, Molecular Weight

    Separation of 0.1–1 kb RNA molecules in the 3D nanowire structures. The electropherogram was obtained at 250 μm from the entrance of the 3D nanowire structures. The applied electric field in the separation channel was 300 V/cm.
    Figure Legend Snippet: Separation of 0.1–1 kb RNA molecules in the 3D nanowire structures. The electropherogram was obtained at 250 μm from the entrance of the 3D nanowire structures. The applied electric field in the separation channel was 300 V/cm.

    Techniques Used:

    24) Product Images from "DNA methylation profiling in nanochannels"

    Article Title: DNA methylation profiling in nanochannels

    Journal: Biomicrofluidics

    doi: 10.1063/1.3613671

    (a) Schematic of possible outcomes of DNA concatemer formation with 5-cytosine methylated (5mC) and non-methylated segments. (b) Schematic of Alexa568-MBD to DNA concatemer. The entire molecule is stained using the green stain YOYO-1 and Alexa568-MBD binds to methylated stretches.
    Figure Legend Snippet: (a) Schematic of possible outcomes of DNA concatemer formation with 5-cytosine methylated (5mC) and non-methylated segments. (b) Schematic of Alexa568-MBD to DNA concatemer. The entire molecule is stained using the green stain YOYO-1 and Alexa568-MBD binds to methylated stretches.

    Techniques Used: Methylation, Staining

    (a) Fluorescence images of concatenated methylated and non-methylated λ-DNA labeled with Alexa568MBD (red) and YOYO-1 (green), stretched out in nanochannels. Within each panel colors are split for clarity; (left) YOYO-1 only (DNA), (center) composite, (right) Alexa568 only (Alexa568-MBD). Schematic drawings in each panel illustrate the spatial position of the Alexa Fluor 568 MBD and the length of the λ-DNA. The scale bar in panel (b) is 5 microns.
    Figure Legend Snippet: (a) Fluorescence images of concatenated methylated and non-methylated λ-DNA labeled with Alexa568MBD (red) and YOYO-1 (green), stretched out in nanochannels. Within each panel colors are split for clarity; (left) YOYO-1 only (DNA), (center) composite, (right) Alexa568 only (Alexa568-MBD). Schematic drawings in each panel illustrate the spatial position of the Alexa Fluor 568 MBD and the length of the λ-DNA. The scale bar in panel (b) is 5 microns.

    Techniques Used: Fluorescence, Methylation, Labeling

    25) Product Images from "DNA methylation profiling in nanochannels"

    Article Title: DNA methylation profiling in nanochannels

    Journal: Biomicrofluidics

    doi: 10.1063/1.3613671

    Schematic of a device with two microchannel feeds (top and bottom) that are bridged by a nanochannel (inflowing arrows) containing an Alexa568-MBD labeled DNA concatemer. A shallow central shunt channel (outflowing arrows) allows the use of pressure-driven flow.
    Figure Legend Snippet: Schematic of a device with two microchannel feeds (top and bottom) that are bridged by a nanochannel (inflowing arrows) containing an Alexa568-MBD labeled DNA concatemer. A shallow central shunt channel (outflowing arrows) allows the use of pressure-driven flow.

    Techniques Used: Labeling, Flow Cytometry

    (a) Schematic of possible outcomes of DNA concatemer formation with 5-cytosine methylated (5mC) and non-methylated segments. (b) Schematic of Alexa568-MBD to DNA concatemer. The entire molecule is stained using the green stain YOYO-1 and Alexa568-MBD binds to methylated stretches.
    Figure Legend Snippet: (a) Schematic of possible outcomes of DNA concatemer formation with 5-cytosine methylated (5mC) and non-methylated segments. (b) Schematic of Alexa568-MBD to DNA concatemer. The entire molecule is stained using the green stain YOYO-1 and Alexa568-MBD binds to methylated stretches.

    Techniques Used: Methylation, Staining

    (a) Fluorescence images of concatenated methylated and non-methylated λ-DNA labeled with Alexa568MBD (red) and YOYO-1 (green), stretched out in nanochannels. Within each panel colors are split for clarity; (left) YOYO-1 only (DNA), (center) composite, (right) Alexa568 only (Alexa568-MBD). Schematic drawings in each panel illustrate the spatial position of the Alexa Fluor 568 MBD and the length of the λ-DNA. The scale bar in panel (b) is 5 microns.
    Figure Legend Snippet: (a) Fluorescence images of concatenated methylated and non-methylated λ-DNA labeled with Alexa568MBD (red) and YOYO-1 (green), stretched out in nanochannels. Within each panel colors are split for clarity; (left) YOYO-1 only (DNA), (center) composite, (right) Alexa568 only (Alexa568-MBD). Schematic drawings in each panel illustrate the spatial position of the Alexa Fluor 568 MBD and the length of the λ-DNA. The scale bar in panel (b) is 5 microns.

    Techniques Used: Fluorescence, Methylation, Labeling

    26) Product Images from "Defense activation and enhanced pathogen tolerance induced by H2O2 in transgenic tobacco"

    Article Title: Defense activation and enhanced pathogen tolerance induced by H2O2 in transgenic tobacco

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

    doi:

    Necrosis-independent expression of defense proteins in Cat1AS plants. ( A ) Western blot analysis with PR-1, bPR-2, and GPx antibodies. Cat1AS and control plants were exposed to HL for various times (2–8 h) and then returned to LL for 2 weeks before leaf sampling. Expression of PR-1, bPR-2, and GPx was induced by 4 h of HL in Cat1AS, whereas necrosis required 8 h of exposure. PR-1 expression in the control line was not increased by HL. bPR-2 and GPx showed HL induction in the control line but not to the same level as in Cat1AS. Leaf damage was assessed at the time of harvest: no damage indicates that none of the leaves of that plant had any visible sign of injury. ( B ) Optical sections of a YOYO-1 iodide-stained leaf from a Cat1AS plant treated with HL for 4 h ( Left ) or 8 h ( Right ). YOYO-1 iodide is a nuclear dye that is membrane-impermeant and therefore stains only nuclei of damaged cells. (×700.)
    Figure Legend Snippet: Necrosis-independent expression of defense proteins in Cat1AS plants. ( A ) Western blot analysis with PR-1, bPR-2, and GPx antibodies. Cat1AS and control plants were exposed to HL for various times (2–8 h) and then returned to LL for 2 weeks before leaf sampling. Expression of PR-1, bPR-2, and GPx was induced by 4 h of HL in Cat1AS, whereas necrosis required 8 h of exposure. PR-1 expression in the control line was not increased by HL. bPR-2 and GPx showed HL induction in the control line but not to the same level as in Cat1AS. Leaf damage was assessed at the time of harvest: no damage indicates that none of the leaves of that plant had any visible sign of injury. ( B ) Optical sections of a YOYO-1 iodide-stained leaf from a Cat1AS plant treated with HL for 4 h ( Left ) or 8 h ( Right ). YOYO-1 iodide is a nuclear dye that is membrane-impermeant and therefore stains only nuclei of damaged cells. (×700.)

    Techniques Used: Expressing, Western Blot, Sampling, Staining

    27) Product Images from "Visualization of bidirectional initiation of chromosomal DNA replication in a human cell free system"

    Article Title: Visualization of bidirectional initiation of chromosomal DNA replication in a human cell free system

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki994

    Visualization of DNA breaks in the vicinity of replication tracks. Total DNA was visualized by staining with YOYO-1 (faint green) and replicated DNA was visualized as detailed in the legend to Figure 3 . Two representative fields are shown. Note that DNA breaks are observed at the growing yellow end of a replication track, but not at the green end where replication in vitro initiated.
    Figure Legend Snippet: Visualization of DNA breaks in the vicinity of replication tracks. Total DNA was visualized by staining with YOYO-1 (faint green) and replicated DNA was visualized as detailed in the legend to Figure 3 . Two representative fields are shown. Note that DNA breaks are observed at the growing yellow end of a replication track, but not at the green end where replication in vitro initiated.

