Structured Review

Thermo Fisher yoyo 1
Force-dependent DNA intercalation of a mono- (SbG, open symbols) and a bis-intercalator <t>(YOYO,</t> solid symbols) at 1,000 mM NaCl. ( a ) Total fluorescence intensity as a function of DNA elongation. Solid lines: linear fits through origin. ( b , c ) DNA elongation and representative fluorescence images (normalized intensity) as a function of tension. Solid curves: fits to data ( equation (5) and Methods). The DNA tension for each fluorescence image corresponds to the force axis below. Scale bars, 5 μm. ( d ) Binding constant as a function of tension, calculated using elongation data of b , c . Solid lines: exponential fits. ( a – d ) Open red squares: 1.6 nM SbG; open black circles, 10 nM SbG, filled red squares: 0.1 nM YOYO; filled black circles: 0.45 nM YOYO; triangles/diamonds: DNA elongation calculated from total fluorescence intensity. Error bars s.e.m., n ≥5.
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1) Product Images from "The impact of DNA intercalators on DNA and DNA-processing enzymes elucidated through force-dependent binding kinetics"

Article Title: The impact of DNA intercalators on DNA and DNA-processing enzymes elucidated through force-dependent binding kinetics

Journal: Nature Communications

doi: 10.1038/ncomms8304

Force-dependent DNA intercalation of a mono- (SbG, open symbols) and a bis-intercalator (YOYO, solid symbols) at 1,000 mM NaCl. ( a ) Total fluorescence intensity as a function of DNA elongation. Solid lines: linear fits through origin. ( b , c ) DNA elongation and representative fluorescence images (normalized intensity) as a function of tension. Solid curves: fits to data ( equation (5) and Methods). The DNA tension for each fluorescence image corresponds to the force axis below. Scale bars, 5 μm. ( d ) Binding constant as a function of tension, calculated using elongation data of b , c . Solid lines: exponential fits. ( a – d ) Open red squares: 1.6 nM SbG; open black circles, 10 nM SbG, filled red squares: 0.1 nM YOYO; filled black circles: 0.45 nM YOYO; triangles/diamonds: DNA elongation calculated from total fluorescence intensity. Error bars s.e.m., n ≥5.
Figure Legend Snippet: Force-dependent DNA intercalation of a mono- (SbG, open symbols) and a bis-intercalator (YOYO, solid symbols) at 1,000 mM NaCl. ( a ) Total fluorescence intensity as a function of DNA elongation. Solid lines: linear fits through origin. ( b , c ) DNA elongation and representative fluorescence images (normalized intensity) as a function of tension. Solid curves: fits to data ( equation (5) and Methods). The DNA tension for each fluorescence image corresponds to the force axis below. Scale bars, 5 μm. ( d ) Binding constant as a function of tension, calculated using elongation data of b , c . Solid lines: exponential fits. ( a – d ) Open red squares: 1.6 nM SbG; open black circles, 10 nM SbG, filled red squares: 0.1 nM YOYO; filled black circles: 0.45 nM YOYO; triangles/diamonds: DNA elongation calculated from total fluorescence intensity. Error bars s.e.m., n ≥5.

Techniques Used: Fluorescence, Binding Assay

Intercalation kinetics of bis-intercalator YOYO ( a , c , e , g ) and mono-intercalator SbG ( b , d , f ). ( a ) Kymograph of YOYO-binding events, acquired at a tension that increases from 18 to 60 pN (indicated in pN above the kymograph). The average event duration can be clearly seen to increase with tension. Scale bar, 25 s (horizontal), 6 μm (vertical). ( b , c ) De-intercalation of the dye in intercalator-free buffer can be followed by the decrease in DNA elongation over time at constant tension. The displayed decay curves are measurements at different tension at 1,000 mM NaCl (increasing from 6 to 60 pN in 6 pN increments, in the direction indicated by the arrow). ( d , e ) Force dependence of 1/ k off (black circles), K (blue pentagons) and 1/[ I ] k on (red squares) for SbG ( d ) and YOYO ( e ) at 1,000 mM NaCl. ( f , g ) Force dependence of 1/ k off ( F ) at varying [NaCl]. Green diamonds, 100 mM NaCl; magenta triangles, 300 mM NaCl; black circles, 1,000 mM NaCl. Solid grey lines represent single-exponential fits to the data. Error bars, s.e.m., n ≥5 ( d – g ).
Figure Legend Snippet: Intercalation kinetics of bis-intercalator YOYO ( a , c , e , g ) and mono-intercalator SbG ( b , d , f ). ( a ) Kymograph of YOYO-binding events, acquired at a tension that increases from 18 to 60 pN (indicated in pN above the kymograph). The average event duration can be clearly seen to increase with tension. Scale bar, 25 s (horizontal), 6 μm (vertical). ( b , c ) De-intercalation of the dye in intercalator-free buffer can be followed by the decrease in DNA elongation over time at constant tension. The displayed decay curves are measurements at different tension at 1,000 mM NaCl (increasing from 6 to 60 pN in 6 pN increments, in the direction indicated by the arrow). ( d , e ) Force dependence of 1/ k off (black circles), K (blue pentagons) and 1/[ I ] k on (red squares) for SbG ( d ) and YOYO ( e ) at 1,000 mM NaCl. ( f , g ) Force dependence of 1/ k off ( F ) at varying [NaCl]. Green diamonds, 100 mM NaCl; magenta triangles, 300 mM NaCl; black circles, 1,000 mM NaCl. Solid grey lines represent single-exponential fits to the data. Error bars, s.e.m., n ≥5 ( d – g ).

Techniques Used: Binding Assay

Impact of intercalators on DNA mechanics. ( a , b ) Nonequilibrium force-extension curves of SxG ( a ) and YOYO ( b ) at 10 and 25 mM NaCl, respectively, at increasing dye coverage (direction of increase indicated by arrows). For each curve, the dye coverage is essentially constant (Methods). Insets, curves shifted horizontally to overlay low-force regions. ( c , d ) Equilibrium force-extension curves in absence (black) and presence (blue) of YO ( c ) and POPO ( d ) at 1,000 mM NaCl. Cyan, fits to data compiled from the black curve using equation (4) .
Figure Legend Snippet: Impact of intercalators on DNA mechanics. ( a , b ) Nonequilibrium force-extension curves of SxG ( a ) and YOYO ( b ) at 10 and 25 mM NaCl, respectively, at increasing dye coverage (direction of increase indicated by arrows). For each curve, the dye coverage is essentially constant (Methods). Insets, curves shifted horizontally to overlay low-force regions. ( c , d ) Equilibrium force-extension curves in absence (black) and presence (blue) of YO ( c ) and POPO ( d ) at 1,000 mM NaCl. Cyan, fits to data compiled from the black curve using equation (4) .

Techniques Used:

Experimental approach. ( a ) Multichannel laminar flow cell used for (I), optical trapping of two microspheres (1), tethering of DNA between the microspheres (2) and mechanical characterization of the tethered DNA (3) and for (II), immersing the DNA dumbbell in an intercalator-containing buffer by moving the flow cell with respect to the optical traps using stage motion. ( b ) Force-extension curves of bare DNA (blue) and DNA with an equilibrated coverage of SxO (red). Black curve and inset: equilibration dynamics monitored by determining DNA elongation going from bare DNA to intercalated DNA at a constant tension of 48 pN. ( c , d ) Fluorescence microscopy images of individual DNA-bound SxO ( c ) and YOYO ( d ) dyes. Scale bars, 3 μm. ( e , f ) Kymographs show real-time binding and dissociation dynamics of individual SxO ( e ) and YOYO ( f ) molecules. Scale bars, 50 s (horizontal), 5 μm (vertical). ( g ) DNA elongation (measured with optical tweezers) as a function of the number of bound intercalators (obtained from fluorescence intensity as detailed in Methods). A linear fit yields the length increase per dye (Δ x eq ), 0.68±0.04 nm (solid) and 0.34±0.03 nm per dye (dashed line).
Figure Legend Snippet: Experimental approach. ( a ) Multichannel laminar flow cell used for (I), optical trapping of two microspheres (1), tethering of DNA between the microspheres (2) and mechanical characterization of the tethered DNA (3) and for (II), immersing the DNA dumbbell in an intercalator-containing buffer by moving the flow cell with respect to the optical traps using stage motion. ( b ) Force-extension curves of bare DNA (blue) and DNA with an equilibrated coverage of SxO (red). Black curve and inset: equilibration dynamics monitored by determining DNA elongation going from bare DNA to intercalated DNA at a constant tension of 48 pN. ( c , d ) Fluorescence microscopy images of individual DNA-bound SxO ( c ) and YOYO ( d ) dyes. Scale bars, 3 μm. ( e , f ) Kymographs show real-time binding and dissociation dynamics of individual SxO ( e ) and YOYO ( f ) molecules. Scale bars, 50 s (horizontal), 5 μm (vertical). ( g ) DNA elongation (measured with optical tweezers) as a function of the number of bound intercalators (obtained from fluorescence intensity as detailed in Methods). A linear fit yields the length increase per dye (Δ x eq ), 0.68±0.04 nm (solid) and 0.34±0.03 nm per dye (dashed line).

Techniques Used: Flow Cytometry, Fluorescence, Microscopy, Binding Assay

Impact of intercalators on DNA overstretching and DNA-enzyme activity. ( a – c ) Kymographs and corresponding force-extension curves (red) in the presence of YOYO ( a ), SbG ( b ) and YO ( c ) at 100 mM NaCl. The kymograph image and corresponding force-extension curves are co-aligned along the horizontal axis, which is possible because of the constant stretching speed of the moving bead (visible as the bright, upward tilted bar in the image). Thus, the fluorescence pattern in the vertical direction in the kymograph is a DNA-staining pattern at the DNA extension indicated on the horizontal axis. Cyan curves, no intercalator present. ( d ) Time traces recorded at 40 pN showing dsDNA digestion by the exonuclease activity of T7 DNA polymerase. Black, no intercalator; red, YO; blue, SxO; green, SbG. Under these experimental conditions, YOYO abolished all exonuclease activities. ( e ) Histograms of the resulting average digestion rates obtained at the four different conditions (Methods).
Figure Legend Snippet: Impact of intercalators on DNA overstretching and DNA-enzyme activity. ( a – c ) Kymographs and corresponding force-extension curves (red) in the presence of YOYO ( a ), SbG ( b ) and YO ( c ) at 100 mM NaCl. The kymograph image and corresponding force-extension curves are co-aligned along the horizontal axis, which is possible because of the constant stretching speed of the moving bead (visible as the bright, upward tilted bar in the image). Thus, the fluorescence pattern in the vertical direction in the kymograph is a DNA-staining pattern at the DNA extension indicated on the horizontal axis. Cyan curves, no intercalator present. ( d ) Time traces recorded at 40 pN showing dsDNA digestion by the exonuclease activity of T7 DNA polymerase. Black, no intercalator; red, YO; blue, SxO; green, SbG. Under these experimental conditions, YOYO abolished all exonuclease activities. ( e ) Histograms of the resulting average digestion rates obtained at the four different conditions (Methods).

Techniques Used: Activity Assay, Fluorescence, Staining

2) Product Images from "Compression of the DNA substrate by a viral packaging motor is supported by removal of intercalating dye during translocation"

Article Title: Compression of the DNA substrate by a viral packaging motor is supported by removal of intercalating dye during translocation

Journal:

doi: 10.1073/pnas.1214318109

IDs inhibit in vitro DNA packaging. ( A ) Nuclease assays showing inhibition of packaging in the presence of YOYO-1, 9AA, and EthBr dyes (1 μM). ( B ) SDS/PAGE gel image showing purified ac , acq , and wt terminases. ( C ) The acq terminase mutant more
Figure Legend Snippet: IDs inhibit in vitro DNA packaging. ( A ) Nuclease assays showing inhibition of packaging in the presence of YOYO-1, 9AA, and EthBr dyes (1 μM). ( B ) SDS/PAGE gel image showing purified ac , acq , and wt terminases. ( C ) The acq terminase mutant more

Techniques Used: In Vitro, Inhibition, SDS Page, Purification, Mutagenesis

DNA is packaged after removal of ID from the DNA–dye complex. ( A ) Packaging of 5 kb DNA in the presence of different concentrations of YOYO-1. ( A , i ) Typhoon image, ( A , ii ) same gel stained with EthBr, and ( A , iii ) nuclease assay gel. ( B ) Nuclease
Figure Legend Snippet: DNA is packaged after removal of ID from the DNA–dye complex. ( A ) Packaging of 5 kb DNA in the presence of different concentrations of YOYO-1. ( A , i ) Typhoon image, ( A , ii ) same gel stained with EthBr, and ( A , iii ) nuclease assay gel. ( B ) Nuclease

Techniques Used: Staining, Nuclease Assay

Terminase releases IDs from DNA. ( A ) Gel mobility shift assays showing the shift in YOYO-1–bound (500 nM) 70 bp, 280 bp, and 5 kb DNAs in the presence of terminase. ( B ) Decrease in fluorescence intensity of the 70 bp DNA–YOYO-1 complex
Figure Legend Snippet: Terminase releases IDs from DNA. ( A ) Gel mobility shift assays showing the shift in YOYO-1–bound (500 nM) 70 bp, 280 bp, and 5 kb DNAs in the presence of terminase. ( B ) Decrease in fluorescence intensity of the 70 bp DNA–YOYO-1 complex

Techniques Used: Mobility Shift, Fluorescence

3) Product Images from "Integrated systems biology analysis of KSHV latent infection reveals viral induction and reliance on peroxisome mediated lipid metabolism"

Article Title: Integrated systems biology analysis of KSHV latent infection reveals viral induction and reliance on peroxisome mediated lipid metabolism

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006256

KSHV latently infected endothelial cells require peroxisome proteins. (A.) Overview of Essential Fatty Acids (EFA) and peroxisome pathway. Numbers indicate genes altered by KSHV (time post infection in black and fold change in red) as identified by RNA-seq in orange ( PLA2G4A ), a previously published metabolomics screen [ 19 ] in blue and proteomic screen in red. siRNA treatments of ABCD3 and ACOX1 for panel B are indicated in blocked red sign. (B.) TIME cells were transfected with a control siRNA (siSCRB) or siRNA to ABCD3 or ACOX1. siABCD3 and siACOX1 treatments lead to greater than 70% reduction in ABCD3 and ACOX1 expression as determined by qRT-PCR normalized to the housekeeping genes GAPDH and HPRT. (C.) TIME cells were transfected with siRNAs as in panel B and 24 later were Mock- or KSHV-infected. 96 hpi (120 hours post transfection) cells were harvested and % cell death was measured using Trypan blue stain. In parallel, cells were treated with 20 μM QVD, a pan-caspase inhibitor. Data shown is from three independent experiments. Student’s t-test ( D .) Data shows the average fold change in % dead cells over control siRNA transfected cells from three independent experiments from panel C. (E.) IncuCyte microscopy images identifying dead cell nuclei (YOYO-1) for Mock- and KSHV-infected cells transfected with siSCRB, siABCD3 or siACOX1 at 96 hpi. Essen software was used to identify cell nuclei by size and fluorescent intensity, with background subtracted. YOYO-1 positive nuclei are in fluorescent green. All the data are represented as mean +/- SEM.
Figure Legend Snippet: KSHV latently infected endothelial cells require peroxisome proteins. (A.) Overview of Essential Fatty Acids (EFA) and peroxisome pathway. Numbers indicate genes altered by KSHV (time post infection in black and fold change in red) as identified by RNA-seq in orange ( PLA2G4A ), a previously published metabolomics screen [ 19 ] in blue and proteomic screen in red. siRNA treatments of ABCD3 and ACOX1 for panel B are indicated in blocked red sign. (B.) TIME cells were transfected with a control siRNA (siSCRB) or siRNA to ABCD3 or ACOX1. siABCD3 and siACOX1 treatments lead to greater than 70% reduction in ABCD3 and ACOX1 expression as determined by qRT-PCR normalized to the housekeeping genes GAPDH and HPRT. (C.) TIME cells were transfected with siRNAs as in panel B and 24 later were Mock- or KSHV-infected. 96 hpi (120 hours post transfection) cells were harvested and % cell death was measured using Trypan blue stain. In parallel, cells were treated with 20 μM QVD, a pan-caspase inhibitor. Data shown is from three independent experiments. Student’s t-test ( D .) Data shows the average fold change in % dead cells over control siRNA transfected cells from three independent experiments from panel C. (E.) IncuCyte microscopy images identifying dead cell nuclei (YOYO-1) for Mock- and KSHV-infected cells transfected with siSCRB, siABCD3 or siACOX1 at 96 hpi. Essen software was used to identify cell nuclei by size and fluorescent intensity, with background subtracted. YOYO-1 positive nuclei are in fluorescent green. All the data are represented as mean +/- SEM.