    Techniques Used: Staining, In Vitro

    Heterogeneity of replication fork progression in vitro . ( A ) Heterogeneity of individual replication fork progression rates during the incubation in vitro . Progression rates of individual forks calculated from replication track lengths obtained during the first digoxigenin label and during the second biotin label were plotted against each other. Data from G 1 phase and S phase template nuclei are shown on the left and right panels as indicated. ( B ) Replication track length distribution. The lengths of all measured digoxigenin labelled DNA replication tracks were divided into 10 kb classes and their frequency of occurrence was plotted. Data from G 1 phase and S phase template nuclei are shown in the left and right panels, respectively. ( C ) Visualization of divergently moving bidirectional replication forks in G 1 phase template nuclei. Representative patterns of replicating unbroken DNA fibres counterstained with YOYO-1 (faint green) are shown. Note that asymmetric fork progression is detected by different yellow track lengths for every pattern.
    Figure Legend Snippet: Heterogeneity of replication fork progression in vitro . ( A ) Heterogeneity of individual replication fork progression rates during the incubation in vitro . Progression rates of individual forks calculated from replication track lengths obtained during the first digoxigenin label and during the second biotin label were plotted against each other. Data from G 1 phase and S phase template nuclei are shown on the left and right panels as indicated. ( B ) Replication track length distribution. The lengths of all measured digoxigenin labelled DNA replication tracks were divided into 10 kb classes and their frequency of occurrence was plotted. Data from G 1 phase and S phase template nuclei are shown in the left and right panels, respectively. ( C ) Visualization of divergently moving bidirectional replication forks in G 1 phase template nuclei. Representative patterns of replicating unbroken DNA fibres counterstained with YOYO-1 (faint green) are shown. Note that asymmetric fork progression is detected by different yellow track lengths for every pattern.

    Techniques Used: In Vitro, Incubation

    28) Product Images from "Optimization of Tet1 ligand density in HPMA-co-oligolysine copolymers for targeted neuronal gene delivery"

    Article Title: Optimization of Tet1 ligand density in HPMA-co-oligolysine copolymers for targeted neuronal gene delivery

    Journal: Biomaterials

    doi: 10.1016/j.biomaterials.2013.08.045

    YOYO-1 fluorescence quenching assay as a measure of DNA complexation and condensation in ddH 2 O.
    Figure Legend Snippet: YOYO-1 fluorescence quenching assay as a measure of DNA complexation and condensation in ddH 2 O.

    Techniques Used: Fluorescence

    29) Product Images from "DNA is a co-factor for its own replication in Xenopus egg extracts"

    Article Title: DNA is a co-factor for its own replication in Xenopus egg extracts

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq739

    Efficient DNA replication of immobilized plasmids. ( A ) The 1.8% agarose blocks (5 µl) containing 0.1 nM p10.4 were incubated with two volumes of HSS, with or without Geminin. After 30 min, the supernatant was exchanged with two volumes of NPE containing [α- 32 P]dATP. Reactions in solution containing a final concentration of 0.1 or 0.5 nM p10.4 were carried out in parallel. In all cases, DNA replication efficiency was determined 90 min after NPE addition. In lanes 1 and 2, the DNA was not released from the block and thus remained in the well, where the block was loaded. In lane 4, one-fifth of the 0.5 nM reaction was loaded. ( B ) Cartoon illustrating procedure to determine plasmid position and replication in an agarose block. ( C ) The 1.8% agarose blocks containing 0.1 nM p10.4 and 2.8 µm beads (to provide reference points) were prepared. Plasmids were stained with YOYO-1, photographed and de-stained. Subsequently, HSS was added, followed by NPE containing biotin-dUTP. After 90 min, the blocks were stained again with YOYO-1 and the biotin-dUTP was detected with AlexaFluor 647 conjugated streptavidin (SA-647). The same position in the block that was imaged before extract addition was located and images were acquired by fluorescence microscopy. The green and blue channels represent the initial and final plasmid positions as determined by YOYO-1 staining, respectively. The red channel represents SA-647 staining of incorporated biotin-dUTP. The blue and red channels were shifted in the y-axis to facilitate analysis. Plasmid classification is shown below the shifted image. (bar = 5 µm) ( D ) The average percentage of plasmids that was immobile, shifted, or mobile from eight areas of a single block was calculated and graphed. The subset of plasmids in each group that replicated is shown in red. Error bars indicate the standard deviation of the eight areas that were scanned. ( E ) Replication in diluted extracts. p10.4 (0.1 nM) was replicated in agarose blocks or in solution as described in (A). In the ‘diluted’ condition, HSS and NPE were each diluted 5- and 4-fold with ELB, respectively.
    Figure Legend Snippet: Efficient DNA replication of immobilized plasmids. ( A ) The 1.8% agarose blocks (5 µl) containing 0.1 nM p10.4 were incubated with two volumes of HSS, with or without Geminin. After 30 min, the supernatant was exchanged with two volumes of NPE containing [α- 32 P]dATP. Reactions in solution containing a final concentration of 0.1 or 0.5 nM p10.4 were carried out in parallel. In all cases, DNA replication efficiency was determined 90 min after NPE addition. In lanes 1 and 2, the DNA was not released from the block and thus remained in the well, where the block was loaded. In lane 4, one-fifth of the 0.5 nM reaction was loaded. ( B ) Cartoon illustrating procedure to determine plasmid position and replication in an agarose block. ( C ) The 1.8% agarose blocks containing 0.1 nM p10.4 and 2.8 µm beads (to provide reference points) were prepared. Plasmids were stained with YOYO-1, photographed and de-stained. Subsequently, HSS was added, followed by NPE containing biotin-dUTP. After 90 min, the blocks were stained again with YOYO-1 and the biotin-dUTP was detected with AlexaFluor 647 conjugated streptavidin (SA-647). The same position in the block that was imaged before extract addition was located and images were acquired by fluorescence microscopy. The green and blue channels represent the initial and final plasmid positions as determined by YOYO-1 staining, respectively. The red channel represents SA-647 staining of incorporated biotin-dUTP. The blue and red channels were shifted in the y-axis to facilitate analysis. Plasmid classification is shown below the shifted image. (bar = 5 µm) ( D ) The average percentage of plasmids that was immobile, shifted, or mobile from eight areas of a single block was calculated and graphed. The subset of plasmids in each group that replicated is shown in red. Error bars indicate the standard deviation of the eight areas that were scanned. ( E ) Replication in diluted extracts. p10.4 (0.1 nM) was replicated in agarose blocks or in solution as described in (A). In the ‘diluted’ condition, HSS and NPE were each diluted 5- and 4-fold with ELB, respectively.