Techniques Used: Infection, RNA Sequencing Assay, Transfection, Expressing, Quantitative RT-PCR, Staining, Microscopy, Software

4) Product Images from "Nuclear import of HIV-1 intracellular reverse transcription complexes is mediated by importin 7"

Article Title: Nuclear import of HIV-1 intracellular reverse transcription complexes is mediated by importin 7

Journal:

doi: 10.1093/emboj/cdg357

Fig. 1. Analyses of purified HIV-1 RTCs by confocal microscopy and endogenous reverse transcription assay. ( A ) Purified RTCs were adsorbed onto a plastic tissue culture dish, fixed and labelled with the nucleic acid dye YOYO-1 and anti-Vpr or anti-IN antibodies. Images were acquired sequentially and merged using the Confocal Assistant software. Mutant RTCs lacking Vpr (RTC Vpr–), samples from uninfected cells (CTR–) and non- immune rabbit sera were used as controls. Scale bar, 15 µm. ( B ) Endogenous RT assay on the equilibrium density fractions containing the peak of the viral DNA. Samples were incubated in the presence or absence of exogenous dNTPs and then subjected to PCR with primers specific for the (+) strand DNA (expected band size is 600 bp). MW, DNA molecular weight markers; 1, RTC fraction incubated in the presence of exogenous dNTPs; 2, same fraction without dNTPs; 3, fraction from uninfected cells; 4–6, same conditions as in 1–3 but RTCs were extracted in 160 mM KCl buffer after the first extraction in hypotonic buffer; 7, positive control; 8, no DNA.
Figure Legend Snippet: Fig. 1. Analyses of purified HIV-1 RTCs by confocal microscopy and endogenous reverse transcription assay. ( A ) Purified RTCs were adsorbed onto a plastic tissue culture dish, fixed and labelled with the nucleic acid dye YOYO-1 and anti-Vpr or anti-IN antibodies. Images were acquired sequentially and merged using the Confocal Assistant software. Mutant RTCs lacking Vpr (RTC Vpr–), samples from uninfected cells (CTR–) and non- immune rabbit sera were used as controls. Scale bar, 15 µm. ( B ) Endogenous RT assay on the equilibrium density fractions containing the peak of the viral DNA. Samples were incubated in the presence or absence of exogenous dNTPs and then subjected to PCR with primers specific for the (+) strand DNA (expected band size is 600 bp). MW, DNA molecular weight markers; 1, RTC fraction incubated in the presence of exogenous dNTPs; 2, same fraction without dNTPs; 3, fraction from uninfected cells; 4–6, same conditions as in 1–3 but RTCs were extracted in 160 mM KCl buffer after the first extraction in hypotonic buffer; 7, positive control; 8, no DNA.

Techniques Used: Purification, Confocal Microscopy, Software, Mutagenesis, Incubation, Polymerase Chain Reaction, Molecular Weight, Positive Control

5) Product Images from "Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy"

Article Title: Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy

Journal:

doi: 10.1073/pnas.1516811113

Polar-dSTORM imaging in DNA. ( A ) Polar-dSTORM images (superimposed to traditional dSTORM images) of local regions in dsDNA fibers labeled with YOYO-1, immobilized in vitro on a sample surface (Intensity threshold of 12,000 counts, expected σ P
Figure Legend Snippet: Polar-dSTORM imaging in DNA. ( A ) Polar-dSTORM images (superimposed to traditional dSTORM images) of local regions in dsDNA fibers labeled with YOYO-1, immobilized in vitro on a sample surface (Intensity threshold of 12,000 counts, expected σ P

Techniques Used: Imaging, Labeling, In Vitro

6) Product Images from "Stem–loop binding protein expressed in growing oocytes is required for accumulation of mRNAs encoding histones H3 and H4 and for early embryonic development in the mouse"

Article Title: Stem–loop binding protein expressed in growing oocytes is required for accumulation of mRNAs encoding histones H3 and H4 and for early embryonic development in the mouse

Journal:

doi: 10.1016/j.ydbio.2007.10.032

Expression of SLBP in wild-type oocytes. Ovarian sections were stained using YOYO-1 to label DNA (left panels) and anti-SLBP (right panels), except in panel A where right panel shows overlaid images. (A) Ovary of 5-day animal showing primordial and primary
Figure Legend Snippet: Expression of SLBP in wild-type oocytes. Ovarian sections were stained using YOYO-1 to label DNA (left panels) and anti-SLBP (right panels), except in panel A where right panel shows overlaid images. (A) Ovary of 5-day animal showing primordial and primary

Techniques Used: Expressing, Staining

7) Product Images from "Assay Development and Multivariate Scoring for High-Content Discovery of Chemoprotectants of Endoplasmic-Reticulum-Stress-Mediated Amylin-Induced Cytotoxicity in Pancreatic Beta Cells"

Article Title: Assay Development and Multivariate Scoring for High-Content Discovery of Chemoprotectants of Endoplasmic-Reticulum-Stress-Mediated Amylin-Induced Cytotoxicity in Pancreatic Beta Cells

Journal:

doi: 10.1089/adt.2014.591

Pipeline images used for identifying and categorizing HCS data at the cell level with live nuclei stained with cell-permeant Hoechst-33342 ( blue ) and dead nuclei stained with both Hoechst-33342 and YoYo-1 ( green ). (A) Amylin/tunicamycin-treated entire-well
Figure Legend Snippet: Pipeline images used for identifying and categorizing HCS data at the cell level with live nuclei stained with cell-permeant Hoechst-33342 ( blue ) and dead nuclei stained with both Hoechst-33342 and YoYo-1 ( green ). (A) Amylin/tunicamycin-treated entire-well

Techniques Used: Staining

8) Product Images from "An Integrated Chromosome Map of Microsatellite Markers and Inversion Breakpoints for an Asian Malaria Mosquito, Anopheles stephensi"

Article Title: An Integrated Chromosome Map of Microsatellite Markers and Inversion Breakpoints for an Asian Malaria Mosquito, Anopheles stephensi

Journal:

doi: 10.1093/jhered/esr072

FISH performed on the chromosomes of Anopheles stephensi . Chromosomes counterstained with the fluorophore YOYO-1 and hybridized with fluorescently labeled probes Cy5 (blue) and Cy3 (red) are shown.
Figure Legend Snippet: FISH performed on the chromosomes of Anopheles stephensi . Chromosomes counterstained with the fluorophore YOYO-1 and hybridized with fluorescently labeled probes Cy5 (blue) and Cy3 (red) are shown.

Techniques Used: Fluorescence In Situ Hybridization, Labeling

9) Product Images from "Maximizing gene delivery efficiencies of cationic helical polypeptides via balanced membrane penetration and cellular targeting"

Article Title: Maximizing gene delivery efficiencies of cationic helical polypeptides via balanced membrane penetration and cellular targeting

Journal:

doi: 10.1016/j.biomaterials.2013.09.090

Uptake level of ternary complexes containing YOYO-1-DNA in HeLa, COS-7, and Raw 264.7 cell following 4-h incubation at 37 °C (n=3). PVBLG-8/DNA weight ratio was maintained constant at 15. N represents naked DNA.
Figure Legend Snippet: Uptake level of ternary complexes containing YOYO-1-DNA in HeLa, COS-7, and Raw 264.7 cell following 4-h incubation at 37 °C (n=3). PVBLG-8/DNA weight ratio was maintained constant at 15. N represents naked DNA.

Techniques Used: Incubation

10) Product Images from "Super-resolution microscopy reveals decondensed chromatin structure at transcription sites"

Article Title: Super-resolution microscopy reveals decondensed chromatin structure at transcription sites

Journal: Scientific Reports

doi: 10.1038/srep04477

BALM images of chromatin fibers | (a) A representative BALM image of chromatin fibers stained with YOYO-1. Scale bar: 10 μm. (b) Histogram showing the distribution of chromatin width (Cw). Inset i shows the chromatin width (Cw) and λDNA width (Lw) in BALM image and TIRF image (n ≥ 20) (***P
Figure Legend Snippet: BALM images of chromatin fibers | (a) A representative BALM image of chromatin fibers stained with YOYO-1. Scale bar: 10 μm. (b) Histogram showing the distribution of chromatin width (Cw). Inset i shows the chromatin width (Cw) and λDNA width (Lw) in BALM image and TIRF image (n ≥ 20) (***P

Techniques Used: Staining

Functionality of chromatin fibers | (a) Schematic of chromatin fiber preparation (b) Representative TIRFM images of the colocalization of DNA and histone proteins on chromatin fibers. Scale bar: 10 μm. (c) Line profile for determination of FWHM (representing chromatin width (Cw) in the inset red box) (d) Bar graph showing chromatin width (Cw) in four labeling ways: DNA stained with hoechst (DNA-hoechst), DNA stained with YOYO-1 (DNA-YOYO-1), H2B tagged with EGFP (H2B-EGFP), and H1 immunolabeled with antibodies (H1-AB) (n ≥ 20, all the ‘n’ in the following text refers to the number of fibers) (*P
Figure Legend Snippet: Functionality of chromatin fibers | (a) Schematic of chromatin fiber preparation (b) Representative TIRFM images of the colocalization of DNA and histone proteins on chromatin fibers. Scale bar: 10 μm. (c) Line profile for determination of FWHM (representing chromatin width (Cw) in the inset red box) (d) Bar graph showing chromatin width (Cw) in four labeling ways: DNA stained with hoechst (DNA-hoechst), DNA stained with YOYO-1 (DNA-YOYO-1), H2B tagged with EGFP (H2B-EGFP), and H1 immunolabeled with antibodies (H1-AB) (n ≥ 20, all the ‘n’ in the following text refers to the number of fibers) (*P

Techniques Used: Labeling, Staining, Immunolabeling

11) Product Images from "Surface Charge, Electroosmotic Flow and DNA Extension in Chemically Modified Thermoplastic Nanoslits and Nanochannels"

Article Title: Surface Charge, Electroosmotic Flow and DNA Extension in Chemically Modified Thermoplastic Nanoslits and Nanochannels

Journal:

doi: 10.1039/c4an01439a

(a) Representative fluorescence intensity profile of an individual YOYO-1 stained λ-DNA molecule after injection (red line) and confinement (blue line) in the plasma modified nanochannel filled with 2X TBE buffer. Complete injection into the nanochannel
Figure Legend Snippet: (a) Representative fluorescence intensity profile of an individual YOYO-1 stained λ-DNA molecule after injection (red line) and confinement (blue line) in the plasma modified nanochannel filled with 2X TBE buffer. Complete injection into the nanochannel

Techniques Used: Fluorescence, Staining, Injection, Modification

12) Product Images from "Sorting of small infectious virus particles by flow virometry reveals distinct infectivity profiles"

Article Title: Sorting of small infectious virus particles by flow virometry reveals distinct infectivity profiles

Journal: Nature communications

doi: 10.1038/ncomms7022

Correlation between Glycoprotein (GPC) levels and RNA content. Flow virometry assay of JUNV particles stained with Alexa 647 GPC antibody only (a) or YOYO-1 only (b) . Dot plots represent nucleic acid dye fluorescence (x axis) as a function of the Alexa 647 GPC fluorescence (y axis). (c) Flow virometry analysis of particles stained with both Alexa 647 GPC antibody and the nucleic acid dye YOYO-1. (d) Pie charts representing the percentage of potentially infectious particles (GPC + RNA + ) in the low and high fractions. The high fraction contains about 2.5 times more virions with both genetic material and surface GPC than the low fraction. (e, f) Relative amount of RNA contained in 20,000 sorted particles from total RNA + or GPC + RNA + viral particles. Reverse transcriptase RT-qPCR with primers targeting the Short (e) or the Large (f) segment of JUNV is shown. Error bars are the mean +/− s.d. of triplicates.
Figure Legend Snippet: Correlation between Glycoprotein (GPC) levels and RNA content. Flow virometry assay of JUNV particles stained with Alexa 647 GPC antibody only (a) or YOYO-1 only (b) . Dot plots represent nucleic acid dye fluorescence (x axis) as a function of the Alexa 647 GPC fluorescence (y axis). (c) Flow virometry analysis of particles stained with both Alexa 647 GPC antibody and the nucleic acid dye YOYO-1. (d) Pie charts representing the percentage of potentially infectious particles (GPC + RNA + ) in the low and high fractions. The high fraction contains about 2.5 times more virions with both genetic material and surface GPC than the low fraction. (e, f) Relative amount of RNA contained in 20,000 sorted particles from total RNA + or GPC + RNA + viral particles. Reverse transcriptase RT-qPCR with primers targeting the Short (e) or the Large (f) segment of JUNV is shown. Error bars are the mean +/− s.d. of triplicates.

Techniques Used: Gel Permeation Chromatography, Flow Cytometry, Staining, Fluorescence, Quantitative RT-PCR

13) Product Images from "Mesencephalic origin of the inferior lobe in zebrafish"

Article Title: Mesencephalic origin of the inferior lobe in zebrafish

Journal: BMC Biology

doi: 10.1186/s12915-019-0631-y

Localization of the mCherry-positive cells in young larval brains of Tg(her5:ERT2CreERT2;βact:lox-stop-lox-hmgb1:mCherry) zebrafish treated with tamoxifen at 24 hpf. Anterior to the left for a – d , e , h , and j . a – d 3D reconstruction from confocal images of a whole head of 3 dpf larva. mCherry-positive cells are shown in magenta, and YOYO-1, a nuclear marker, is shown in green. a , b Side view of the larval head with ( a ) and without ( b ) YOYO-1 labeling. c , d Top view of the larval head with ( c ) and without ( d ) YOYO-1 labeling. The mCherry-positive cells are still close to the MHB at this stage. Some cells are starting to migrate anteriorly, but there are no mCherry-positive cells in the forebrain or in other brain areas. e – k 3D reconstruction from confocal images of dissected brains of 3 dpf ( e – g ), 5 dpf ( h , i ), and 7 dpf ( j , k ) larvae. mCherry-positive cells are shown in magenta, and DiD fiber labeling is shown in gray. e A whole brain at 3 dpf is shown in lateral view. f A sagittal section through the same specimen. g A frontal section. The hypothalamus (Hy) is extending in ventral position below the midbrain and is devoid of mCherry-positive cells. h A whole brain at 5 dpf is shown in lateral view. i A frontal section from the same brain showing the first appearance of the inferior lobe (IL; arrow), with a few mCherry-positive cells at the periphery of the structure. j A whole brain at 7 dpf is shown in lateral view. k A frontal section from the same brain showing the growing IL (arrow), with more mCherry-positive cells added laterally. Abbreviations: Cb cerebellum, Hy hypothalamus, IL inferior lobe, TeO optic tectum, Tel telencephalon. Scale bars: a – d , 100 μm. e – k , 50 μm
Figure Legend Snippet: Localization of the mCherry-positive cells in young larval brains of Tg(her5:ERT2CreERT2;βact:lox-stop-lox-hmgb1:mCherry) zebrafish treated with tamoxifen at 24 hpf. Anterior to the left for a – d , e , h , and j . a – d 3D reconstruction from confocal images of a whole head of 3 dpf larva. mCherry-positive cells are shown in magenta, and YOYO-1, a nuclear marker, is shown in green. a , b Side view of the larval head with ( a ) and without ( b ) YOYO-1 labeling. c , d Top view of the larval head with ( c ) and without ( d ) YOYO-1 labeling. The mCherry-positive cells are still close to the MHB at this stage. Some cells are starting to migrate anteriorly, but there are no mCherry-positive cells in the forebrain or in other brain areas. e – k 3D reconstruction from confocal images of dissected brains of 3 dpf ( e – g ), 5 dpf ( h , i ), and 7 dpf ( j , k ) larvae. mCherry-positive cells are shown in magenta, and DiD fiber labeling is shown in gray. e A whole brain at 3 dpf is shown in lateral view. f A sagittal section through the same specimen. g A frontal section. The hypothalamus (Hy) is extending in ventral position below the midbrain and is devoid of mCherry-positive cells. h A whole brain at 5 dpf is shown in lateral view. i A frontal section from the same brain showing the first appearance of the inferior lobe (IL; arrow), with a few mCherry-positive cells at the periphery of the structure. j A whole brain at 7 dpf is shown in lateral view. k A frontal section from the same brain showing the growing IL (arrow), with more mCherry-positive cells added laterally. Abbreviations: Cb cerebellum, Hy hypothalamus, IL inferior lobe, TeO optic tectum, Tel telencephalon. Scale bars: a – d , 100 μm. e – k , 50 μm