    Techniques Used: Incubation, Concentration Assay, Blocking Assay, Plasmid Preparation, Staining, Fluorescence, Microscopy, Standard Deviation

    30) Product Images from "A transient ischemic environment induces reversible compaction of chromatin"

    Article Title: A transient ischemic environment induces reversible compaction of chromatin

    Journal: Genome Biology

    doi: 10.1186/s13059-015-0802-2

    Oxygen and nutrient deprivation induces compaction of chromatin. HL-1 cells were fixed, permeabilized, and immunostained with anti-acetylated histone H3K14 and then counter-stained with Vybrant DyeCycle Violet. Two-color SMLM was performed on untreated HL-1 cells ( a , b ) or on cells exposed to 1 hour of OND ( d , e ). The dashed boxes in ( a , d ) are shown as zoomed views in ( b ) and ( e ), respectively. For comparison, wide-field images of the inset regions are shown in ( c , f ). Chromatin voids are indicated by asterisks and atolls marked by the arrow . Representative SMLM images of Vybrant Dyecycle Violet-stained nuclei, either untreated, subjected to 1 hour of OND or 5, 15, 60 and 240 minutes after release from OND are shown in ( g ). A discriminatory threshold (pixel intensity ≤ 50) was applied to the experimental set of SMLM imaged nuclei (a minimum of nine cells were imaged), with box plots and representative images describing the median and range of the proportion of the nucleus with chromatin shown in ( h ). P values compared with untreated are reported above the box plots. UT untreated
    Figure Legend Snippet: Oxygen and nutrient deprivation induces compaction of chromatin. HL-1 cells were fixed, permeabilized, and immunostained with anti-acetylated histone H3K14 and then counter-stained with Vybrant DyeCycle Violet. Two-color SMLM was performed on untreated HL-1 cells ( a , b ) or on cells exposed to 1 hour of OND ( d , e ). The dashed boxes in ( a , d ) are shown as zoomed views in ( b ) and ( e ), respectively. For comparison, wide-field images of the inset regions are shown in ( c , f ). Chromatin voids are indicated by asterisks and atolls marked by the arrow . Representative SMLM images of Vybrant Dyecycle Violet-stained nuclei, either untreated, subjected to 1 hour of OND or 5, 15, 60 and 240 minutes after release from OND are shown in ( g ). A discriminatory threshold (pixel intensity ≤ 50) was applied to the experimental set of SMLM imaged nuclei (a minimum of nine cells were imaged), with box plots and representative images describing the median and range of the proportion of the nucleus with chromatin shown in ( h ). P values compared with untreated are reported above the box plots. UT untreated

    Techniques Used: Staining

    31) Product Images from "The p53 activator overcomes resistance to ALK inhibitors by regulating p53-target selectivity in ALK-driven neuroblastomas"

    Article Title: The p53 activator overcomes resistance to ALK inhibitors by regulating p53-target selectivity in ALK-driven neuroblastomas

    Journal: Cell Death Discovery

    doi: 10.1038/s41420-018-0059-0

    Combination treatment with the two ALK inhibitors and a p53 activator induces activation of the intrinsic apoptosis pathway. a , b Activation of mitochondria-mediated apoptotic pathway by combination treatment of the two ALK inhibitors with the p53 activator Nutlin-3. NB39-nu or NB1 cells were treated with 1000 nM of either of the two ALK inhibitors and 10 µM of Nutlin-3 for 16 h. The activation of the caspase cascade as a result of combination treatment was detected by immunoblot analysis ( a ). The expression levels of caspases-8 and -9 in NB39-nu or NB1 cells are shown in ( b ). c The increase in cytosolic cytochrome c following combination treatment. NB1 cells were treated with 1000 nM of either of the two ALK inhibitors and 10 µM of Nutlin-3 for 16 h. Cytosolic fractions were prepared by subcellular fractionation and the amount of cytochrome c present was quantified by ELISA. Data show the mean ± SEM ( n = 3). * p
    Figure Legend Snippet: Combination treatment with the two ALK inhibitors and a p53 activator induces activation of the intrinsic apoptosis pathway. a , b Activation of mitochondria-mediated apoptotic pathway by combination treatment of the two ALK inhibitors with the p53 activator Nutlin-3. NB39-nu or NB1 cells were treated with 1000 nM of either of the two ALK inhibitors and 10 µM of Nutlin-3 for 16 h. The activation of the caspase cascade as a result of combination treatment was detected by immunoblot analysis ( a ). The expression levels of caspases-8 and -9 in NB39-nu or NB1 cells are shown in ( b ). c The increase in cytosolic cytochrome c following combination treatment. NB1 cells were treated with 1000 nM of either of the two ALK inhibitors and 10 µM of Nutlin-3 for 16 h. Cytosolic fractions were prepared by subcellular fractionation and the amount of cytochrome c present was quantified by ELISA. Data show the mean ± SEM ( n = 3). * p

    Techniques Used: Activation Assay, Expressing, Fractionation, Enzyme-linked Immunosorbent Assay

    32) Product Images from "Multiplexed Supramolecular Self-Assembly For Non-Viral Gene Delivery"

    Article Title: Multiplexed Supramolecular Self-Assembly For Non-Viral Gene Delivery

    Journal: Biomaterials

    doi: 10.1016/j.biomaterials.2010.08.024

    (A) In vitro transfection of COS-7 cells with complexes formed at various N/P ratios. Lipofectamine 2000 (LFA) was used as a control. (B) In vitro transfection of HeLa cells with complexes formed at various N/P ratios. Lipofectamine 2000 (LFA) was used
    Figure Legend Snippet: (A) In vitro transfection of COS-7 cells with complexes formed at various N/P ratios. Lipofectamine 2000 (LFA) was used as a control. (B) In vitro transfection of HeLa cells with complexes formed at various N/P ratios. Lipofectamine 2000 (LFA) was used

    Techniques Used: In Vitro, Transfection

    33) Product Images from "Light-Mediated Activation of siRNA Release in Diblock Copolymer Assemblies for Controlled Gene Silencing"

    Article Title: Light-Mediated Activation of siRNA Release in Diblock Copolymer Assemblies for Controlled Gene Silencing

    Journal: Advanced healthcare materials

    doi: 10.1002/adhm.201400671

    Representative western blot analysis of NIH/3T3 cell extracts collected 48 h post-transfection (n = 4). Cells were treated with GAPDH targeted siRNA/mPEG- b -P(APNBMA) 23.6 polyplexes with (+) or without (−) UV exposure (20 min), or with Lipofectamine
    Figure Legend Snippet: Representative western blot analysis of NIH/3T3 cell extracts collected 48 h post-transfection (n = 4). Cells were treated with GAPDH targeted siRNA/mPEG- b -P(APNBMA) 23.6 polyplexes with (+) or without (−) UV exposure (20 min), or with Lipofectamine

    Techniques Used: Western Blot, Transfection

    34) Product Images from "Methylated DNA-Binding Proteins from Arabidopsis 1"

    Article Title: Methylated DNA-Binding Proteins from Arabidopsis 1

    Journal: Plant Physiology

    doi: 10.1104/pp.103.026708

    Histochemical analysis. Maize ( Zea mays ) nuclei and chromosomes were prepared on slide glasses and stained with 4′,6 diamidino-2-phenylindole (DAPI; A and C). Nucleus at interphase and metaphase is seen at left and right side, respectively. Samples were then subjected to binding with GST (B) or AtMBD5-GST fusion protein (D) and visualized with rhodamine-labeled anti-GST antibodies. Extended DNA fibers were simultaneously visualized with YOYO staining (E) and AtMBD5-GST fusion protein (F). A separately prepared extended DNA fiber was stained with monoclonal anti-m 5 C antibodies and visualized with the labeled secondary anti-mouse antibodies (G). Bar = 10 μm.
    Figure Legend Snippet: Histochemical analysis. Maize ( Zea mays ) nuclei and chromosomes were prepared on slide glasses and stained with 4′,6 diamidino-2-phenylindole (DAPI; A and C). Nucleus at interphase and metaphase is seen at left and right side, respectively. Samples were then subjected to binding with GST (B) or AtMBD5-GST fusion protein (D) and visualized with rhodamine-labeled anti-GST antibodies. Extended DNA fibers were simultaneously visualized with YOYO staining (E) and AtMBD5-GST fusion protein (F). A separately prepared extended DNA fiber was stained with monoclonal anti-m 5 C antibodies and visualized with the labeled secondary anti-mouse antibodies (G). Bar = 10 μm.