Techniques Used: Marker, Labeling

14) Product Images from "Improving the population genetics toolbox for the study of the African malaria vector Anopheles nili: microsatellite mapping to chromosomes"

Article Title: Improving the population genetics toolbox for the study of the African malaria vector Anopheles nili: microsatellite mapping to chromosomes

Journal: Parasites & Vectors

doi: 10.1186/1756-3305-4-202

FISH of microsatellites performed on the An. nili chromosomes . Hybridizations of microsatellite markers 1G13 (A), 2Ateta and 1F43 (B), and B115 (C) with polytene chromosomes are shown. Chromosomes were counterstained with the fluorophore YOYO-1 and hybridized with fluorescently labeled probes Cy5 (blue) and Cy3 (red). The top panel shows fluorescent images of chromosomes after FISH. The bottom panel shows inverted grayscale chromosome images with color labels and distinct banding patterns.
Figure Legend Snippet: FISH of microsatellites performed on the An. nili chromosomes . Hybridizations of microsatellite markers 1G13 (A), 2Ateta and 1F43 (B), and B115 (C) with polytene chromosomes are shown. Chromosomes were counterstained with the fluorophore YOYO-1 and hybridized with fluorescently labeled probes Cy5 (blue) and Cy3 (red). The top panel shows fluorescent images of chromosomes after FISH. The bottom panel shows inverted grayscale chromosome images with color labels and distinct banding patterns.

Techniques Used: Fluorescence In Situ Hybridization, Labeling

15) Product Images from "Novel polyethyleneimine-R8-heparin nanogel for high-efficiency gene delivery in vitro and in vivo"

Article Title: Novel polyethyleneimine-R8-heparin nanogel for high-efficiency gene delivery in vitro and in vivo

Journal: Drug Delivery

doi: 10.1080/10717544.2017.1417512

LSCM images of HCT-116 cells following incubation with HPR/pDNA nanoparticles at 37 °C for 0.5, 1, 2, 4 h. Plasmid DNA was labeled with YOYO-1. Endolysosomes were stained with LysoTracker-Red and cell nuclei were stained with DAPI.
Figure Legend Snippet: LSCM images of HCT-116 cells following incubation with HPR/pDNA nanoparticles at 37 °C for 0.5, 1, 2, 4 h. Plasmid DNA was labeled with YOYO-1. Endolysosomes were stained with LysoTracker-Red and cell nuclei were stained with DAPI.

Techniques Used: Incubation, Plasmid Preparation, Labeling, Staining

16) Product Images from "DNA of a circular minichromosome linearized by restriction enzymes or other reagents is resistant to further cleavage: an influence of chromatin topology on the accessibility of DNA"

Article Title: DNA of a circular minichromosome linearized by restriction enzymes or other reagents is resistant to further cleavage: an influence of chromatin topology on the accessibility of DNA

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks723

( A ) The circular DNA of the EBV minichromosome. TR is the terminal repeated sequence through which linear EBV DNA is circularized to form the minichromosome, oriP is the preferred but not unique origin of replication, MAR is the nuclear matrix attachment region which coincides with a micrococcal nuclease-hypersensitive region. Approximate positions of regions transcribed in Raji cells are shown by arrows; black, highest level; white, intermediate; dashed, lowest ( 22 ). ( B ) Forms of minichromosome DNA considered in this work and ( C ) their migration in a PFGE gel of total cell DNA shown by hybridizing with an EBV DNA probe. In all gel images, the top coincides with the sample wells and each panel shows lanes from the same gel. Cells encapsulated in agarose beads and deproteinized were incubated with: C, no addition (2 h); PacI (100 U/ml, 2 h) which cuts minichromosome DNA at a single site; NbB, nicking endonuclease Nb.BbvCI (100 U/ml, 1 h). Lane virus DNA, linear EBV DNA; lane λ, oligomers of λ DNA (48.5 kb). ( D ) Representative DNA molecules extracted from the region close to the origin after incubating cells with Nb.BbvCI, stained with YOYO-1 and spread by molecular combing; these are believed to be nicked circular minichromosome DNA (see text).
Figure Legend Snippet: ( A ) The circular DNA of the EBV minichromosome. TR is the terminal repeated sequence through which linear EBV DNA is circularized to form the minichromosome, oriP is the preferred but not unique origin of replication, MAR is the nuclear matrix attachment region which coincides with a micrococcal nuclease-hypersensitive region. Approximate positions of regions transcribed in Raji cells are shown by arrows; black, highest level; white, intermediate; dashed, lowest ( 22 ). ( B ) Forms of minichromosome DNA considered in this work and ( C ) their migration in a PFGE gel of total cell DNA shown by hybridizing with an EBV DNA probe. In all gel images, the top coincides with the sample wells and each panel shows lanes from the same gel. Cells encapsulated in agarose beads and deproteinized were incubated with: C, no addition (2 h); PacI (100 U/ml, 2 h) which cuts minichromosome DNA at a single site; NbB, nicking endonuclease Nb.BbvCI (100 U/ml, 1 h). Lane virus DNA, linear EBV DNA; lane λ, oligomers of λ DNA (48.5 kb). ( D ) Representative DNA molecules extracted from the region close to the origin after incubating cells with Nb.BbvCI, stained with YOYO-1 and spread by molecular combing; these are believed to be nicked circular minichromosome DNA (see text).

Techniques Used: Sequencing, Migration, Incubation, Staining

17) Product Images from "The avian malaria parasite Plasmodium gallinaceum causes marked structural changes on the surface of its host erythrocyte"

Article Title: The avian malaria parasite Plasmodium gallinaceum causes marked structural changes on the surface of its host erythrocyte

Journal:

doi: 10.1016/j.jsb.2008.03.005

Composite bright field and epifluorescence microscopy images of (a) P. falciparum -infected erythrocytes, (b) P. gallinaceum -infected erythrocytes, and (c) P. gallinaceum -infected erythrocytes treated with DNase I prior to staining with YOYO-1. Bar in
Figure Legend Snippet: Composite bright field and epifluorescence microscopy images of (a) P. falciparum -infected erythrocytes, (b) P. gallinaceum -infected erythrocytes, and (c) P. gallinaceum -infected erythrocytes treated with DNase I prior to staining with YOYO-1. Bar in

Techniques Used: Epifluorescence Microscopy, Infection, Staining

Representative example of P. gallinaceum -infected erythrocytes stained with Giemsa (a–d) or YOYO-1 following DAase I treatment (e–h). A typical early stage of development is shown in (a) and (e). As the parasite develops from the trophozoite
Figure Legend Snippet: Representative example of P. gallinaceum -infected erythrocytes stained with Giemsa (a–d) or YOYO-1 following DAase I treatment (e–h). A typical early stage of development is shown in (a) and (e). As the parasite develops from the trophozoite

Techniques Used: Infection, Staining

AFM images of P. gallinaceum -infected erythrocytes at various stages of development. The insert shows the same cell imaged by combined LM and YOYO-1 fluorescence following DNase I treatment. The cell surface lesion resulting from merozoite invasion (arrows
Figure Legend Snippet: AFM images of P. gallinaceum -infected erythrocytes at various stages of development. The insert shows the same cell imaged by combined LM and YOYO-1 fluorescence following DNase I treatment. The cell surface lesion resulting from merozoite invasion (arrows

Techniques Used: Infection, Fluorescence

18) 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

19) 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

20) Product Images from "Environmental enrichment strengthens corticocortical interactions and reduces amyloid-? oligomers in aged mice"

Article Title: Environmental enrichment strengthens corticocortical interactions and reduces amyloid-? oligomers in aged mice

Journal: Frontiers in Aging Neuroscience

doi: 10.3389/fnagi.2014.00001

Identification of anatomical projections from A1 to V1. (A) Representative image of the CTB injection site and YoYo-1 nuclear counterstaining, which remained confined within the boundaries of V1. (B) Left; immunofluorescence images showing retrograde transport of CTB injected in V1 to ipsilateral layers IV-VI of the primary auditory cortex (red), with a few scattered somata in layer II-III. Right; in the hemisphere contralateral to the injection site, a few cells are labeled in layers V–VI. Nuclei are counterstained with YoYo-1 (green). Scale bar is 500 μm.
Figure Legend Snippet: Identification of anatomical projections from A1 to V1. (A) Representative image of the CTB injection site and YoYo-1 nuclear counterstaining, which remained confined within the boundaries of V1. (B) Left; immunofluorescence images showing retrograde transport of CTB injected in V1 to ipsilateral layers IV-VI of the primary auditory cortex (red), with a few scattered somata in layer II-III. Right; in the hemisphere contralateral to the injection site, a few cells are labeled in layers V–VI. Nuclei are counterstained with YoYo-1 (green). Scale bar is 500 μm.

Techniques Used: CtB Assay, Injection, Immunofluorescence, Labeling

Related Articles

Luciferase:

Article Title: A Guanidinylated Bioreducible Polymer with High Nuclear Localization Ability for Gene Delivery Systems
Article Snippet: Luciferase assay system and reporter lysis buffer were purchased from Promega (Madison, WI). .. YOYO-1 iodide (1 mM solution in DMSO) was purchased from Molecular Probes (Eugene, OR).

Article Title: Reduction-responsive cross-linked stearyl peptide for effective delivery of plasmid DNA
Article Snippet: For this purpose, we first synthesized and characterized C-SHR, examined the cytotoxicity and transfection efficiency of C-SHR/pDNA in HEK293 and HeLa cells, and finally evaluated the gene transfection efficiency in vivo. .. Materials used in this study were l -histidine hydrochloride, l -arginine, stearic acid, and hydrogen peroxide (30% w/v; Sangon Biotech, Shanghai, People’s Republic of China); polyethylenimine (PEI, branched, molecular weight 25 kDa), Lipofectamine™ 2000, l -buthionine-sulfoximine, 4′,6-diamidino-2-phenylindole dihydrochloride, l -cysteine hydrochloride monohydrate, ethidium bromide, dithiothreitol (DTT, Sigma-Aldrich, St Louis, MO, USA); a luciferase assay kit (Promega, Madison, WI, USA); pDNA (pGL3 and pEGFP) (Shanghai Innovation Biotechnology Co Ltd, Shanghai, People’s Republic of China); enhanced bicinchoninic acid protein assay kit (Beyotime, Nanjing, People’s Republic of China); Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum, YOYO-1 (Y3601) and penicillin–streptomycin solution 5 kU/mL (Life Technologies, Carlsbad, CA, USA); a Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies Inc, Nanjing, People’s Republic of China); and HEK293 cells and HeLa cells (Cell Culture Center of the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences, Shanghai, People’s Republic of China). .. All animal experiments were performed in accordance with the ethics and regulations of animal experiments of Second Military Medical University (Shanghai, People’s Republic of China).

Article Title: Polymer Transfected Primary Myoblasts Mediated Efficient Gene Expression and Angiogenic Proliferation
Article Snippet: The luciferase assay system with reporter lysis buffer was purchased from Promega (Madison, WI). .. YOYO-1 iodide (1 mM solution in DMSO) was purchased from Molecular Probes (Eugene, OR).

Article Title: A Family of Bioreducible Poly(disulfide amine)s for Gene Delivery
Article Snippet: Luciferase assay system with reporter lysis buffer was purchased from Promega (Madison, WI). .. YOYO-1 iodide (1 mM solution in DMSO) was purchased from Molecular Probes (Eugene, OR).

Synthesized:

Article Title: DNA condensation by the nucleocapsid protein of HIV-1: a mechanism ensuring DNA protection
Article Snippet: Branched chain polyethylenimine (PEI) (25 kDa) was a gift from Professor J.-P. Behr, Illkirch, France. .. YOYO-1 (491/509) was obtained from Molecular Probes Inc. NCp7 and the related peptides were synthesized by solid-phase chemistry as described ( ). .. Purity of the peptides analyzed by mass spectrometry was > 98%.

Article Title: DNA Directed Self-Assembly of Single Walled Carbon Nanotubes into Three-Way Junction Nanostructures
Article Snippet: The powder form of SWNTs with 1–2 nm diameter, 500 nm length, and 60% purity was purchased from Nanostructured and Amorphous Materials Inc. in Los Alamos, New Mexico, USA. ssDNA strands to prepare DNA-3WJ nanostructures were synthesized by Sentromer DNA Technologies, Istanbul, Turkey. .. Asymmetrical cyanine dye, YOYO-1 (Y3601), was purchased from Life Technologies.

Lambda DNA Preparation:

Article Title: Controlled generation of nanopatterned electrical DNA interface
Article Snippet: Paragraph title: Lambda DNA Preparation ... To observe the characteristics of the attached lambda DNAs according to the electrophoresis conditions, YOYO-1 (491/509 1 mM Solution in DMSO, Molecular Probes, OR, USA) was used for the fluorescent staining, described as follows.

Cytometry:

Article Title: Human Erythropoietin Gene Delivery Using an Arginine-grafted Bioreducible Polymer System
Article Snippet: YOYO-1 iodide- (1 mmol/l solution in DMSO; Molecular Probes, Eugene, OR) tagged phEPO (1 molecule dye per 50 bp nucleotide) was prepared in the dark for 30 minutes. .. The polyplexes were incubated with cells at 37 °C for 4 hours in serum free media.

Article Title: Radical cure of experimental babesiosis in immunodeficient mice using a combination of an endochin-like quinolone and atovaquone
Article Snippet: Paragraph title: Flow cytometry ... After RNase treatment, cells were stained with 20 nM YOYO-1 (Invitrogen; #Y3601) for 1 h at room temperature and in the dark.

Electrophoresis:

Article Title: Chk1 and DNA-PK mediate TPEN-induced DNA damage in a ROS dependent manner in human colon cancer cells
Article Snippet: Electrophoresis was performed in electrophoresis buffer (300 mM NaOH, 1 mM EDTA) for 30 min at 25 V and 250 mA. .. Slides were then neutralized with neutralizing solution (0.4 M Tris-HCl, pH 7.5), and stained with 50 µl of YOYO-1 (Thermofisher, Y3601) stain (0.25 µM YOYO-1, 2.5% DMSO, 0.5% sucrose).

Article Title: Controlled generation of nanopatterned electrical DNA interface
Article Snippet: DNA (Lambda DNA (0.3 μg/μL), Thermo Scientific, MA, USA) was tested in this study. .. To observe the characteristics of the attached lambda DNAs according to the electrophoresis conditions, YOYO-1 (491/509 1 mM Solution in DMSO, Molecular Probes, OR, USA) was used for the fluorescent staining, described as follows. .. After mixing 1 μL of YOYO-1 in 21.5 μL of lambda DNAs in an Eppendorf tube, the tube was wrapped with foil to block out light, then incubated in an oven at 55 °C for 2 h, ensuring the bonding of the YOYO-1 particles with the lambda DNAs; 100 μL of the TBE buffer (Tris-Borate-EDTA buffer, Sigma Aldrich, MO, USA) was added to the lambda DNA-YOYO-1 mixture solution and washed three times.

Incubation:

Article Title: In vitro effects of PI3K/mTOR inhibition in canine hemangiosarcoma
Article Snippet: NucLight Red transfected cells were seeded in quintuplicate at a density of 2,000 cells/well in a 96 well plate and incubated overnight in standard conditions. .. Vehicle-containing C/10 or C/10 with various concentrations of VDC-597 plus YOYO®-1 iodide reagent (Life Technologies, Cat #: Y3601) at concentration of 50 nM (per manufacturer’s protocol) was added to the wells.

Article Title: Human Erythropoietin Gene Delivery Using an Arginine-grafted Bioreducible Polymer System
Article Snippet: YOYO-1 iodide- (1 mmol/l solution in DMSO; Molecular Probes, Eugene, OR) tagged phEPO (1 molecule dye per 50 bp nucleotide) was prepared in the dark for 30 minutes. .. Polyplexes were prepared by mixing YOYO-1 iodide-labeled phEPO (2 µg) with ABP polymer at wt/wt ratios of 1/10, 1/20, and 1/40 in 20 mmol/l HEPES/5% glucose solution, and incubated at room temperature for 30 minutes before transfection. phEPO /PEI (wt/wt 1/1) was used as a positive control.

Article Title: Cyclooxygenase-2, prostaglandin E2 glycerol ester and nitric oxide are involved in muscarine-induced presynaptic enhancement at the vertebrate neuromuscular junction
Article Snippet: To visualize nerve terminals, either: (1) preparations were incubated with 2 μg ml−1 mouse anti-synaptotagmin monoclonal antibody (mAb 48, Developmental Studies Hybridoma Bank at the University of Iowa) and either goat anti-mouse secondary antibody conjugated to Alexa Fluor 555 or chicken anti-mouse secondary antibody conjugated to Alexa Fluor 647 (5 μg ml−1 ; Invitrogen); or (2) the cut end of the motor axon was dipped into a small (1–2 μl) well containing 20 m m Texas Red conjugated to 10,000 molecular weight dextran (Molecular Probes, Carlsbad, CA, USA) in 10 m m Hepes buffer (pH 7.2) and incubated overnight at 9°C to allow the nerve terminals to fill with the Texas Red dextran. .. To visualize the perisynaptic Schwann cells (PSCs), preparations were either (1) incubated with YOYO-1 Iodide (125 n m , Y3601; Invitrogen) for 5 min at 24°C just prior to mounting or (2) incubated with 2 μg ml−1 mouse anti-HNK-1 IgM monoclonal antibody (C6680; Sigma-Aldrich) and goat anti-mouse IgM secondary antibody conjugated to TRITC (5 μg ml−1 ; American Qualex). .. After being stained, NMJs were imaged with an Olympus IX81 microscope, 60× objective (numerical aperture 1.4), with a DSU confocal attachment (disc no. 2) and a Hamamatsu Orca EM camera.

Article Title: Mechanical and structural properties of YOYO-1 complexed DNA
Article Snippet: A 1 mM YOYO-1 stock solution (Invitrogen Y3601) was diluted 400- to 6700-fold in 10 mM phosphate buffer (sodium phosphate buffer: 1.88 mM NaH2 PO4 ·H2 O, 8.13 mM Na2 HPO4 ·2H2 O, resulting in a pH of 7.5) depending on the desired final staining ratio. .. The DNA was either a mixture of unmodified and a small amount of modified molecules (used later on for tethering the DNA to magnetic beads and the flow cell) or unmodified DNA only (used later on for flushing).

Amplification:

Article Title: A Guanidinylated Bioreducible Polymer with High Nuclear Localization Ability for Gene Delivery Systems
Article Snippet: The plasmid pCMV-Luc, containing a firefly luciferase reporter gene was amplified in E.coli DH5α and isolated by standard Maxiprep kit (Invitrogen, Carlsbad, CA). .. YOYO-1 iodide (1 mM solution in DMSO) was purchased from Molecular Probes (Eugene, OR).

Article Title: Polymer Transfected Primary Myoblasts Mediated Efficient Gene Expression and Angiogenic Proliferation
Article Snippet: The plasmids, pCMV-Luc, pCMV-GFP, and pCMV-VEGF165 , containing a firefly luciferase reporter gene, green fluorescent protein gene and vascular endothelial growth factor gene 165, respectively, were amplified in E. coli DH5α and purified by standard Maxiprep kit (Invitrogen, Carlsbad, CA), separately. .. YOYO-1 iodide (1 mM solution in DMSO) was purchased from Molecular Probes (Eugene, OR).

Article Title: A Family of Bioreducible Poly(disulfide amine)s for Gene Delivery
Article Snippet: The plasmid pCMV-Luc, containing a firefly luciferase reporter gene, was amplified in E.coli DH5α and purified by standard Maxiprep kit (Invitrogen, Carlsbad, CA). .. YOYO-1 iodide (1 mM solution in DMSO) was purchased from Molecular Probes (Eugene, OR).

Single Cell Gel Electrophoresis:

Article Title: Chk1 and DNA-PK mediate TPEN-induced DNA damage in a ROS dependent manner in human colon cancer cells
Article Snippet: Paragraph title: Comet assay ... Slides were then neutralized with neutralizing solution (0.4 M Tris-HCl, pH 7.5), and stained with 50 µl of YOYO-1 (Thermofisher, Y3601) stain (0.25 µM YOYO-1, 2.5% DMSO, 0.5% sucrose).

Apoptosis Assay:

Article Title: In vitro effects of PI3K/mTOR inhibition in canine hemangiosarcoma
Article Snippet: Paragraph title: Cell growth and apoptosis assay ... Vehicle-containing C/10 or C/10 with various concentrations of VDC-597 plus YOYO®-1 iodide reagent (Life Technologies, Cat #: Y3601) at concentration of 50 nM (per manufacturer’s protocol) was added to the wells.

Expressing:

Article Title: Gas-liquid interfacial plasmas producing reactive species for cell membrane permeabilization
Article Snippet: Cell membrane permeabilization is an important technique in molecular biology and medical procedures such as cancer pharmacotherapy,( ) gene therapy,( ) RNA-mediated suppression of gene expression,( ) and the creation of induced pluripotent stem (iPS) cells. However, electroporation,( ) a conventional method for permeabilizing cells, is problematic in terms of the low survival fraction, which reduces the efficacy of minimally invasive in vivo treatments. .. In this experiment, we use the non-membrane-permeative fluorescent dye YOYO-1 (Y3601, Molecular Probes) as the delivery material to investigate the effect of plasma irradiation on cell membrane permeability.

BIA-KA:

Article Title: A Guanidinylated Bioreducible Polymer with High Nuclear Localization Ability for Gene Delivery Systems
Article Snippet: BCA™ protein assay kit was purchased from PIERCE (Rocford, Il). .. YOYO-1 iodide (1 mM solution in DMSO) was purchased from Molecular Probes (Eugene, OR).

Article Title: Polymer Transfected Primary Myoblasts Mediated Efficient Gene Expression and Angiogenic Proliferation
Article Snippet: BCA™ protein assay kit was purchased from Thermo Scientific (Rockford, IL). .. YOYO-1 iodide (1 mM solution in DMSO) was purchased from Molecular Probes (Eugene, OR).

Modification:

Article Title: Human Erythropoietin Gene Delivery Using an Arginine-grafted Bioreducible Polymer System
Article Snippet: Briefly, BM cells were isolated from the bilateral femur of 7-week-old SD rats and prepared with Dulbecco's modified Eagle's medium. .. YOYO-1 iodide- (1 mmol/l solution in DMSO; Molecular Probes, Eugene, OR) tagged phEPO (1 molecule dye per 50 bp nucleotide) was prepared in the dark for 30 minutes.

Article Title: A Guanidinylated Bioreducible Polymer with High Nuclear Localization Ability for Gene Delivery Systems
Article Snippet: SYBR safe DNA gel stain (10,000X in DMSO), fetal bovine serum (FBS), Dulbecco’s phosphate buffered saline (DPBS), and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Invitrogen (Carlsbad, Ca). .. YOYO-1 iodide (1 mM solution in DMSO) was purchased from Molecular Probes (Eugene, OR).

Article Title: Reduction-responsive cross-linked stearyl peptide for effective delivery of plasmid DNA
Article Snippet: For this purpose, we first synthesized and characterized C-SHR, examined the cytotoxicity and transfection efficiency of C-SHR/pDNA in HEK293 and HeLa cells, and finally evaluated the gene transfection efficiency in vivo. .. Materials used in this study were l -histidine hydrochloride, l -arginine, stearic acid, and hydrogen peroxide (30% w/v; Sangon Biotech, Shanghai, People’s Republic of China); polyethylenimine (PEI, branched, molecular weight 25 kDa), Lipofectamine™ 2000, l -buthionine-sulfoximine, 4′,6-diamidino-2-phenylindole dihydrochloride, l -cysteine hydrochloride monohydrate, ethidium bromide, dithiothreitol (DTT, Sigma-Aldrich, St Louis, MO, USA); a luciferase assay kit (Promega, Madison, WI, USA); pDNA (pGL3 and pEGFP) (Shanghai Innovation Biotechnology Co Ltd, Shanghai, People’s Republic of China); enhanced bicinchoninic acid protein assay kit (Beyotime, Nanjing, People’s Republic of China); Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum, YOYO-1 (Y3601) and penicillin–streptomycin solution 5 kU/mL (Life Technologies, Carlsbad, CA, USA); a Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies Inc, Nanjing, People’s Republic of China); and HEK293 cells and HeLa cells (Cell Culture Center of the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences, Shanghai, People’s Republic of China). .. All animal experiments were performed in accordance with the ethics and regulations of animal experiments of Second Military Medical University (Shanghai, People’s Republic of China).

Article Title: A Family of Bioreducible Poly(disulfide amine)s for Gene Delivery
Article Snippet: Dulbecco's Modified Eagle's medium (DMEM), penicillin-streptomycin (P/S), fetal bovine serum (FBS), trypsin-like enzyme (TrypLE Express) and Dulbecco's phosphate buffered saline (PBS) were all purchased from Invitrogen-Gibco (Carlsbad, CA). .. YOYO-1 iodide (1 mM solution in DMSO) was purchased from Molecular Probes (Eugene, OR).

Article Title: Mechanical and structural properties of YOYO-1 complexed DNA
Article Snippet: A 1 mM YOYO-1 stock solution (Invitrogen Y3601) was diluted 400- to 6700-fold in 10 mM phosphate buffer (sodium phosphate buffer: 1.88 mM NaH2 PO4 ·H2 O, 8.13 mM Na2 HPO4 ·2H2 O, resulting in a pH of 7.5) depending on the desired final staining ratio. .. An ∼10-fold smaller volume of DNA was added to provide a final DNA concentration of 0.56 ng μl−1 corresponding to a base pair concentration of 860 nM.

Electroporation:

Article Title: Gas-liquid interfacial plasmas producing reactive species for cell membrane permeabilization
Article Snippet: Cell membrane permeabilization is an important technique in molecular biology and medical procedures such as cancer pharmacotherapy,( ) gene therapy,( ) RNA-mediated suppression of gene expression,( ) and the creation of induced pluripotent stem (iPS) cells. However, electroporation,( ) a conventional method for permeabilizing cells, is problematic in terms of the low survival fraction, which reduces the efficacy of minimally invasive in vivo treatments. .. In this experiment, we use the non-membrane-permeative fluorescent dye YOYO-1 (Y3601, Molecular Probes) as the delivery material to investigate the effect of plasma irradiation on cell membrane permeability.

Transfection:

Article Title: In vitro effects of PI3K/mTOR inhibition in canine hemangiosarcoma
Article Snippet: NucLight Red transfected cells were seeded in quintuplicate at a density of 2,000 cells/well in a 96 well plate and incubated overnight in standard conditions. .. Vehicle-containing C/10 or C/10 with various concentrations of VDC-597 plus YOYO®-1 iodide reagent (Life Technologies, Cat #: Y3601) at concentration of 50 nM (per manufacturer’s protocol) was added to the wells.

Cell Culture:

Article Title: Human Erythropoietin Gene Delivery Using an Arginine-grafted Bioreducible Polymer System
Article Snippet: Also, HepG2, HEK293, and NRK cells were cultured. .. YOYO-1 iodide- (1 mmol/l solution in DMSO; Molecular Probes, Eugene, OR) tagged phEPO (1 molecule dye per 50 bp nucleotide) was prepared in the dark for 30 minutes.

Article Title: Reduction-responsive cross-linked stearyl peptide for effective delivery of plasmid DNA
Article Snippet: For this purpose, we first synthesized and characterized C-SHR, examined the cytotoxicity and transfection efficiency of C-SHR/pDNA in HEK293 and HeLa cells, and finally evaluated the gene transfection efficiency in vivo. .. Materials used in this study were l -histidine hydrochloride, l -arginine, stearic acid, and hydrogen peroxide (30% w/v; Sangon Biotech, Shanghai, People’s Republic of China); polyethylenimine (PEI, branched, molecular weight 25 kDa), Lipofectamine™ 2000, l -buthionine-sulfoximine, 4′,6-diamidino-2-phenylindole dihydrochloride, l -cysteine hydrochloride monohydrate, ethidium bromide, dithiothreitol (DTT, Sigma-Aldrich, St Louis, MO, USA); a luciferase assay kit (Promega, Madison, WI, USA); pDNA (pGL3 and pEGFP) (Shanghai Innovation Biotechnology Co Ltd, Shanghai, People’s Republic of China); enhanced bicinchoninic acid protein assay kit (Beyotime, Nanjing, People’s Republic of China); Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum, YOYO-1 (Y3601) and penicillin–streptomycin solution 5 kU/mL (Life Technologies, Carlsbad, CA, USA); a Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies Inc, Nanjing, People’s Republic of China); and HEK293 cells and HeLa cells (Cell Culture Center of the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences, Shanghai, People’s Republic of China). .. All animal experiments were performed in accordance with the ethics and regulations of animal experiments of Second Military Medical University (Shanghai, People’s Republic of China).

Imaging:

Article Title: Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination
Article Snippet: Here, we describe the use of double-tethered DNA curtains to visualize and quantify the diffusive properties of MRN, which rapidly locates DSBs in human cells ( ). .. Imaging buffer: 40m M Tris–HCl [pH 8.0]; 60m M NaCl; 1m M ; MgCl2 ; 2m M DTT; 0.2 mgmL−1 BSA Biotinylated anti-FLAG M2 antibody (F9291; Sigma) Streptavidin-conjugated quantum dots (QDots) 705nm (Q10163MP; Thermo) YOYO-1 stored as a 1m M stock in DMSO (Y3601; Life Technologies) Glucose oxidase type II from Aspergillus niger (G2133; Sigma) Catalase from bovine liver (C9322; Sigma) .. Follow flowcell assembly protocol for double-tethered DNA curtains from Section 2.3.

Recombinant:

Article Title: Cyclooxygenase-2, prostaglandin E2 glycerol ester and nitric oxide are involved in muscarine-induced presynaptic enhancement at the vertebrate neuromuscular junction
Article Snippet: Control experiments were performed by adding the secondary antibody without the primary antibody and by preabsorbing the primary antibody with recombinant human COX-2 (Invitrogen) for 5 h at 4°C prior to being added to the tissue. .. To visualize the perisynaptic Schwann cells (PSCs), preparations were either (1) incubated with YOYO-1 Iodide (125 n m , Y3601; Invitrogen) for 5 min at 24°C just prior to mounting or (2) incubated with 2 μg ml−1 mouse anti-HNK-1 IgM monoclonal antibody (C6680; Sigma-Aldrich) and goat anti-mouse IgM secondary antibody conjugated to TRITC (5 μg ml−1 ; American Qualex).

Crocin Bleaching Assay:

Article Title: A Guanidinylated Bioreducible Polymer with High Nuclear Localization Ability for Gene Delivery Systems
Article Snippet: N,N′-cystaminebisacrylamide (CBA) was purchased from PolySciences, Inc. (Warrington, PA). .. YOYO-1 iodide (1 mM solution in DMSO) was purchased from Molecular Probes (Eugene, OR).