    Techniques Used: Staining, Binding Assay, Labeling

    35) Product Images from "Adsorbed Fibrinogen Enhances Production of Bone- and Angiogenic-Related Factors by Monocytes/Macrophages"

    Article Title: Adsorbed Fibrinogen Enhances Production of Bone- and Angiogenic-Related Factors by Monocytes/Macrophages

    Journal: Tissue Engineering. Part A

    doi: 10.1089/ten.tea.2012.0439

    Monocyte/macrophage morphology on Ch films. (A) Macrophages were differentiated on RGD (a–c) , Ch films (d–f) , and Ch films with adsorbed human Fg (g–i) . (B) Macrophages were differentiated on RGD (a,b) , Ch films (c,d) , and Ch films with adsorbed human Fg (e,f) in the presence of IL-4. At days 3, 7, and 10 cells were fixed and fluorescently stained for F-actin filaments with rhodamine phalloidin (red) and nuclei with YOYO-1 (green). Arrows indicate filopodia structures. Scale bar corresponds to 100 μm.
    Figure Legend Snippet: Monocyte/macrophage morphology on Ch films. (A) Macrophages were differentiated on RGD (a–c) , Ch films (d–f) , and Ch films with adsorbed human Fg (g–i) . (B) Macrophages were differentiated on RGD (a,b) , Ch films (c,d) , and Ch films with adsorbed human Fg (e,f) in the presence of IL-4. At days 3, 7, and 10 cells were fixed and fluorescently stained for F-actin filaments with rhodamine phalloidin (red) and nuclei with YOYO-1 (green). Arrows indicate filopodia structures. Scale bar corresponds to 100 μm.

    Techniques Used: Staining

    36) Product Images from "Multifunctional Nucleus-targeting Nanoparticles with Ultra-high Gene Transfection Efficiency for In Vivo Gene Therapy"

    Article Title: Multifunctional Nucleus-targeting Nanoparticles with Ultra-high Gene Transfection Efficiency for In Vivo Gene Therapy

    Journal: Theranostics

    doi: 10.7150/thno.17588

    Comparison of the transfection efficiency of PF 33 /pGFP (PF 33 ), RRPHC/pGFP (RRPHC) and Lipofectamine 3000/pGFP (Lipo 3000) in medium containing 0% - 30% serum in HCT 116 cell. (A) Fluorescence microscopy images. (B) Analysis of transfection efficiency in serum-free medium by flow cytometry. (C) Analysis of the transfection efficiency in medium containing 30% serum by flow cytometry. (D, E) Quantitative analysis of GFP-positive cells (%) and Mean Fluorescence Intensity (MFI) by flow cytometry. pGFP indicates GFP pDNA. The scale bar indicates 200 μm.
    Figure Legend Snippet: Comparison of the transfection efficiency of PF 33 /pGFP (PF 33 ), RRPHC/pGFP (RRPHC) and Lipofectamine 3000/pGFP (Lipo 3000) in medium containing 0% - 30% serum in HCT 116 cell. (A) Fluorescence microscopy images. (B) Analysis of transfection efficiency in serum-free medium by flow cytometry. (C) Analysis of the transfection efficiency in medium containing 30% serum by flow cytometry. (D, E) Quantitative analysis of GFP-positive cells (%) and Mean Fluorescence Intensity (MFI) by flow cytometry. pGFP indicates GFP pDNA. The scale bar indicates 200 μm.

    Techniques Used: Transfection, Fluorescence, Microscopy, Flow Cytometry, Cytometry

    Transfection efficiencies of various complexes in HCT 116 cells at 24 h. (A) Comparison of various complexes in serum-free medium, PEI 1.8K/pGFP (a), PEI 25K/pGFP (b), PF 33 /pGFP at mass ratio of 5:1 (c), 10:1(d), HAC/pGFP (e), and RRPHC/pGFP (f). (B) Quantitative analysis of transfection efficiency in serum-free medium by flow cytometry. (C) Comparison of PF 33 /pGFP (PF 33 ), RRPHC/pGFP (RRPHC) and Lipofectamine 2000/pGFP (Lipo 2000) in medium containing 10% - 30% serum. (D) Quantitative analysis of transfection efficiency in medium containing 10% - 30% serum by flow cytometry. *p
    Figure Legend Snippet: Transfection efficiencies of various complexes in HCT 116 cells at 24 h. (A) Comparison of various complexes in serum-free medium, PEI 1.8K/pGFP (a), PEI 25K/pGFP (b), PF 33 /pGFP at mass ratio of 5:1 (c), 10:1(d), HAC/pGFP (e), and RRPHC/pGFP (f). (B) Quantitative analysis of transfection efficiency in serum-free medium by flow cytometry. (C) Comparison of PF 33 /pGFP (PF 33 ), RRPHC/pGFP (RRPHC) and Lipofectamine 2000/pGFP (Lipo 2000) in medium containing 10% - 30% serum. (D) Quantitative analysis of transfection efficiency in medium containing 10% - 30% serum by flow cytometry. *p

    Techniques Used: Transfection, HAC Assay, Flow Cytometry, Cytometry

    37) Product Images from "Imaging chromatin nanostructure with binding-activated localization microscopy based on DNA structure fluctuations"

    Article Title: Imaging chromatin nanostructure with binding-activated localization microscopy based on DNA structure fluctuations

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1301

    DNA structure fluctuation-assisted BALM (fBALM) of HL-1 cell nucleus using YOYO-1. ( A ) Super-resolution DNA signal density image reconstructed from single molecule signals of YOYO-1 transiently fluorescing at locally renaturing dsDNA. The image was acquired 5 h after immersion with glucose oxidase containing buffer (pH∼3.7). The gradual drop in pH over time ensured preservation of nuclear structure (Figure 2 ). A part of a conventional wide field image presented in grey for comparison. ( B ) Left: 3× magnification of a rectangular region of interest indicated in (A). Asterisks show void regions with very low signal density, likely an interchromatin compartment ( 3 ). Right: high magnification of the small inset shown in (B) on the left depicts a progressively enlarged part of the image illustrating structure details at nanometer scale. ( C ) Signal intensity profile between arrow-heads marked in B, inset lower right. The red line corresponds to multiple Gaussian fits. Black squares correspond to the actual signal intensity in the DNA density image. The numbers correspond to the full-width-at-half-maximum (FWHM) for each of the peaks fitted with Gaussian curve. ( D ) Fourier ring correlation (FRC) analysis of the super-resolution image with the resolution value estimate obtained from the intersection of the polynomial fit with the empirical threshold. ( E ) Comparison of single molecule localization microscopy on HL-1 cells labeled with anti-H3 primary and Alexa 647 secondary antibody (left), or with a 10 min EdU pulse followed by Alexa 488 ‘click-it’ reaction to stain chromatin surrounding replication factories.
    Figure Legend Snippet: DNA structure fluctuation-assisted BALM (fBALM) of HL-1 cell nucleus using YOYO-1. ( A ) Super-resolution DNA signal density image reconstructed from single molecule signals of YOYO-1 transiently fluorescing at locally renaturing dsDNA. The image was acquired 5 h after immersion with glucose oxidase containing buffer (pH∼3.7). The gradual drop in pH over time ensured preservation of nuclear structure (Figure 2 ). A part of a conventional wide field image presented in grey for comparison. ( B ) Left: 3× magnification of a rectangular region of interest indicated in (A). Asterisks show void regions with very low signal density, likely an interchromatin compartment ( 3 ). Right: high magnification of the small inset shown in (B) on the left depicts a progressively enlarged part of the image illustrating structure details at nanometer scale. ( C ) Signal intensity profile between arrow-heads marked in B, inset lower right. The red line corresponds to multiple Gaussian fits. Black squares correspond to the actual signal intensity in the DNA density image. The numbers correspond to the full-width-at-half-maximum (FWHM) for each of the peaks fitted with Gaussian curve. ( D ) Fourier ring correlation (FRC) analysis of the super-resolution image with the resolution value estimate obtained from the intersection of the polynomial fit with the empirical threshold. ( E ) Comparison of single molecule localization microscopy on HL-1 cells labeled with anti-H3 primary and Alexa 647 secondary antibody (left), or with a 10 min EdU pulse followed by Alexa 488 ‘click-it’ reaction to stain chromatin surrounding replication factories.