Article Title: A Family of Bioreducible Poly(disulfide amine)s for Gene Delivery
Article Snippet: All chemicals: spermine (SP, Sigma, St. Louis, MO), N,N ′-bis(3-aminopropyl)-1,3-propanediamine (APPD, Sigma-Aldrich, St. Louis, MO), N,N ′-bis(3-aminopropyl)-ethylenediamine (APED, Acros Organics, Fair Lawn, NJ), N,N ′-bis(2-aminoethyl)-1,3-propanediamine (AEPD, Sigma-Aldrich, St. Louis, MO), triethylenetetramine (TETA, Sigma-Fluka, St. Louis, MO), N,N ′-cystaminebisacrylamide (CBA, PolySciences, Warrington, PA), branced polyethylenimine (bPEI, Mw =25kDa, Sigma, St. Louis, MO), ethylenediamine (EDA, Sigma-Aldrich, St. Louis, MO), 2-acetyldimedone (Dde-OH, EMD Chemicals, Inc. Gibbstown, NJ), hydroxylamine hydrochloride (NH2 OH·HCl, Sigma-Aldrich, St. Louis, MO), imidazole (Sigma-Aldrich, St. Louis, MO), N -methyl-2-pyrrolidinone (NMP, Sigma-Aldrich, St. Louis, MO), N,N -dimethylformamide (DMF, Sigma-Aldrich, St. Louis, MO), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma, St. Louis, MO), dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO), dithiothreitol (DTT, Sigma-Aldrich, St. Louis, MO), and SYBR® Safe DNA gel stain (10,000×concentrate in DMSO, Invitrogn, Carlsbad, CA) were purchased in the highest purity and used without further purification. .. YOYO-1 iodide (1 mM solution in DMSO) was purchased from Molecular Probes (Eugene, OR).

Immunofluorescence:

Article Title: Cyclooxygenase-2, prostaglandin E2 glycerol ester and nitric oxide are involved in muscarine-induced presynaptic enhancement at the vertebrate neuromuscular junction
Article Snippet: Paragraph title: Immunofluorescence ... To visualize the perisynaptic Schwann cells (PSCs), preparations were either (1) incubated with YOYO-1 Iodide (125 n m , Y3601; Invitrogen) for 5 min at 24°C just prior to mounting or (2) incubated with 2 μg ml−1 mouse anti-HNK-1 IgM monoclonal antibody (C6680; Sigma-Aldrich) and goat anti-mouse IgM secondary antibody conjugated to TRITC (5 μg ml−1 ; American Qualex).

Molecular Weight:

Article Title: Cyclooxygenase-2, prostaglandin E2 glycerol ester and nitric oxide are involved in muscarine-induced presynaptic enhancement at the vertebrate neuromuscular junction
Article Snippet: To visualize nerve terminals, either: (1) preparations were incubated with 2 μg ml−1 mouse anti-synaptotagmin monoclonal antibody (mAb 48, Developmental Studies Hybridoma Bank at the University of Iowa) and either goat anti-mouse secondary antibody conjugated to Alexa Fluor 555 or chicken anti-mouse secondary antibody conjugated to Alexa Fluor 647 (5 μg ml−1 ; Invitrogen); or (2) the cut end of the motor axon was dipped into a small (1–2 μl) well containing 20 m m Texas Red conjugated to 10,000 molecular weight dextran (Molecular Probes, Carlsbad, CA, USA) in 10 m m Hepes buffer (pH 7.2) and incubated overnight at 9°C to allow the nerve terminals to fill with the Texas Red dextran. .. To visualize the perisynaptic Schwann cells (PSCs), preparations were either (1) incubated with YOYO-1 Iodide (125 n m , Y3601; Invitrogen) for 5 min at 24°C just prior to mounting or (2) incubated with 2 μg ml−1 mouse anti-HNK-1 IgM monoclonal antibody (C6680; Sigma-Aldrich) and goat anti-mouse IgM secondary antibody conjugated to TRITC (5 μg ml−1 ; American Qualex).

Article Title: Reduction-responsive cross-linked stearyl peptide for effective delivery of plasmid DNA
Article Snippet: For this purpose, we first synthesized and characterized C-SHR, examined the cytotoxicity and transfection efficiency of C-SHR/pDNA in HEK293 and HeLa cells, and finally evaluated the gene transfection efficiency in vivo. .. Materials used in this study were l -histidine hydrochloride, l -arginine, stearic acid, and hydrogen peroxide (30% w/v; Sangon Biotech, Shanghai, People’s Republic of China); polyethylenimine (PEI, branched, molecular weight 25 kDa), Lipofectamine™ 2000, l -buthionine-sulfoximine, 4′,6-diamidino-2-phenylindole dihydrochloride, l -cysteine hydrochloride monohydrate, ethidium bromide, dithiothreitol (DTT, Sigma-Aldrich, St Louis, MO, USA); a luciferase assay kit (Promega, Madison, WI, USA); pDNA (pGL3 and pEGFP) (Shanghai Innovation Biotechnology Co Ltd, Shanghai, People’s Republic of China); enhanced bicinchoninic acid protein assay kit (Beyotime, Nanjing, People’s Republic of China); Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum, YOYO-1 (Y3601) and penicillin–streptomycin solution 5 kU/mL (Life Technologies, Carlsbad, CA, USA); a Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies Inc, Nanjing, People’s Republic of China); and HEK293 cells and HeLa cells (Cell Culture Center of the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences, Shanghai, People’s Republic of China). .. All animal experiments were performed in accordance with the ethics and regulations of animal experiments of Second Military Medical University (Shanghai, People’s Republic of China).

Staining:

Article Title: Fanconi anemia signaling and Mus81 cooperate to safeguard development and crosslink repair
Article Snippet: CIdU and IdU staining of labeled DNA fibers from primary murine embryonic fibroblasts (MEFs) was carried out as described ( ) with the following modifications in the plug washing and melting steps: following proteinase K digestions plugs were washed 5 × 10 min in 10-ml TE50 buffer (10-mM Tris-HCL pH 7.0, 50-mM ethylenediaminetetraacetic acid). .. One plug was transferred to a round bottom polycarbonate tube with 100-μl 6.7-μM YOYO-1 (Y3601; Invitrogen) in TE50 for 30 min at room temperature in the dark to stain genomic DNA. .. Plugs were washed 3 × 5 min in 10-ml TE50 and incubated in for 5 min in 5 ml of 50-mM MES buffer at pH 5.7.

Article Title: Gas-liquid interfacial plasmas producing reactive species for cell membrane permeabilization
Article Snippet: Recently, it was reported that genes can be efficiently introduced into cells using APP and that the method enables spatially selective membrane permeabilization. ( – ) Furthermore, the use of fluorescent dyes such as YOYO-1 and LIVE/DEAD Stain indicated that APP irradiation enhances the permeability of the cell membrane without killing the cell. ( – ) To understand the underlying mechanism of cell membrane permeabilization, we have investigated the effects of plasma-produced reactive species in the liquid phase region on cell activity. .. In this experiment, we use the non-membrane-permeative fluorescent dye YOYO-1 (Y3601, Molecular Probes) as the delivery material to investigate the effect of plasma irradiation on cell membrane permeability.

Article Title: Radical cure of experimental babesiosis in immunodeficient mice using a combination of an endochin-like quinolone and atovaquone
Article Snippet: The cells were washed again with PBS and treated with 0.25 mg/ml RNase (Invitrogen; #12091–021) for 1 h at 37°C. .. After RNase treatment, cells were stained with 20 nM YOYO-1 (Invitrogen; #Y3601) for 1 h at room temperature and in the dark. .. The cells were washed and resuspended in PBS for flow analysis.

Article Title: A Guanidinylated Bioreducible Polymer with High Nuclear Localization Ability for Gene Delivery Systems
Article Snippet: SYBR safe DNA gel stain (10,000X in DMSO), fetal bovine serum (FBS), Dulbecco’s phosphate buffered saline (DPBS), and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Invitrogen (Carlsbad, Ca). .. YOYO-1 iodide (1 mM solution in DMSO) was purchased from Molecular Probes (Eugene, OR).

Article Title: Single molecule linear analysis of DNA in nano-channel labeled with sequence specific fluorescent probes
Article Snippet: To increase the probe TM, modified LNA bases are incorporated in the probes, for example +C and +T are modified LNA bases in the probe ATT + CTCCTGCC + TCA. .. All the DNA (4 ng/µl) samples were stained with intercalating dye YOYO-1 iodide (Invitrogen, Carlsbad, CA; Cat #Y3601) (1 molecule/10 bp) in presence of 0.4 M DTT (Promega Inc, Madison, WI, USA; Cat #V3151). .. The sample was diluted by two times using the flow buffer consisting of 1× TBE, 3.6% Tween and 10% polyvinyl pyrrolidone (PVP).

Article Title: Disruption of Apicoplast Biogenesis by Chemical Stabilization of an Imported Protein Evades the Delayed-Death Phenotype in Malaria Parasites
Article Snippet: For dose-response assays, sorbitol-synchronized ring-stage parasites were grown in 96-well plates containing 2-fold serial dilutions of WR99210 (Jacobus Pharmaceutical Company) in the presence or absence of 200 μM IPP. .. After 3 days of growth, parasites were fixed in 1% paraformaldehyde in PBS and were stained with 50 nM YOYO-1 idide (ThermoFisher Y3601). .. Parasitemia was analyzed on a BD Accuri C6 flow cytometer.

Article Title: Chk1 and DNA-PK mediate TPEN-induced DNA damage in a ROS dependent manner in human colon cancer cells
Article Snippet: Electrophoresis was performed in electrophoresis buffer (300 mM NaOH, 1 mM EDTA) for 30 min at 25 V and 250 mA. .. Slides were then neutralized with neutralizing solution (0.4 M Tris-HCl, pH 7.5), and stained with 50 µl of YOYO-1 (Thermofisher, Y3601) stain (0.25 µM YOYO-1, 2.5% DMSO, 0.5% sucrose). .. A fluorescent microscope illuminated with blue light (490nm) and KS 300 V3 image analysis software was used to analyze the slides.

Article Title: A Family of Bioreducible Poly(disulfide amine)s for Gene Delivery
Article Snippet: All chemicals: spermine (SP, Sigma, St. Louis, MO), N,N ′-bis(3-aminopropyl)-1,3-propanediamine (APPD, Sigma-Aldrich, St. Louis, MO), N,N ′-bis(3-aminopropyl)-ethylenediamine (APED, Acros Organics, Fair Lawn, NJ), N,N ′-bis(2-aminoethyl)-1,3-propanediamine (AEPD, Sigma-Aldrich, St. Louis, MO), triethylenetetramine (TETA, Sigma-Fluka, St. Louis, MO), N,N ′-cystaminebisacrylamide (CBA, PolySciences, Warrington, PA), branced polyethylenimine (bPEI, Mw =25kDa, Sigma, St. Louis, MO), ethylenediamine (EDA, Sigma-Aldrich, St. Louis, MO), 2-acetyldimedone (Dde-OH, EMD Chemicals, Inc. Gibbstown, NJ), hydroxylamine hydrochloride (NH2 OH·HCl, Sigma-Aldrich, St. Louis, MO), imidazole (Sigma-Aldrich, St. Louis, MO), N -methyl-2-pyrrolidinone (NMP, Sigma-Aldrich, St. Louis, MO), N,N -dimethylformamide (DMF, Sigma-Aldrich, St. Louis, MO), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma, St. Louis, MO), dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO), dithiothreitol (DTT, Sigma-Aldrich, St. Louis, MO), and SYBR® Safe DNA gel stain (10,000×concentrate in DMSO, Invitrogn, Carlsbad, CA) were purchased in the highest purity and used without further purification. .. YOYO-1 iodide (1 mM solution in DMSO) was purchased from Molecular Probes (Eugene, OR).

Article Title: Controlled generation of nanopatterned electrical DNA interface
Article Snippet: DNA (Lambda DNA (0.3 μg/μL), Thermo Scientific, MA, USA) was tested in this study. .. To observe the characteristics of the attached lambda DNAs according to the electrophoresis conditions, YOYO-1 (491/509 1 mM Solution in DMSO, Molecular Probes, OR, USA) was used for the fluorescent staining, described as follows. .. After mixing 1 μL of YOYO-1 in 21.5 μL of lambda DNAs in an Eppendorf tube, the tube was wrapped with foil to block out light, then incubated in an oven at 55 °C for 2 h, ensuring the bonding of the YOYO-1 particles with the lambda DNAs; 100 μL of the TBE buffer (Tris-Borate-EDTA buffer, Sigma Aldrich, MO, USA) was added to the lambda DNA-YOYO-1 mixture solution and washed three times.

Article Title: Mechanical and structural properties of YOYO-1 complexed DNA
Article Snippet: DNA was stained with YOYO-1 as described elsewhere ( ). .. A 1 mM YOYO-1 stock solution (Invitrogen Y3601) was diluted 400- to 6700-fold in 10 mM phosphate buffer (sodium phosphate buffer: 1.88 mM NaH2 PO4 ·H2 O, 8.13 mM Na2 HPO4 ·2H2 O, resulting in a pH of 7.5) depending on the desired final staining ratio. .. An ∼10-fold smaller volume of DNA was added to provide a final DNA concentration of 0.56 ng μl−1 corresponding to a base pair concentration of 860 nM.

MTT Assay:

Article Title: A Family of Bioreducible Poly(disulfide amine)s for Gene Delivery
Article Snippet: All chemicals: spermine (SP, Sigma, St. Louis, MO), N,N ′-bis(3-aminopropyl)-1,3-propanediamine (APPD, Sigma-Aldrich, St. Louis, MO), N,N ′-bis(3-aminopropyl)-ethylenediamine (APED, Acros Organics, Fair Lawn, NJ), N,N ′-bis(2-aminoethyl)-1,3-propanediamine (AEPD, Sigma-Aldrich, St. Louis, MO), triethylenetetramine (TETA, Sigma-Fluka, St. Louis, MO), N,N ′-cystaminebisacrylamide (CBA, PolySciences, Warrington, PA), branced polyethylenimine (bPEI, Mw =25kDa, Sigma, St. Louis, MO), ethylenediamine (EDA, Sigma-Aldrich, St. Louis, MO), 2-acetyldimedone (Dde-OH, EMD Chemicals, Inc. Gibbstown, NJ), hydroxylamine hydrochloride (NH2 OH·HCl, Sigma-Aldrich, St. Louis, MO), imidazole (Sigma-Aldrich, St. Louis, MO), N -methyl-2-pyrrolidinone (NMP, Sigma-Aldrich, St. Louis, MO), N,N -dimethylformamide (DMF, Sigma-Aldrich, St. Louis, MO), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma, St. Louis, MO), dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO), dithiothreitol (DTT, Sigma-Aldrich, St. Louis, MO), and SYBR® Safe DNA gel stain (10,000×concentrate in DMSO, Invitrogn, Carlsbad, CA) were purchased in the highest purity and used without further purification. .. YOYO-1 iodide (1 mM solution in DMSO) was purchased from Molecular Probes (Eugene, OR).

In Vivo:

Article Title: Gas-liquid interfacial plasmas producing reactive species for cell membrane permeabilization
Article Snippet: Cell membrane permeabilization is an important technique in molecular biology and medical procedures such as cancer pharmacotherapy,( ) gene therapy,( ) RNA-mediated suppression of gene expression,( ) and the creation of induced pluripotent stem (iPS) cells. However, electroporation,( ) a conventional method for permeabilizing cells, is problematic in terms of the low survival fraction, which reduces the efficacy of minimally invasive in vivo treatments. .. In this experiment, we use the non-membrane-permeative fluorescent dye YOYO-1 (Y3601, Molecular Probes) as the delivery material to investigate the effect of plasma irradiation on cell membrane permeability.

Fluorescence:

Article Title: Fanconi anemia signaling and Mus81 cooperate to safeguard development and crosslink repair
Article Snippet: One plug was transferred to a round bottom polycarbonate tube with 100-μl 6.7-μM YOYO-1 (Y3601; Invitrogen) in TE50 for 30 min at room temperature in the dark to stain genomic DNA. .. One plug was transferred to a round bottom polycarbonate tube with 100-μl 6.7-μM YOYO-1 (Y3601; Invitrogen) in TE50 for 30 min at room temperature in the dark to stain genomic DNA.