    Techniques Used: Preserving, Microscopy, Labeling, Staining

    38) Product Images from "Compaction and condensation of DNA mediated by the C-terminal domain of Hfq"

    Article Title: Compaction and condensation of DNA mediated by the C-terminal domain of Hfq

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx431

    Long axis of unconstrained T4–DNA in T-buffer versus the concentration of Hfq–CTR (red, ○) and Hfq–NTR (green, ⋄). The dashed curves are drawn as an aid to the eye and the arrow denotes the condensation threshold (for Hfq–CTR only). The inset shows fluorescence images of a non-condensed (left) and condensed (right) T4–DNA molecule.
    Figure Legend Snippet: Long axis of unconstrained T4–DNA in T-buffer versus the concentration of Hfq–CTR (red, ○) and Hfq–NTR (green, ⋄). The dashed curves are drawn as an aid to the eye and the arrow denotes the condensation threshold (for Hfq–CTR only). The inset shows fluorescence images of a non-condensed (left) and condensed (right) T4–DNA molecule.

    Techniques Used: Concentration Assay, Fluorescence

    ( A ) Relative extension R ∥ / L of T4–DNA inside 200 × 300 nm 2 channels and in T-buffer versus the concentration of Hfq–CTR (red, ○) and Hfq–NTR (green, ⋄). ( B ) As in panel A, but for T4–DNA inside 150 × 250 nm 2 channels and in T-buffer with 30 mM KGlu. The dashed curves are drawn as an aid to the eye and the arrows denote the condensation thresholds.
    Figure Legend Snippet: ( A ) Relative extension R ∥ / L of T4–DNA inside 200 × 300 nm 2 channels and in T-buffer versus the concentration of Hfq–CTR (red, ○) and Hfq–NTR (green, ⋄). ( B ) As in panel A, but for T4–DNA inside 150 × 250 nm 2 channels and in T-buffer with 30 mM KGlu. The dashed curves are drawn as an aid to the eye and the arrows denote the condensation thresholds.

    Techniques Used: Concentration Assay

    In vitro analysis of Hfq protein–DNA binding properties. ( A ) Gel shift showing the binding of wild-type Hfq to DNA. ( B ) Binding of Hfq–CTR. ( C ) Binding of Hfq–NTR. The intensities of the asterisk-marked bands are set out in the graphs versus the concentration of monomeric Hfq, Hfq–CTR and Hfq–NTR, respectively. Note that a significant supershifted species is observed only in the case of wild-type Hfq. Curves represent non-linear least-squares fits of cooperative (Hfq, Hfq–NTR) and non-cooperative (Hfq–CTR) binding models.
    Figure Legend Snippet: In vitro analysis of Hfq protein–DNA binding properties. ( A ) Gel shift showing the binding of wild-type Hfq to DNA. ( B ) Binding of Hfq–CTR. ( C ) Binding of Hfq–NTR. The intensities of the asterisk-marked bands are set out in the graphs versus the concentration of monomeric Hfq, Hfq–CTR and Hfq–NTR, respectively. Note that a significant supershifted species is observed only in the case of wild-type Hfq. Curves represent non-linear least-squares fits of cooperative (Hfq, Hfq–NTR) and non-cooperative (Hfq–CTR) binding models.

    Techniques Used: In Vitro, Binding Assay, Electrophoretic Mobility Shift Assay, Concentration Assay

    39) Product Images from "Spatial separation between replisome‐ and template‐induced replication stress signaling"

    Article Title: Spatial separation between replisome‐ and template‐induced replication stress signaling

    Journal: The EMBO Journal

    doi: 10.15252/embj.201798369

    ss DNA accumulates within tracts of newly synthesized DNA in response to polymerase‐blocking lesions DNA fibers, prepared by combing of genomic DNA isolated from cells synchronized in G1 and released into S phase in the presence of EdU (added 15 min before release). Cells were harvested 20 min after release for control; 30 min after UV irradiation (20 J/m 2 ); 30 min after treatment with 0.04% MMS for 30 min prior to release; and 60 min after release into 120 mM HU. Fibers were stained with YOYO‐1 for total DNA (blue). EdU incorporation was visualized by a click reaction with Alexa Fluor 647 (red), and ssDNA was detected by means of an antibody (green). Scale bar = 10 kbp. Quantification of the fraction of ssDNA within newly replicated DNA, determined for individual EdU‐stained tracts by measuring total tract length and total length of ssDNA within that tract. Evidence for EdU‐stained regions representing replication tracts is shown in Appendix Fig S3D and E . Number of replication tracts analyzed: Control = 186; UV = 204; MMS = 168; HU = 198. Quantification of the fraction of ssDNA within or outside of EdU‐stained replication tracts derived from WT or exo1Δ cells, determined as above. G1‐arrested cells were incubated with or without 0.02% MMS for 30 min and released into EdU for 30 min. Number of replication tracts analyzed: WT control = 63; exo1Δ control = 151; WT MMS = 124; exo1Δ MMS = 122. Number of EdU‐negative tracts analyzed: WT control = 96; WT MMS = 171. Density of ssDNA tracts within or outside of individual replication tracts from the experiment shown in panel (C), calculated by dividing the number of ssDNA tracts by the length (in kb) of the corresponding EdU‐stained or EdU‐negative region. Data information: (B–D) Significance was calculated by the Mann–Whitney test (ns: not significant; * P
    Figure Legend Snippet: ss DNA accumulates within tracts of newly synthesized DNA in response to polymerase‐blocking lesions DNA fibers, prepared by combing of genomic DNA isolated from cells synchronized in G1 and released into S phase in the presence of EdU (added 15 min before release). Cells were harvested 20 min after release for control; 30 min after UV irradiation (20 J/m 2 ); 30 min after treatment with 0.04% MMS for 30 min prior to release; and 60 min after release into 120 mM HU. Fibers were stained with YOYO‐1 for total DNA (blue). EdU incorporation was visualized by a click reaction with Alexa Fluor 647 (red), and ssDNA was detected by means of an antibody (green). Scale bar = 10 kbp. Quantification of the fraction of ssDNA within newly replicated DNA, determined for individual EdU‐stained tracts by measuring total tract length and total length of ssDNA within that tract. Evidence for EdU‐stained regions representing replication tracts is shown in Appendix Fig S3D and E . Number of replication tracts analyzed: Control = 186; UV = 204; MMS = 168; HU = 198. Quantification of the fraction of ssDNA within or outside of EdU‐stained replication tracts derived from WT or exo1Δ cells, determined as above. G1‐arrested cells were incubated with or without 0.02% MMS for 30 min and released into EdU for 30 min. Number of replication tracts analyzed: WT control = 63; exo1Δ control = 151; WT MMS = 124; exo1Δ MMS = 122. Number of EdU‐negative tracts analyzed: WT control = 96; WT MMS = 171. Density of ssDNA tracts within or outside of individual replication tracts from the experiment shown in panel (C), calculated by dividing the number of ssDNA tracts by the length (in kb) of the corresponding EdU‐stained or EdU‐negative region. Data information: (B–D) Significance was calculated by the Mann–Whitney test (ns: not significant; * P

    Techniques Used: Synthesized, Blocking Assay, Isolation, Irradiation, Staining, Derivative Assay, Incubation, MANN-WHITNEY

    40) Product Images from "Additive manufacturing of laminar flow cells for single-molecule experiments"

    Article Title: Additive manufacturing of laminar flow cells for single-molecule experiments