Magnetic Beads:

Article Title: Mechanical and structural properties of YOYO-1 complexed DNA
Article Snippet: A 1 mM YOYO-1 stock solution (Invitrogen Y3601) was diluted 400- to 6700-fold in 10 mM phosphate buffer (sodium phosphate buffer: 1.88 mM NaH2 PO4 ·H2 O, 8.13 mM Na2 HPO4 ·2H2 O, resulting in a pH of 7.5) depending on the desired final staining ratio. .. An ∼10-fold smaller volume of DNA was added to provide a final DNA concentration of 0.56 ng μl−1 corresponding to a base pair concentration of 860 nM.

Isolation:

Article Title: Human Erythropoietin Gene Delivery Using an Arginine-grafted Bioreducible Polymer System
Article Snippet: Briefly, BM cells were isolated from the bilateral femur of 7-week-old SD rats and prepared with Dulbecco's modified Eagle's medium. .. YOYO-1 iodide- (1 mmol/l solution in DMSO; Molecular Probes, Eugene, OR) tagged phEPO (1 molecule dye per 50 bp nucleotide) was prepared in the dark for 30 minutes.

Article Title: A Guanidinylated Bioreducible Polymer with High Nuclear Localization Ability for Gene Delivery Systems
Article Snippet: The plasmid pCMV-Luc, containing a firefly luciferase reporter gene was amplified in E.coli DH5α and isolated by standard Maxiprep kit (Invitrogen, Carlsbad, CA). .. YOYO-1 iodide (1 mM solution in DMSO) was purchased from Molecular Probes (Eugene, OR).

Bicinchoninic Acid Protein Assay:

Article Title: Reduction-responsive cross-linked stearyl peptide for effective delivery of plasmid DNA
Article Snippet: For this purpose, we first synthesized and characterized C-SHR, examined the cytotoxicity and transfection efficiency of C-SHR/pDNA in HEK293 and HeLa cells, and finally evaluated the gene transfection efficiency in vivo. .. Materials used in this study were l -histidine hydrochloride, l -arginine, stearic acid, and hydrogen peroxide (30% w/v; Sangon Biotech, Shanghai, People’s Republic of China); polyethylenimine (PEI, branched, molecular weight 25 kDa), Lipofectamine™ 2000, l -buthionine-sulfoximine, 4′,6-diamidino-2-phenylindole dihydrochloride, l -cysteine hydrochloride monohydrate, ethidium bromide, dithiothreitol (DTT, Sigma-Aldrich, St Louis, MO, USA); a luciferase assay kit (Promega, Madison, WI, USA); pDNA (pGL3 and pEGFP) (Shanghai Innovation Biotechnology Co Ltd, Shanghai, People’s Republic of China); enhanced bicinchoninic acid protein assay kit (Beyotime, Nanjing, People’s Republic of China); Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum, YOYO-1 (Y3601) and penicillin–streptomycin solution 5 kU/mL (Life Technologies, Carlsbad, CA, USA); a Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies Inc, Nanjing, People’s Republic of China); and HEK293 cells and HeLa cells (Cell Culture Center of the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences, Shanghai, People’s Republic of China). .. All animal experiments were performed in accordance with the ethics and regulations of animal experiments of Second Military Medical University (Shanghai, People’s Republic of China).

Flow Cytometry:

Article Title: Human Erythropoietin Gene Delivery Using an Arginine-grafted Bioreducible Polymer System
Article Snippet: YOYO-1 iodide- (1 mmol/l solution in DMSO; Molecular Probes, Eugene, OR) tagged phEPO (1 molecule dye per 50 bp nucleotide) was prepared in the dark for 30 minutes. .. The polyplexes were incubated with cells at 37 °C for 4 hours in serum free media.

Article Title: Radical cure of experimental babesiosis in immunodeficient mice using a combination of an endochin-like quinolone and atovaquone
Article Snippet: Paragraph title: Flow cytometry ... After RNase treatment, cells were stained with 20 nM YOYO-1 (Invitrogen; #Y3601) for 1 h at room temperature and in the dark.

Article Title: Mechanical and structural properties of YOYO-1 complexed DNA
Article Snippet: A 1 mM YOYO-1 stock solution (Invitrogen Y3601) was diluted 400- to 6700-fold in 10 mM phosphate buffer (sodium phosphate buffer: 1.88 mM NaH2 PO4 ·H2 O, 8.13 mM Na2 HPO4 ·2H2 O, resulting in a pH of 7.5) depending on the desired final staining ratio. .. An ∼10-fold smaller volume of DNA was added to provide a final DNA concentration of 0.56 ng μl−1 corresponding to a base pair concentration of 860 nM.

Microscopy:

Article Title: Fanconi anemia signaling and Mus81 cooperate to safeguard development and crosslink repair
Article Snippet: One plug was transferred to a round bottom polycarbonate tube with 100-μl 6.7-μM YOYO-1 (Y3601; Invitrogen) in TE50 for 30 min at room temperature in the dark to stain genomic DNA. .. One plug was transferred to a round bottom polycarbonate tube with 100-μl 6.7-μM YOYO-1 (Y3601; Invitrogen) in TE50 for 30 min at room temperature in the dark to stain genomic DNA.

Purification:

Article Title: DNA condensation by the nucleocapsid protein of HIV-1: a mechanism ensuring DNA protection
Article Snippet: The plasmid DNA pCMV-luc (5.2 kb) was propagated and purified as described ( ). .. YOYO-1 (491/509) was obtained from Molecular Probes Inc. NCp7 and the related peptides were synthesized by solid-phase chemistry as described ( ).

Article Title: Human Erythropoietin Gene Delivery Using an Arginine-grafted Bioreducible Polymer System
Article Snippet: The suspension was centrifuged, erythrocytes were lysed using RBC lysis buffer, and the purified cells were seeded in RPMI-1640 medium. .. YOYO-1 iodide- (1 mmol/l solution in DMSO; Molecular Probes, Eugene, OR) tagged phEPO (1 molecule dye per 50 bp nucleotide) was prepared in the dark for 30 minutes.

Article Title: Polymer Transfected Primary Myoblasts Mediated Efficient Gene Expression and Angiogenic Proliferation
Article Snippet: The plasmids, pCMV-Luc, pCMV-GFP, and pCMV-VEGF165 , containing a firefly luciferase reporter gene, green fluorescent protein gene and vascular endothelial growth factor gene 165, respectively, were amplified in E. coli DH5α and purified by standard Maxiprep kit (Invitrogen, Carlsbad, CA), separately. .. YOYO-1 iodide (1 mM solution in DMSO) was purchased from Molecular Probes (Eugene, OR).

Article Title: A Family of Bioreducible Poly(disulfide amine)s for Gene Delivery
Article Snippet: The plasmid pCMV-Luc, containing a firefly luciferase reporter gene, was amplified in E.coli DH5α and purified by standard Maxiprep kit (Invitrogen, Carlsbad, CA). .. YOYO-1 iodide (1 mM solution in DMSO) was purchased from Molecular Probes (Eugene, OR).

Labeling:

Article Title: Fanconi anemia signaling and Mus81 cooperate to safeguard development and crosslink repair
Article Snippet: CIdU and IdU staining of labeled DNA fibers from primary murine embryonic fibroblasts (MEFs) was carried out as described ( ) with the following modifications in the plug washing and melting steps: following proteinase K digestions plugs were washed 5 × 10 min in 10-ml TE50 buffer (10-mM Tris-HCL pH 7.0, 50-mM ethylenediaminetetraacetic acid). .. One plug was transferred to a round bottom polycarbonate tube with 100-μl 6.7-μM YOYO-1 (Y3601; Invitrogen) in TE50 for 30 min at room temperature in the dark to stain genomic DNA.

Article Title: Fabrication of long poly(dimethyl siloxane) nanochannels by replicating protein deposit from confined solution evaporation
Article Snippet: Poly(dimethyl siloxane) (PDMS) (Sylgard 184) was purchased from Dow Corning. .. 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. .. Figure shows the schematic of fabricating the PDMS nanochannels.

FACS:

Article Title: Human Erythropoietin Gene Delivery Using an Arginine-grafted Bioreducible Polymer System
Article Snippet: YOYO-1 iodide- (1 mmol/l solution in DMSO; Molecular Probes, Eugene, OR) tagged phEPO (1 molecule dye per 50 bp nucleotide) was prepared in the dark for 30 minutes. .. The polyplexes were incubated with cells at 37 °C for 4 hours in serum free media.

Chromatin Immunoprecipitation:

Article Title: Single molecule linear analysis of DNA in nano-channel labeled with sequence specific fluorescent probes
Article Snippet: All the DNA (4 ng/µl) samples were stained with intercalating dye YOYO-1 iodide (Invitrogen, Carlsbad, CA; Cat #Y3601) (1 molecule/10 bp) in presence of 0.4 M DTT (Promega Inc, Madison, WI, USA; Cat #V3151). .. All the DNA (4 ng/µl) samples were stained with intercalating dye YOYO-1 iodide (Invitrogen, Carlsbad, CA; Cat #Y3601) (1 molecule/10 bp) in presence of 0.4 M DTT (Promega Inc, Madison, WI, USA; Cat #V3151).

Plasmid Preparation:

Article Title: DNA condensation by the nucleocapsid protein of HIV-1: a mechanism ensuring DNA protection
Article Snippet: The plasmid DNA pCMV-luc (5.2 kb) was propagated and purified as described ( ). .. YOYO-1 (491/509) was obtained from Molecular Probes Inc. NCp7 and the related peptides were synthesized by solid-phase chemistry as described ( ).

Article Title: A Guanidinylated Bioreducible Polymer with High Nuclear Localization Ability for Gene Delivery Systems
Article Snippet: The plasmid pCMV-Luc, containing a firefly luciferase reporter gene was amplified in E.coli DH5α and isolated by standard Maxiprep kit (Invitrogen, Carlsbad, CA). .. YOYO-1 iodide (1 mM solution in DMSO) was purchased from Molecular Probes (Eugene, OR).

Article Title: A Family of Bioreducible Poly(disulfide amine)s for Gene Delivery
Article Snippet: The plasmid pCMV-Luc, containing a firefly luciferase reporter gene, was amplified in E.coli DH5α and purified by standard Maxiprep kit (Invitrogen, Carlsbad, CA). .. YOYO-1 iodide (1 mM solution in DMSO) was purchased from Molecular Probes (Eugene, OR).

Software:

Article Title: Fanconi anemia signaling and Mus81 cooperate to safeguard development and crosslink repair
Article Snippet: One plug was transferred to a round bottom polycarbonate tube with 100-μl 6.7-μM YOYO-1 (Y3601; Invitrogen) in TE50 for 30 min at room temperature in the dark to stain genomic DNA. .. One plug was transferred to a round bottom polycarbonate tube with 100-μl 6.7-μM YOYO-1 (Y3601; Invitrogen) in TE50 for 30 min at room temperature in the dark to stain genomic DNA.

Irradiation:

Article Title: Gas-liquid interfacial plasmas producing reactive species for cell membrane permeabilization
Article Snippet: We also investigated the relationship between plasma-induced cell membrane permeabilization and the spatial distributions of reactive species in the liquid phase region. .. In this experiment, we use the non-membrane-permeative fluorescent dye YOYO-1 (Y3601, Molecular Probes) as the delivery material to investigate the effect of plasma irradiation on cell membrane permeability. .. The green fluorescence of YOYO-1 increases 1,000-fold when the dye is transferred into cells and binds double-stranded DNA (dsDNA) in the nucleus, allowing transfected cells to be easily identified. ( , ) In addition, YOYO-1 exhibits strong fluorescence within 0.5 s of binding to dsDNA, enabling rapid analysis of the fluorescence intensity of the cells and enumeration of the transfected cells as soon as the sample is irradiated with plasma.

Negative Control:

Article Title: Human Erythropoietin Gene Delivery Using an Arginine-grafted Bioreducible Polymer System
Article Snippet: YOYO-1 iodide- (1 mmol/l solution in DMSO; Molecular Probes, Eugene, OR) tagged phEPO (1 molecule dye per 50 bp nucleotide) was prepared in the dark for 30 minutes. .. Samples were analyzed by flow cytometry (FACS Caliber; BD Biosciences, San Jose, CA) at a minimum of 1 × 104 cells using the FL1-height channel for YOYO-1 dye.

Next-Generation Sequencing:

Article Title: Microfluidic-assisted analysis of replicating DNA molecules
Article Snippet: Solutions of MMS in water inactivate over time and can be disposed. .. · β-agarase (New England Biolabs m0392S) · proteinase K powder (Invitrogen 25530-015) · YOYO-1dye (Invitrogen Y3601) · rat anti-CldU/BrdU (bromodeoxyuridine) antibody (Serotec MA2060 or Accurate Chemical OBT0030G) · normal goat serum (NGS; Accurate Chemical ACL1200-100) · BSA, bovine serum albumin (Sigma a3294) · goat anti-rat Alexa 594-conjugated antibody (Invitrogen Molecular Dynamics A1100) · mouse anti-IdU/BrdU antibody (Becton Dickinson 347580) · goat anti-mouse Alexa 488-conjugated antibody (Invitrogen Molecular Dynamics A11001) · mouse anti-DNA antibody (anti-deoxythymidine) (Chemicon MAB3034). .. · low gelling temperature (LGT) agarose (any brand, but must be DNAse-free) · EDTA, disodium salt (any high quality supplier can be used for this and the chemicals below) · deoxycholic acid, sodium salt · N-lauroylsarcosine, sodium salt · NaCl · Tris base · Tween 20 non-ionic detergent · phosphate-buffered saline (PBS) · HCL, concentrated · ‘Hard-as-Nails’ clear nail polish (Sally Hansen 2103, from your local drugstore)

CCK-8 Assay:

Article Title: Reduction-responsive cross-linked stearyl peptide for effective delivery of plasmid DNA
Article Snippet: For this purpose, we first synthesized and characterized C-SHR, examined the cytotoxicity and transfection efficiency of C-SHR/pDNA in HEK293 and HeLa cells, and finally evaluated the gene transfection efficiency in vivo. .. Materials used in this study were l -histidine hydrochloride, l -arginine, stearic acid, and hydrogen peroxide (30% w/v; Sangon Biotech, Shanghai, People’s Republic of China); polyethylenimine (PEI, branched, molecular weight 25 kDa), Lipofectamine™ 2000, l -buthionine-sulfoximine, 4′,6-diamidino-2-phenylindole dihydrochloride, l -cysteine hydrochloride monohydrate, ethidium bromide, dithiothreitol (DTT, Sigma-Aldrich, St Louis, MO, USA); a luciferase assay kit (Promega, Madison, WI, USA); pDNA (pGL3 and pEGFP) (Shanghai Innovation Biotechnology Co Ltd, Shanghai, People’s Republic of China); enhanced bicinchoninic acid protein assay kit (Beyotime, Nanjing, People’s Republic of China); Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum, YOYO-1 (Y3601) and penicillin–streptomycin solution 5 kU/mL (Life Technologies, Carlsbad, CA, USA); a Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies Inc, Nanjing, People’s Republic of China); and HEK293 cells and HeLa cells (Cell Culture Center of the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences, Shanghai, People’s Republic of China). .. All animal experiments were performed in accordance with the ethics and regulations of animal experiments of Second Military Medical University (Shanghai, People’s Republic of China).

Spectrophotometry:

Article Title: DNA condensation by the nucleocapsid protein of HIV-1: a mechanism ensuring DNA protection
Article Snippet: YOYO-1 (491/509) was obtained from Molecular Probes Inc. NCp7 and the related peptides were synthesized by solid-phase chemistry as described ( ). .. YOYO-1 (491/509) was obtained from Molecular Probes Inc. NCp7 and the related peptides were synthesized by solid-phase chemistry as described ( ).