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-53151-z

    Single-channel LFC. ( a ) Single-channel LFC mounted on the microscope stage and connected to the pumping system. ( b ) The 3D design of the single-channel LFC. ( c ) The depiction of immobilization and linearization of a single DNA molecule (green line) interacting with a fluorescently labelled protein (red circle). The DNA is held in place using an optical trap (red cone) to control a polystyrene microsphere (black circle) attached to one end of the DNA. ( d ) Bright-field microscope image of the trapped polystyrene microsphere (top; position of DNA indicated with dashed line), pseudo-coloured fluorescent image of DNA labelled with YOYO (middle), pseudo-coloured fluorescent image of a single protein molecule labelled with ATTO647N while scanning along the DNA with gamma adjusted to 1.3 (bottom). White scale bars equal 1 µm. ( e ) A kymograph of the movement of protein along DNA with a duration of around 2 seconds; the horizontal and vertical white scale bars equal 1 µm and 200 ms, respectively. ( f ) Density distribution of proteins’ movement within frame intervals of 13.5 m for a collection of 49 trajectories of AlkF as exemplified in ( e ). ( g ) Mean squared displacement (MSD) of 49 scanning trajectories of AlkF along DNA. Error bars represent standard error of the mean (SEM).
    Figure Legend Snippet: Single-channel LFC. ( a ) Single-channel LFC mounted on the microscope stage and connected to the pumping system. ( b ) The 3D design of the single-channel LFC. ( c ) The depiction of immobilization and linearization of a single DNA molecule (green line) interacting with a fluorescently labelled protein (red circle). The DNA is held in place using an optical trap (red cone) to control a polystyrene microsphere (black circle) attached to one end of the DNA. ( d ) Bright-field microscope image of the trapped polystyrene microsphere (top; position of DNA indicated with dashed line), pseudo-coloured fluorescent image of DNA labelled with YOYO (middle), pseudo-coloured fluorescent image of a single protein molecule labelled with ATTO647N while scanning along the DNA with gamma adjusted to 1.3 (bottom). White scale bars equal 1 µm. ( e ) A kymograph of the movement of protein along DNA with a duration of around 2 seconds; the horizontal and vertical white scale bars equal 1 µm and 200 ms, respectively. ( f ) Density distribution of proteins’ movement within frame intervals of 13.5 m for a collection of 49 trajectories of AlkF as exemplified in ( e ). ( g ) Mean squared displacement (MSD) of 49 scanning trajectories of AlkF along DNA. Error bars represent standard error of the mean (SEM).

    Techniques Used: Microscopy, Mass Spectrometry

    DNA dumbbell construction. Step-by-step process of DNA dumbbell construction and exposure to intercalating dye in a flow-free environment using reservoir-based LFC. (1) Individual trapping of DNA-attached streptavidin coated (diameter: 1.76 µm) and free anti-digoxigenin coated beads (diameter: 0.9 µm) within the reservoir and translocating into the main channel; (top-right inset: bright-field image of the trapped beads) (2) applying gentle flow (0.05 mm/s) for a few seconds to elongate DNA and attachment to second bead (3) Incubation of the DNA dumbbell with YOYO-1 in a flow-free reservoir. (4) Visualization of the DNA in the flow-free main channel; bottom-left inset: fluorescent image of DNA dumbbell.
    Figure Legend Snippet: DNA dumbbell construction. Step-by-step process of DNA dumbbell construction and exposure to intercalating dye in a flow-free environment using reservoir-based LFC. (1) Individual trapping of DNA-attached streptavidin coated (diameter: 1.76 µm) and free anti-digoxigenin coated beads (diameter: 0.9 µm) within the reservoir and translocating into the main channel; (top-right inset: bright-field image of the trapped beads) (2) applying gentle flow (0.05 mm/s) for a few seconds to elongate DNA and attachment to second bead (3) Incubation of the DNA dumbbell with YOYO-1 in a flow-free reservoir. (4) Visualization of the DNA in the flow-free main channel; bottom-left inset: fluorescent image of DNA dumbbell.

    Techniques Used: Flow Cytometry, Incubation

    Related Articles

    Flow Cytometry:

    Article Title: Phage Mu Gam protein promotes NHEJ in concert with Escherichia coli ligase
    Article Snippet: .. After all free FLAG-Gam was washed out, the flow cell was switched to imaging buffer containing 0.5 nM YOYO1 (Invitrogen), 1.4 mM glucose, glucose oxidase, and catalase to visualize DNA. .. Following injection of YOYO-1, 20 nM RecBCD was injected into the flow cell at 0.4 mL⋅min−1 .

    Article Title: DNA interrogation by the CRISPR RNA-guided endonuclease Cas9
    Article Snippet: .. The flow cell was then washed with 3–5 mL of Imaging Buffer containing 40 mM Tris-HCl, 25 mM KCl, 1mg mL−1 BSA, 1 mM MgCl2 , 1 mM DTT, 0.75 nM YOYO1 (Life Technologies), 0.8% glucose, and 0.2X glucose oxidase/catalase. .. Finally, 0.5 nM anti-FLAG antibody-coated QDs were incubated in the flow cell for 5 min, followed by a wash of 1–2 mL of Imaging Buffer.

    Sample Prep:

    Article Title: Method to identify and minimize artifacts induced by fluorescent impurities in single-molecule localization microscopy
    Article Snippet: .. 2.16 DNA Sample Preparation To further demonstrate our spectral fitting method, we imaged stretched lambda phage DNA (Thermo Scientific, SD0011) labeled with YOYO-1 (Invitrogen, Y3601). ..

    Labeling:

    Article Title: Method to identify and minimize artifacts induced by fluorescent impurities in single-molecule localization microscopy
    Article Snippet: .. 2.16 DNA Sample Preparation To further demonstrate our spectral fitting method, we imaged stretched lambda phage DNA (Thermo Scientific, SD0011) labeled with YOYO-1 (Invitrogen, Y3601). ..

    Article Title: Fabrication of long poly(dimethyl siloxane) nanochannels by replicating protein deposit from confined solution evaporation
    Article Snippet: .. Bovine serum albumin (BSA) was purchased from Sigma-Aldrich. λ-DNA was purchased from New England BioLab and labeled with YOYO-1 (Y3601, purchased from Invitrogen) for observation. ..

    Imaging:

    Article Title: Phage Mu Gam protein promotes NHEJ in concert with Escherichia coli ligase
    Article Snippet: .. After all free FLAG-Gam was washed out, the flow cell was switched to imaging buffer containing 0.5 nM YOYO1 (Invitrogen), 1.4 mM glucose, glucose oxidase, and catalase to visualize DNA. .. Following injection of YOYO-1, 20 nM RecBCD was injected into the flow cell at 0.4 mL⋅min−1 .

    Article Title: DNA interrogation by the CRISPR RNA-guided endonuclease Cas9
    Article Snippet: .. The flow cell was then washed with 3–5 mL of Imaging Buffer containing 40 mM Tris-HCl, 25 mM KCl, 1mg mL−1 BSA, 1 mM MgCl2 , 1 mM DTT, 0.75 nM YOYO1 (Life Technologies), 0.8% glucose, and 0.2X glucose oxidase/catalase. .. Finally, 0.5 nM anti-FLAG antibody-coated QDs were incubated in the flow cell for 5 min, followed by a wash of 1–2 mL of Imaging Buffer.

    BIA-KA:

    Article Title: Macropinocytosis activated by oncogenic Dbl enables specific targeted delivery of Tat/pDNA nano-complexes into ovarian cancer cells
    Article Snippet: .. The YOYO-1 (Y3601), Dextran-AF647 (D22914), Tfn-AF647 (T-23366), CTxB-AF647 (C-34778), goat anti-mouse IgG-AF488 (A-11001), anti-mouse IgG-AF647 (A32728), anti-rabbit IgG-AF488 (A-11070), 5-(N-ethyl-N-isopropyl) amiloride (E3111), Lipofectamine 3000 (L3000-015), Lipofectamine® RNAiMax (13778075), and the BCA protein assay Kit (23225) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). .. Rhodamine-phalloidin (PHDR1) was purchased from Cytoskeleton, Inc (Denver, CO, USA).