Produced:

Article Title: Gas-liquid interfacial plasmas producing reactive species for cell membrane permeabilization
Article Snippet: In this experiment, we use the non-membrane-permeative fluorescent dye YOYO-1 (Y3601, Molecular Probes) as the delivery material to investigate the effect of plasma irradiation on cell membrane permeability. .. The green fluorescence of YOYO-1 increases 1,000-fold when the dye is transferred into cells and binds double-stranded DNA (dsDNA) in the nucleus, allowing transfected cells to be easily identified. ( , ) In addition, YOYO-1 exhibits strong fluorescence within 0.5 s of binding to dsDNA, enabling rapid analysis of the fluorescence intensity of the cells and enumeration of the transfected cells as soon as the sample is irradiated with plasma.

Concentration Assay:

Article Title: DNA condensation by the nucleocapsid protein of HIV-1: a mechanism ensuring DNA protection
Article Snippet: YOYO-1 (491/509) was obtained from Molecular Probes Inc. NCp7 and the related peptides were synthesized by solid-phase chemistry as described ( ). .. YOYO-1 (491/509) was obtained from Molecular Probes Inc. NCp7 and the related peptides were synthesized by solid-phase chemistry as described ( ).

Article Title: In vitro effects of PI3K/mTOR inhibition in canine hemangiosarcoma
Article Snippet: NucLight Red transfected cells were seeded in quintuplicate at a density of 2,000 cells/well in a 96 well plate and incubated overnight in standard conditions. .. Vehicle-containing C/10 or C/10 with various concentrations of VDC-597 plus YOYO®-1 iodide reagent (Life Technologies, Cat #: Y3601) at concentration of 50 nM (per manufacturer’s protocol) was added to the wells. .. The plate was placed within the Incucyte Live Cell Imaging apparatus (Essen BioScience, Ann Arbor, MI) and the number of viable cells (red fluorescing) and apoptotic cells (green fluorescing) were measured over time.

Cell Counting:

Article Title: Reduction-responsive cross-linked stearyl peptide for effective delivery of plasmid DNA
Article Snippet: For this purpose, we first synthesized and characterized C-SHR, examined the cytotoxicity and transfection efficiency of C-SHR/pDNA in HEK293 and HeLa cells, and finally evaluated the gene transfection efficiency in vivo. .. Materials used in this study were l -histidine hydrochloride, l -arginine, stearic acid, and hydrogen peroxide (30% w/v; Sangon Biotech, Shanghai, People’s Republic of China); polyethylenimine (PEI, branched, molecular weight 25 kDa), Lipofectamine™ 2000, l -buthionine-sulfoximine, 4′,6-diamidino-2-phenylindole dihydrochloride, l -cysteine hydrochloride monohydrate, ethidium bromide, dithiothreitol (DTT, Sigma-Aldrich, St Louis, MO, USA); a luciferase assay kit (Promega, Madison, WI, USA); pDNA (pGL3 and pEGFP) (Shanghai Innovation Biotechnology Co Ltd, Shanghai, People’s Republic of China); enhanced bicinchoninic acid protein assay kit (Beyotime, Nanjing, People’s Republic of China); Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum, YOYO-1 (Y3601) and penicillin–streptomycin solution 5 kU/mL (Life Technologies, Carlsbad, CA, USA); a Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies Inc, Nanjing, People’s Republic of China); and HEK293 cells and HeLa cells (Cell Culture Center of the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences, Shanghai, People’s Republic of China). .. All animal experiments were performed in accordance with the ethics and regulations of animal experiments of Second Military Medical University (Shanghai, People’s Republic of China).

Lysis:

Article Title: Human Erythropoietin Gene Delivery Using an Arginine-grafted Bioreducible Polymer System
Article Snippet: The suspension was centrifuged, erythrocytes were lysed using RBC lysis buffer, and the purified cells were seeded in RPMI-1640 medium. .. YOYO-1 iodide- (1 mmol/l solution in DMSO; Molecular Probes, Eugene, OR) tagged phEPO (1 molecule dye per 50 bp nucleotide) was prepared in the dark for 30 minutes.

Article Title: A Guanidinylated Bioreducible Polymer with High Nuclear Localization Ability for Gene Delivery Systems
Article Snippet: Luciferase assay system and reporter lysis buffer were purchased from Promega (Madison, WI). .. YOYO-1 iodide (1 mM solution in DMSO) was purchased from Molecular Probes (Eugene, OR).

Article Title: Polymer Transfected Primary Myoblasts Mediated Efficient Gene Expression and Angiogenic Proliferation
Article Snippet: The luciferase assay system with reporter lysis buffer was purchased from Promega (Madison, WI). .. YOYO-1 iodide (1 mM solution in DMSO) was purchased from Molecular Probes (Eugene, OR).

Article Title: A Family of Bioreducible Poly(disulfide amine)s for Gene Delivery
Article Snippet: Luciferase assay system with reporter lysis buffer was purchased from Promega (Madison, WI). .. YOYO-1 iodide (1 mM solution in DMSO) was purchased from Molecular Probes (Eugene, OR).

Activity Assay:

Article Title: Gas-liquid interfacial plasmas producing reactive species for cell membrane permeabilization
Article Snippet: Recently, it was reported that genes can be efficiently introduced into cells using APP and that the method enables spatially selective membrane permeabilization. ( – ) Furthermore, the use of fluorescent dyes such as YOYO-1 and LIVE/DEAD Stain indicated that APP irradiation enhances the permeability of the cell membrane without killing the cell. ( – ) To understand the underlying mechanism of cell membrane permeabilization, we have investigated the effects of plasma-produced reactive species in the liquid phase region on cell activity. .. In this experiment, we use the non-membrane-permeative fluorescent dye YOYO-1 (Y3601, Molecular Probes) as the delivery material to investigate the effect of plasma irradiation on cell membrane permeability.

Permeability:

Article Title: Gas-liquid interfacial plasmas producing reactive species for cell membrane permeabilization
Article Snippet: We also investigated the relationship between plasma-induced cell membrane permeabilization and the spatial distributions of reactive species in the liquid phase region. .. In this experiment, we use the non-membrane-permeative fluorescent dye YOYO-1 (Y3601, Molecular Probes) as the delivery material to investigate the effect of plasma irradiation on cell membrane permeability. .. The green fluorescence of YOYO-1 increases 1,000-fold when the dye is transferred into cells and binds double-stranded DNA (dsDNA) in the nucleus, allowing transfected cells to be easily identified. ( , ) In addition, YOYO-1 exhibits strong fluorescence within 0.5 s of binding to dsDNA, enabling rapid analysis of the fluorescence intensity of the cells and enumeration of the transfected cells as soon as the sample is irradiated with plasma.

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  • 99
    Thermo Fisher yoyo 1 dye
    NF-κB signaling blocks NLRC4/caspase-8-dependent apoptosis pathway. Casp1 −/− Casp11 −/− BMDMs were pre-stimulated for with or without various stimuli – Pam3CSK4 (1 μg ml −1 ), LPS (1 μg ml −1 ), R837 (2 μg ml −1 ), TNFα (100 ng ml −1 ), IFN-α (100 U ml −1 ), or IFN-β (100 U ml −1 ), then electroporated with flagellin (0.5 μg ml −1 ) or stimulated with FasL (100 ng ml −1 ). ( a ) LDH release after 16 h. Data is represented as mean ± SD; n = 3. ( b ) % <t>YOYO-1</t> positive BMDMs from live cell imaging taken every 45 min for 16 h.
    Yoyo 1 Dye, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/yoyo 1 dye/product/Thermo Fisher
    Average 99 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    yoyo 1 dye - by Bioz Stars, 2019-10
    99/100 stars
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    75
    Thermo Fisher yoyo 1
    DNA intercalators tilt and twirl beyond the OST. ( A ) Polarized image pairs of a single <t>YOYO-1</t> molecule bound to DNA oriented at ω = 45° in regime 3 alternately excited with x - and y -polarized light (scale bar, 500 nm). A single molecule is detected in image pairs 2 to 5. Illustration: When a dipole twirls, its mean azimuthal orientation with respect to the DNA axis changes. Thus, the dipole is only efficiently excited when it rotates through the axis of the excitation polarization. Hence, toggling between x / y -polarized excitation strongly biases the emission polarization, as seen in, e.g., polarized image pairs 3 and 4. ( B and C ) Polarization scatterplots of single YOYO-1 (B) and SYTOX Orange (C) dyes bound to DNA oriented at ω = 0° and ω = 45° in regime 3.
    Yoyo 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 75/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/yoyo 1/product/Thermo Fisher
    Average 75 stars, based on 0 article reviews
    Price from $9.99 to $1999.99
    yoyo 1 - by Bioz Stars, 2019-10
    75/100 stars
      Buy from Supplier

    99
    Thermo Fisher yoyo 1 iodide
    DNA intercalators tilt and twirl beyond the OST. ( A ) Polarized image pairs of a single <t>YOYO-1</t> molecule bound to DNA oriented at ω = 45° in regime 3 alternately excited with x - and y -polarized light (scale bar, 500 nm). A single molecule is detected in image pairs 2 to 5. Illustration: When a dipole twirls, its mean azimuthal orientation with respect to the DNA axis changes. Thus, the dipole is only efficiently excited when it rotates through the axis of the excitation polarization. Hence, toggling between x / y -polarized excitation strongly biases the emission polarization, as seen in, e.g., polarized image pairs 3 and 4. ( B and C ) Polarization scatterplots of single YOYO-1 (B) and SYTOX Orange (C) dyes bound to DNA oriented at ω = 0° and ω = 45° in regime 3.
    Yoyo 1 Iodide, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/yoyo 1 iodide/product/Thermo Fisher
    Average 99 stars, based on 31 article reviews
    Price from $9.99 to $1999.99
    yoyo 1 iodide - by Bioz Stars, 2019-10
    99/100 stars
      Buy from Supplier

    Image Search Results


    NF-κB signaling blocks NLRC4/caspase-8-dependent apoptosis pathway. Casp1 −/− Casp11 −/− BMDMs were pre-stimulated for with or without various stimuli – Pam3CSK4 (1 μg ml −1 ), LPS (1 μg ml −1 ), R837 (2 μg ml −1 ), TNFα (100 ng ml −1 ), IFN-α (100 U ml −1 ), or IFN-β (100 U ml −1 ), then electroporated with flagellin (0.5 μg ml −1 ) or stimulated with FasL (100 ng ml −1 ). ( a ) LDH release after 16 h. Data is represented as mean ± SD; n = 3. ( b ) % YOYO-1 positive BMDMs from live cell imaging taken every 45 min for 16 h.

    Journal: Scientific Reports

    Article Title: ASC- and caspase-8-dependent apoptotic pathway diverges from the NLRC4 inflammasome in macrophages

    doi: 10.1038/s41598-018-21998-3

    Figure Lengend Snippet: NF-κB signaling blocks NLRC4/caspase-8-dependent apoptosis pathway. Casp1 −/− Casp11 −/− BMDMs were pre-stimulated for with or without various stimuli – Pam3CSK4 (1 μg ml −1 ), LPS (1 μg ml −1 ), R837 (2 μg ml −1 ), TNFα (100 ng ml −1 ), IFN-α (100 U ml −1 ), or IFN-β (100 U ml −1 ), then electroporated with flagellin (0.5 μg ml −1 ) or stimulated with FasL (100 ng ml −1 ). ( a ) LDH release after 16 h. Data is represented as mean ± SD; n = 3. ( b ) % YOYO-1 positive BMDMs from live cell imaging taken every 45 min for 16 h.

    Article Snippet: Ultra-pure flagellin (Psuedomonas aeruginosa ), Pam3CSK4, R837 (Imiquimod), ultra-pure LPS (E. coli O111:B4), Poly(dA:dT), Nigericin were purchased from Invivogen, Mega FasL from AdipoGen, IFN-α and IFN-β from PBL Assay Science, TNFα from Genentech, ATP and bovine cytochrome-c from Sigma, YOYO-1 dye from Thermofisher Scientific, Nuclear-ID DNA stain from Enzo Life Sciences.

    Techniques: Live Cell Imaging

    ASC and caspase-8 are identified through a genome-wide CRISPR/Cas9 screen for caspase-1-independent NLRC4-mediated cell death. ( a ) % YOYO-1 positive iMac cell lines from live imaging taken every hour up to 16 h after flagellin electroporation. Data is represented as mean ± SD; n = 3 images. ( b ) Scatter plot showing relative fold-change enrichment of genes ( x-axis ) with their corresponding enrichment p value ( y-axis ) from n = 3 biological replicates. Counts are log2 transformed and normalized using median scaling. Top scoring genes are highlighted. ( c ) Box-plot showing the distribution of individual sgRNA frequencies of flagellin- over control-treated populations, ordered left-to-right by increasing p-value.

    Journal: Scientific Reports

    Article Title: ASC- and caspase-8-dependent apoptotic pathway diverges from the NLRC4 inflammasome in macrophages

    doi: 10.1038/s41598-018-21998-3

    Figure Lengend Snippet: ASC and caspase-8 are identified through a genome-wide CRISPR/Cas9 screen for caspase-1-independent NLRC4-mediated cell death. ( a ) % YOYO-1 positive iMac cell lines from live imaging taken every hour up to 16 h after flagellin electroporation. Data is represented as mean ± SD; n = 3 images. ( b ) Scatter plot showing relative fold-change enrichment of genes ( x-axis ) with their corresponding enrichment p value ( y-axis ) from n = 3 biological replicates. Counts are log2 transformed and normalized using median scaling. Top scoring genes are highlighted. ( c ) Box-plot showing the distribution of individual sgRNA frequencies of flagellin- over control-treated populations, ordered left-to-right by increasing p-value.

    Article Snippet: Ultra-pure flagellin (Psuedomonas aeruginosa ), Pam3CSK4, R837 (Imiquimod), ultra-pure LPS (E. coli O111:B4), Poly(dA:dT), Nigericin were purchased from Invivogen, Mega FasL from AdipoGen, IFN-α and IFN-β from PBL Assay Science, TNFα from Genentech, ATP and bovine cytochrome-c from Sigma, YOYO-1 dye from Thermofisher Scientific, Nuclear-ID DNA stain from Enzo Life Sciences.

    Techniques: Genome Wide, CRISPR, Imaging, Electroporation, Transformation Assay

    ASC and caspase-8 are required for caspase-1-independent NLRC4 activated cell death in BMDMs. ( a – d ) BMDMs with or without Pam3CSK4 (1 μg ml −1 ) pre-stimulation were electroporated with flagellin (0.5 μg ml −1 ) or cytochrome-c (50 μg ml −1 ). ( a ) and ( c ) LDH release after 16 h. Data is represented as mean ± SD; n = 3. ( b ) and ( d ) % YOYO-1 positive BMDMs from two mice/genotype after flagellin electroporation. Live cell images taken every 30 min up to 16 h. ( e ) Immunoblot of caspase-8, caspase-1, caspase-3 and GSDMD in combined cell extract (ext) and supernatant (sup) from BMDMs 3 hrs after flagellin electroporation (no pre-stimulation). Pro-forms (pro) and cleaved forms are represented in blots. ( f ) BMDCs and thioglycollate-elicited peritoneal macrophages with no pre-stimulation were electroporated with flagellin (0.5 μg ml −1 ), cytochrome-c (50 μg ml −1 ), or FasL (100 ng ml −1 ) and measured for LDH release after 16 h. Data is represented as mean ± SD; n = 3.

    Journal: Scientific Reports

    Article Title: ASC- and caspase-8-dependent apoptotic pathway diverges from the NLRC4 inflammasome in macrophages

    doi: 10.1038/s41598-018-21998-3

    Figure Lengend Snippet: ASC and caspase-8 are required for caspase-1-independent NLRC4 activated cell death in BMDMs. ( a – d ) BMDMs with or without Pam3CSK4 (1 μg ml −1 ) pre-stimulation were electroporated with flagellin (0.5 μg ml −1 ) or cytochrome-c (50 μg ml −1 ). ( a ) and ( c ) LDH release after 16 h. Data is represented as mean ± SD; n = 3. ( b ) and ( d ) % YOYO-1 positive BMDMs from two mice/genotype after flagellin electroporation. Live cell images taken every 30 min up to 16 h. ( e ) Immunoblot of caspase-8, caspase-1, caspase-3 and GSDMD in combined cell extract (ext) and supernatant (sup) from BMDMs 3 hrs after flagellin electroporation (no pre-stimulation). Pro-forms (pro) and cleaved forms are represented in blots. ( f ) BMDCs and thioglycollate-elicited peritoneal macrophages with no pre-stimulation were electroporated with flagellin (0.5 μg ml −1 ), cytochrome-c (50 μg ml −1 ), or FasL (100 ng ml −1 ) and measured for LDH release after 16 h. Data is represented as mean ± SD; n = 3.