    Article Title: Macropinocytosis activated by oncogenic Dbl enables specific targeted delivery of Tat/pDNA nano-complexes into ovarian cancer cells
    Article Snippet: .. The YOYO-1 (Y3601), Dextran-AF647 , Tfn-AF647 (T-23366), CTxB-AF647 (C-34778), goat anti-mouse IgG-AF488 (A-11001), anti-mouse IgG-AF647 , anti-rabbit IgG-AF488 (A-11070), 5-(N-ethyl-N-isopropyl) amiloride (E3111), Lipofectamine 3000 (L3000-015), Lipofectamine® RNAiMax (13778075), and the BCA protein assay Kit (23225) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). .. Rhodamine-phalloidin (PHDR1) was purchased from Cytoskeleton, Inc (Denver, CO, USA).

    Staining:

    Article Title: Specific Transfection of Inflamed Brain by Macrophages: A New Therapeutic Strategy for Neurodegenerative Diseases
    Article Snippet: .. A nucleic acid stain, YOYO-1 iodide (491/509), was obtained from Invitrogen (Carlsbad, CA). .. Cells A mouse macrophage cell line (RAW 264.7) was purchased from ATCC (cat # TIB-71), and cultured in Dulbecco's Modified Eagle's Media (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS.

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  • 92
    Thermo Fisher yoyo 1
    The P2X7R permeabilization pathway is cation selective. (a) Fluorescence images of human macrophages incubated in the presence of ATP (2 mM) with the anionic dyes Lucifer yellow (LY, 0.5 mM) or carboxyfluorescein (CF, 0.5 mM) at 37°C. Scale bars: 20 μm. (b) Quantitative comparison of anionic dye uptake by macrophages. Cells were incubated for 15 min with LY or CF in the presence of ATP (2 mM) at 37°C. “n.s.” are not significantly different from ATP (n = 4). (c) Fluorescence images of human macrophages preloaded with anionic calcein-AM (0.5 µM) for 30 min and subsequently stimulated with ATP (2 mM) for 15 min at 37°C. (d) Quantification of change in intracellular calcein fluorescence after 15 min stimulation with ATP (2 mM). There is no significant difference in fluorescence dye after ATP treatment (n = 4 separate experiments). (e) Fluorescence images of human macrophages incubated in the presence of ATP (2 mM) with the cationic dye <t>YOYO-1</t> (5 µM) for 15 mins at 37°C. (f) Quantification of YOYO-1 after macrophages were incubated for 15 min with ATP (2 mM) at 37°C. There is significant uptake of dye by ATP treated cells (p
    Yoyo 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher yoyo 1 label nucleated cells
    Identification of permeable cell types after intracerebral hemorrhage (ICH). (A-C) Detection of cell types with plasmalemma permeability to <t>YOYO-1</t> iodide at 6 h after ICH. (A) Representative photomicrographs of YOYO-1+ cells (a), IBA-1+ (b), and overlay (c) showing no colocalization. (B) Representative photomicrographs of YOYO-1+ cells (d), GFAP+ cells (e), and overlay (f) showing no colocalization. (C) Representative photomicrographs of NeuN+ neurons (g) that colocalized with YOYO-1 (h) at 6 h after ICH (i, overlay) suggesting that neurons are particularly sensitive to plasmalemma damage early after ICH. (D) Detection of cell types with plasmalemma permeability to YOYO-1 at 24 h after ICH. By 24 h, YOYO-1+ cells (j) colocalized with IBA-1+ cells with morphological features of microglia (k) as shown in the overlay (l). YOYO-1+ cells (m) also colocalized with IBA-1+ cells with the morphological appearance of macrophages (n) at 24 h (o, overlay). Scale bar, 10 um for each panel.
    Yoyo 1 Label Nucleated Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher yoyo labeled dna
    <t>DNA</t> focusing into a NZMW. Fluorescence time traces from a single NZMW that contains a 3 nm diameter pore in an array of ZMWs is monitored for the fluorescence from 6000 bp DNA labeled with <t>YOYO-1</t> (9 pixel region of interest for each NZMW, 10.8 ms exposure time, signal-averaged to 100 ms). Inset illustrates DNA entering the illumination volume of a NZMW as it migrates toward the pore, resulting in increased ZMW fluorescence. ZMW arrays are shown in fluorescence images (i)–(iii) with (i) being an averaged image of all frames in the experiment, and (ii) and (iii) being the membrane under respective −850 and 850 mV. Colored arrows identify ZMWs with corresponding colored fluorescence traces in bottom plot. The red arrow identifies a NZMW. Green and red backgrounds in the fluorescence traces correspond to periods of positive and negative voltage, respectively (see Supporting Information for electrical trace).
    Yoyo Labeled Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The P2X7R permeabilization pathway is cation selective. (a) Fluorescence images of human macrophages incubated in the presence of ATP (2 mM) with the anionic dyes Lucifer yellow (LY, 0.5 mM) or carboxyfluorescein (CF, 0.5 mM) at 37°C. Scale bars: 20 μm. (b) Quantitative comparison of anionic dye uptake by macrophages. Cells were incubated for 15 min with LY or CF in the presence of ATP (2 mM) at 37°C. “n.s.” are not significantly different from ATP (n = 4). (c) Fluorescence images of human macrophages preloaded with anionic calcein-AM (0.5 µM) for 30 min and subsequently stimulated with ATP (2 mM) for 15 min at 37°C. (d) Quantification of change in intracellular calcein fluorescence after 15 min stimulation with ATP (2 mM). There is no significant difference in fluorescence dye after ATP treatment (n = 4 separate experiments). (e) Fluorescence images of human macrophages incubated in the presence of ATP (2 mM) with the cationic dye YOYO-1 (5 µM) for 15 mins at 37°C. (f) Quantification of YOYO-1 after macrophages were incubated for 15 min with ATP (2 mM) at 37°C. There is significant uptake of dye by ATP treated cells (p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: ATP-gated P2X7 receptors require chloride channels to promote inflammation in human macrophages

    doi: 10.4049/jimmunol.1801101

    Figure Lengend Snippet: The P2X7R permeabilization pathway is cation selective. (a) Fluorescence images of human macrophages incubated in the presence of ATP (2 mM) with the anionic dyes Lucifer yellow (LY, 0.5 mM) or carboxyfluorescein (CF, 0.5 mM) at 37°C. Scale bars: 20 μm. (b) Quantitative comparison of anionic dye uptake by macrophages. Cells were incubated for 15 min with LY or CF in the presence of ATP (2 mM) at 37°C. “n.s.” are not significantly different from ATP (n = 4). (c) Fluorescence images of human macrophages preloaded with anionic calcein-AM (0.5 µM) for 30 min and subsequently stimulated with ATP (2 mM) for 15 min at 37°C. (d) Quantification of change in intracellular calcein fluorescence after 15 min stimulation with ATP (2 mM). There is no significant difference in fluorescence dye after ATP treatment (n = 4 separate experiments). (e) Fluorescence images of human macrophages incubated in the presence of ATP (2 mM) with the cationic dye YOYO-1 (5 µM) for 15 mins at 37°C. (f) Quantification of YOYO-1 after macrophages were incubated for 15 min with ATP (2 mM) at 37°C. There is significant uptake of dye by ATP treated cells (p

    Article Snippet: MQAE, BAPTA-AM, YO-PRO-1, YOYO-1, pHrodo Red E. coli BioParticles Conjugate, and 35 mm Nunclon surface dishes were purchased from Invitrogen/ThermoFisher (Carlsbad, CA, USA).

    Techniques: Fluorescence, Incubation

    Fluorescent image of YOYO-stained, device-extracted DNA, stretched on silanized glass in 100 µm wide and 5 µm deep PDMS channel. The image is stitched to cover 8 ROIs of an Andor Zyla camera at 100x magnification. The 32 µm scale bar corresponds to 100 kbp.

    Journal: Lab on a chip

    Article Title: Microfluidic long DNA sample preparation from cells

    doi: 10.1039/c8lc01163j

    Figure Lengend Snippet: Fluorescent image of YOYO-stained, device-extracted DNA, stretched on silanized glass in 100 µm wide and 5 µm deep PDMS channel. The image is stitched to cover 8 ROIs of an Andor Zyla camera at 100x magnification. The 32 µm scale bar corresponds to 100 kbp.

    Article Snippet: Cells were lysed with a lysis solution containing 70 µL RIPA buffer (Thermo Fisher Scientific; 0.1% SDS), 10 µL pH 8 TE buffer (Thermo Fisher Scientific), 10 µL SDS lysis buffer (Sigma; 1% SDS), Proteinase K (Qiagen) at a concentration of 2 mg/mL, and YOYO-1 (Thermo Fisher Scientific) at a concentration of 4 µM.

    Techniques: Staining

    Identification of permeable cell types after intracerebral hemorrhage (ICH). (A-C) Detection of cell types with plasmalemma permeability to YOYO-1 iodide at 6 h after ICH. (A) Representative photomicrographs of YOYO-1+ cells (a), IBA-1+ (b), and overlay (c) showing no colocalization. (B) Representative photomicrographs of YOYO-1+ cells (d), GFAP+ cells (e), and overlay (f) showing no colocalization. (C) Representative photomicrographs of NeuN+ neurons (g) that colocalized with YOYO-1 (h) at 6 h after ICH (i, overlay) suggesting that neurons are particularly sensitive to plasmalemma damage early after ICH. (D) Detection of cell types with plasmalemma permeability to YOYO-1 at 24 h after ICH. By 24 h, YOYO-1+ cells (j) colocalized with IBA-1+ cells with morphological features of microglia (k) as shown in the overlay (l). YOYO-1+ cells (m) also colocalized with IBA-1+ cells with the morphological appearance of macrophages (n) at 24 h (o, overlay). Scale bar, 10 um for each panel.

    Journal: Stroke; a journal of cerebral circulation

    Article Title: PLASMALEMMA PERMEABILITY AND NECROTIC CELL DEATH PHENOTYPES AFTER INTRACEREBRAL HEMORRHAGE IN MICE

    doi: 10.1161/STROKEAHA.111.635672

    Figure Lengend Snippet: Identification of permeable cell types after intracerebral hemorrhage (ICH). (A-C) Detection of cell types with plasmalemma permeability to YOYO-1 iodide at 6 h after ICH. (A) Representative photomicrographs of YOYO-1+ cells (a), IBA-1+ (b), and overlay (c) showing no colocalization. (B) Representative photomicrographs of YOYO-1+ cells (d), GFAP+ cells (e), and overlay (f) showing no colocalization. (C) Representative photomicrographs of NeuN+ neurons (g) that colocalized with YOYO-1 (h) at 6 h after ICH (i, overlay) suggesting that neurons are particularly sensitive to plasmalemma damage early after ICH. (D) Detection of cell types with plasmalemma permeability to YOYO-1 at 24 h after ICH. By 24 h, YOYO-1+ cells (j) colocalized with IBA-1+ cells with morphological features of microglia (k) as shown in the overlay (l). YOYO-1+ cells (m) also colocalized with IBA-1+ cells with the morphological appearance of macrophages (n) at 24 h (o, overlay). Scale bar, 10 um for each panel.

    Article Snippet: To show that PI and YOYO-1 label nucleated cells in the ICH model, some brain sections were counterstained with Hoechst 33342 (Thermo Scientific, Rockford, IL) (1:1000) for 15 s.

    Techniques: Permeability

    Plasmalemma resealing after intracerebral hemorrhage. Mice were subjected to ICH and administered YOYO-1 at 24 or 48 h and PI at 48 or 72 h, respectively. (a–d) representative photomicrographs of brain sections showing YOYO-1+ and PI+ cells labeled at 24–48 h. Note that the majority of YOYO-1+ cells are also PI+, however some YOYO-1+ cells are PI-negative (arrows). Scale bars: (a, b) 60 um; (c, d), 20 um.

    Journal: Stroke; a journal of cerebral circulation

    Article Title: PLASMALEMMA PERMEABILITY AND NECROTIC CELL DEATH PHENOTYPES AFTER INTRACEREBRAL HEMORRHAGE IN MICE

    doi: 10.1161/STROKEAHA.111.635672

    Figure Lengend Snippet: Plasmalemma resealing after intracerebral hemorrhage. Mice were subjected to ICH and administered YOYO-1 at 24 or 48 h and PI at 48 or 72 h, respectively. (a–d) representative photomicrographs of brain sections showing YOYO-1+ and PI+ cells labeled at 24–48 h. Note that the majority of YOYO-1+ cells are also PI+, however some YOYO-1+ cells are PI-negative (arrows). Scale bars: (a, b) 60 um; (c, d), 20 um.

    Article Snippet: To show that PI and YOYO-1 label nucleated cells in the ICH model, some brain sections were counterstained with Hoechst 33342 (Thermo Scientific, Rockford, IL) (1:1000) for 15 s.

    Techniques: Mouse Assay, Labeling

    DNA focusing into a NZMW. Fluorescence time traces from a single NZMW that contains a 3 nm diameter pore in an array of ZMWs is monitored for the fluorescence from 6000 bp DNA labeled with YOYO-1 (9 pixel region of interest for each NZMW, 10.8 ms exposure time, signal-averaged to 100 ms). Inset illustrates DNA entering the illumination volume of a NZMW as it migrates toward the pore, resulting in increased ZMW fluorescence. ZMW arrays are shown in fluorescence images (i)–(iii) with (i) being an averaged image of all frames in the experiment, and (ii) and (iii) being the membrane under respective −850 and 850 mV. Colored arrows identify ZMWs with corresponding colored fluorescence traces in bottom plot. The red arrow identifies a NZMW. Green and red backgrounds in the fluorescence traces correspond to periods of positive and negative voltage, respectively (see Supporting Information for electrical trace).

    Journal: Nano Letters

    Article Title: Reversible Positioning of Single Molecules inside Zero-Mode Waveguides

    doi: 10.1021/nl503134x

    Figure Lengend Snippet: DNA focusing into a NZMW. Fluorescence time traces from a single NZMW that contains a 3 nm diameter pore in an array of ZMWs is monitored for the fluorescence from 6000 bp DNA labeled with YOYO-1 (9 pixel region of interest for each NZMW, 10.8 ms exposure time, signal-averaged to 100 ms). Inset illustrates DNA entering the illumination volume of a NZMW as it migrates toward the pore, resulting in increased ZMW fluorescence. ZMW arrays are shown in fluorescence images (i)–(iii) with (i) being an averaged image of all frames in the experiment, and (ii) and (iii) being the membrane under respective −850 and 850 mV. Colored arrows identify ZMWs with corresponding colored fluorescence traces in bottom plot. The red arrow identifies a NZMW. Green and red backgrounds in the fluorescence traces correspond to periods of positive and negative voltage, respectively (see Supporting Information for electrical trace).

    Article Snippet: YOYO-labeled DNA was prepared from 6000 bp DNA (Thermo Scientific, Tewksbury, MA) and YOYO-1 intercalating dye (Life Technologies, Carlsbad, CA).

    Techniques: Fluorescence, Labeling, Mass Spectrometry