    Article Snippet: Ultra-pure flagellin (Psuedomonas aeruginosa ), Pam3CSK4, R837 (Imiquimod), ultra-pure LPS (E. coli O111:B4), Poly(dA:dT), Nigericin were purchased from Invivogen, Mega FasL from AdipoGen, IFN-α and IFN-β from PBL Assay Science, TNFα from Genentech, ATP and bovine cytochrome-c from Sigma, YOYO-1 dye from Thermofisher Scientific, Nuclear-ID DNA stain from Enzo Life Sciences.

    Techniques: Mouse Assay, Electroporation

    Caspase-1 does not compete with ASC for interaction with NLRC4 in enzymatically inactive Casp1 C284A/C284A BMDMs. ( a ) Immunoblot of pro-caspase-1 and actin from wt, Casp1 −/− , Casp1 C284A/C284A BMDMs. ( b ) BMDMs with Pam3CSK4 (1 μg ml −1 ) pre-stimulation were electroporated with flagellin (0.5 μg ml −1 ). IL-1β release and LDH release measured after 16 h. Data is expressed as mean ± SD; n = 3. ( c ) % YOYO-1 positive BMDMs from two mice/genotype after stimulation. BMDMs with no pre-stimulation were electroporated with flagellin (0.5 μg ml −1 ). Live cell images taken every 45 min for 16 h.

    Journal: Scientific Reports

    Article Title: ASC- and caspase-8-dependent apoptotic pathway diverges from the NLRC4 inflammasome in macrophages

    doi: 10.1038/s41598-018-21998-3

    Figure Lengend Snippet: Caspase-1 does not compete with ASC for interaction with NLRC4 in enzymatically inactive Casp1 C284A/C284A BMDMs. ( a ) Immunoblot of pro-caspase-1 and actin from wt, Casp1 −/− , Casp1 C284A/C284A BMDMs. ( b ) BMDMs with Pam3CSK4 (1 μg ml −1 ) pre-stimulation were electroporated with flagellin (0.5 μg ml −1 ). IL-1β release and LDH release measured after 16 h. Data is expressed as mean ± SD; n = 3. ( c ) % YOYO-1 positive BMDMs from two mice/genotype after stimulation. BMDMs with no pre-stimulation were electroporated with flagellin (0.5 μg ml −1 ). Live cell images taken every 45 min for 16 h.

    Article Snippet: Ultra-pure flagellin (Psuedomonas aeruginosa ), Pam3CSK4, R837 (Imiquimod), ultra-pure LPS (E. coli O111:B4), Poly(dA:dT), Nigericin were purchased from Invivogen, Mega FasL from AdipoGen, IFN-α and IFN-β from PBL Assay Science, TNFα from Genentech, ATP and bovine cytochrome-c from Sigma, YOYO-1 dye from Thermofisher Scientific, Nuclear-ID DNA stain from Enzo Life Sciences.

    Techniques: Mouse Assay

    NLRC4 activates a caspase-1-independent cell death pathway in absence of TLR signaling. ( a – c ) BMDMs with or without Pam3CSK4 (1 μg ml −1 ) pre-stimulation were electroporated with flagellin (0.5 μg ml −1 ). ( a ) LDH release measured after 16 h. Data is represented as mean ± SD; n = 3. ( b ) % YOYO-1 positive BMDMs from two mice/genotype after flagellin electroporation. Live cell images taken every 30 min up to 16 h. ( c ) Transmission electron microscopy BMDMs 6 h after flagellin electroporation. Representative images captured at 1000× magnification or 2000× magnification. Black arrows (→) indicate free nuclei. White asterisks (*) indicate chromatin condensation.

    Journal: Scientific Reports

    Article Title: ASC- and caspase-8-dependent apoptotic pathway diverges from the NLRC4 inflammasome in macrophages

    doi: 10.1038/s41598-018-21998-3

    Figure Lengend Snippet: NLRC4 activates a caspase-1-independent cell death pathway in absence of TLR signaling. ( a – c ) BMDMs with or without Pam3CSK4 (1 μg ml −1 ) pre-stimulation were electroporated with flagellin (0.5 μg ml −1 ). ( a ) LDH release measured after 16 h. Data is represented as mean ± SD; n = 3. ( b ) % YOYO-1 positive BMDMs from two mice/genotype after flagellin electroporation. Live cell images taken every 30 min up to 16 h. ( c ) Transmission electron microscopy BMDMs 6 h after flagellin electroporation. Representative images captured at 1000× magnification or 2000× magnification. Black arrows (→) indicate free nuclei. White asterisks (*) indicate chromatin condensation.

    Article Snippet: Ultra-pure flagellin (Psuedomonas aeruginosa ), Pam3CSK4, R837 (Imiquimod), ultra-pure LPS (E. coli O111:B4), Poly(dA:dT), Nigericin were purchased from Invivogen, Mega FasL from AdipoGen, IFN-α and IFN-β from PBL Assay Science, TNFα from Genentech, ATP and bovine cytochrome-c from Sigma, YOYO-1 dye from Thermofisher Scientific, Nuclear-ID DNA stain from Enzo Life Sciences.

    Techniques: Mouse Assay, Electroporation, Transmission Assay, Electron Microscopy

    DFNA5 is dispensable for secondary necrosis after NLRC4/caspase-8-mediated cell death. ( a ) Immunoblot of mouse DFNA5 and actin from iMac whole cell lysates. ( b – e ) Casp1 −/− Casp11 −/− iMac expressing Dfna5 , Asc or luciferase (control) sgRNA were subjected to electroporation with flagellin (0.5 μg ml −1 ) and cytochrome-c (50 μg ml −1 ), or treated with FasL (100 ng ml −1 ). ( b ) and ( e ) LDH release after 16 h. Data is expressed as mean ± SD; n = 3. ( c ) and ( d ) % YOYO-1 positive iMacs from live cell imaging taken every hour for 16 h. Data is expressed as mean ± SD; n = 3. ( f ) Immunoblot for FLAG-tagged DFNA5 (FLAG antibody) and caspase-3 in combined cell extract and supernatant from FLAG-tagged DFNA5 expressing iMacs 3 hrs after electroporation with flagellin (0.5 μg) and cytochrome-c (25 μg), or treated with FasL (100 ng ml −1 ).

    Journal: Scientific Reports

    Article Title: ASC- and caspase-8-dependent apoptotic pathway diverges from the NLRC4 inflammasome in macrophages

    doi: 10.1038/s41598-018-21998-3

    Figure Lengend Snippet: DFNA5 is dispensable for secondary necrosis after NLRC4/caspase-8-mediated cell death. ( a ) Immunoblot of mouse DFNA5 and actin from iMac whole cell lysates. ( b – e ) Casp1 −/− Casp11 −/− iMac expressing Dfna5 , Asc or luciferase (control) sgRNA were subjected to electroporation with flagellin (0.5 μg ml −1 ) and cytochrome-c (50 μg ml −1 ), or treated with FasL (100 ng ml −1 ). ( b ) and ( e ) LDH release after 16 h. Data is expressed as mean ± SD; n = 3. ( c ) and ( d ) % YOYO-1 positive iMacs from live cell imaging taken every hour for 16 h. Data is expressed as mean ± SD; n = 3. ( f ) Immunoblot for FLAG-tagged DFNA5 (FLAG antibody) and caspase-3 in combined cell extract and supernatant from FLAG-tagged DFNA5 expressing iMacs 3 hrs after electroporation with flagellin (0.5 μg) and cytochrome-c (25 μg), or treated with FasL (100 ng ml −1 ).

    Article Snippet: Ultra-pure flagellin (Psuedomonas aeruginosa ), Pam3CSK4, R837 (Imiquimod), ultra-pure LPS (E. coli O111:B4), Poly(dA:dT), Nigericin were purchased from Invivogen, Mega FasL from AdipoGen, IFN-α and IFN-β from PBL Assay Science, TNFα from Genentech, ATP and bovine cytochrome-c from Sigma, YOYO-1 dye from Thermofisher Scientific, Nuclear-ID DNA stain from Enzo Life Sciences.

    Techniques: Expressing, Luciferase, Electroporation, Live Cell Imaging

    DNA intercalators tilt and twirl beyond the OST. ( A ) Polarized image pairs of a single YOYO-1 molecule bound to DNA oriented at ω = 45° in regime 3 alternately excited with x - and y -polarized light (scale bar, 500 nm). A single molecule is detected in image pairs 2 to 5. Illustration: When a dipole twirls, its mean azimuthal orientation with respect to the DNA axis changes. Thus, the dipole is only efficiently excited when it rotates through the axis of the excitation polarization. Hence, toggling between x / y -polarized excitation strongly biases the emission polarization, as seen in, e.g., polarized image pairs 3 and 4. ( B and C ) Polarization scatterplots of single YOYO-1 (B) and SYTOX Orange (C) dyes bound to DNA oriented at ω = 0° and ω = 45° in regime 3.

    Journal: Science Advances

    Article Title: Single-molecule polarization microscopy of DNA intercalators sheds light on the structure of S-DNA

    doi: 10.1126/sciadv.aav1083

    Figure Lengend Snippet: DNA intercalators tilt and twirl beyond the OST. ( A ) Polarized image pairs of a single YOYO-1 molecule bound to DNA oriented at ω = 45° in regime 3 alternately excited with x - and y -polarized light (scale bar, 500 nm). A single molecule is detected in image pairs 2 to 5. Illustration: When a dipole twirls, its mean azimuthal orientation with respect to the DNA axis changes. Thus, the dipole is only efficiently excited when it rotates through the axis of the excitation polarization. Hence, toggling between x / y -polarized excitation strongly biases the emission polarization, as seen in, e.g., polarized image pairs 3 and 4. ( B and C ) Polarization scatterplots of single YOYO-1 (B) and SYTOX Orange (C) dyes bound to DNA oriented at ω = 0° and ω = 45° in regime 3.

    Article Snippet: Both YOYO-1 and SYTOX Orange were purchased from Thermo Fisher Scientific.

    Techniques:

    Intercalated DNA fluorescence changes polarization after the OST. ( A ) Optical tweezers align and stretch a DNA molecule within the microscope image ( x / y ) plane. Polarized laser light excites intercalated dyes, and corresponding fluorescence is resolved into x - or y -polarized components using a polarizing beamsplitter. Dipoles of intercalated dyes are depicted as bidirectional arrows. ( B ) Typical force-extension curves for both bare DNA and DNA in the presence of ~10 nM YOYO-1. While the two curves differ slightly, in both cases, three distinct regimes can be distinguished: before the OST (regime 1), within the OST (regime 2), and beyond the OST (regime 3). ( C and D ) Fluorescence polarization images show DNA densely coated with YOYO-1, extended horizontally to regimes 2 and 3 [(C) and (D), respectively] (scale bar, 5 μm). Insets illustrate the possible intercalator dipole orientations (red) with respect to the DNA axis (green) that could induce the observed fluorescence polarization features. Gray cones depict wobble regions from which dipoles may depart from their mean orientations. Note that in the increased wobble hypothesis [upper inset of (D)], dipoles explore a greater range of orientations due to a wider wobble cone.

    Journal: Science Advances

    Article Title: Single-molecule polarization microscopy of DNA intercalators sheds light on the structure of S-DNA

    doi: 10.1126/sciadv.aav1083

    Figure Lengend Snippet: Intercalated DNA fluorescence changes polarization after the OST. ( A ) Optical tweezers align and stretch a DNA molecule within the microscope image ( x / y ) plane. Polarized laser light excites intercalated dyes, and corresponding fluorescence is resolved into x - or y -polarized components using a polarizing beamsplitter. Dipoles of intercalated dyes are depicted as bidirectional arrows. ( B ) Typical force-extension curves for both bare DNA and DNA in the presence of ~10 nM YOYO-1. While the two curves differ slightly, in both cases, three distinct regimes can be distinguished: before the OST (regime 1), within the OST (regime 2), and beyond the OST (regime 3). ( C and D ) Fluorescence polarization images show DNA densely coated with YOYO-1, extended horizontally to regimes 2 and 3 [(C) and (D), respectively] (scale bar, 5 μm). Insets illustrate the possible intercalator dipole orientations (red) with respect to the DNA axis (green) that could induce the observed fluorescence polarization features. Gray cones depict wobble regions from which dipoles may depart from their mean orientations. Note that in the increased wobble hypothesis [upper inset of (D)], dipoles explore a greater range of orientations due to a wider wobble cone.

    Article Snippet: Both YOYO-1 and SYTOX Orange were purchased from Thermo Fisher Scientific.

    Techniques: Fluorescence, Microscopy

    Intercalator tilting occurs suddenly at the transition between regimes 2 and 3. ( A ) Average emission polarization as a function of force above the OST; data taken at extensions within the transition from regime 2 to regime 3 (vertical bars are SD). Dashed green line indicates P = −0.04, the emission polarization associated with model parameters {θ = 53.9°, α = 29.8°} and unpolarized excitation. Prefixes are omitted, since unpolarized excitation was used. Inset illustrates the “elbow” in the force-extension curve. ( B ) Polarization images demonstrating simultaneous detection of tilted and perpendicular YOYO-1 molecules (force ~7 pN above OST). The dye appearing brightly in both I x and I y channels is tilted (scale bar, 5 μm). ( C to E ) Single-intercalator polarization histograms at forces of 3 pN (C), 7 pN (D), and 35 pN (E) above the onset of the OST. Illustrations depict the proposed mechanism for intercalator tilting: In regime 2 (C), extended tracts of both B-DNA and S-DNA exist, and intercalated sections are primarily flanked by B-DNA and align perpendicular to the DNA axis. At the interface between regimes 2 and 3 (D), where less B-DNA is present, both perpendicular and tilted intercalators are present, flanked by B-DNA and S-DNA, respectively. Beyond the OST (E), when little or no B-DNA remains, all intercalated dipoles are forced to assume a tilted orientation, since they can now only be flanked by S-DNA.

    Journal: Science Advances

    Article Title: Single-molecule polarization microscopy of DNA intercalators sheds light on the structure of S-DNA

    doi: 10.1126/sciadv.aav1083

    Figure Lengend Snippet: Intercalator tilting occurs suddenly at the transition between regimes 2 and 3. ( A ) Average emission polarization as a function of force above the OST; data taken at extensions within the transition from regime 2 to regime 3 (vertical bars are SD). Dashed green line indicates P = −0.04, the emission polarization associated with model parameters {θ = 53.9°, α = 29.8°} and unpolarized excitation. Prefixes are omitted, since unpolarized excitation was used. Inset illustrates the “elbow” in the force-extension curve. ( B ) Polarization images demonstrating simultaneous detection of tilted and perpendicular YOYO-1 molecules (force ~7 pN above OST). The dye appearing brightly in both I x and I y channels is tilted (scale bar, 5 μm). ( C to E ) Single-intercalator polarization histograms at forces of 3 pN (C), 7 pN (D), and 35 pN (E) above the onset of the OST. Illustrations depict the proposed mechanism for intercalator tilting: In regime 2 (C), extended tracts of both B-DNA and S-DNA exist, and intercalated sections are primarily flanked by B-DNA and align perpendicular to the DNA axis. At the interface between regimes 2 and 3 (D), where less B-DNA is present, both perpendicular and tilted intercalators are present, flanked by B-DNA and S-DNA, respectively. Beyond the OST (E), when little or no B-DNA remains, all intercalated dipoles are forced to assume a tilted orientation, since they can now only be flanked by S-DNA.

    Article Snippet: Both YOYO-1 and SYTOX Orange were purchased from Thermo Fisher Scientific.

    Techniques:

    Mechanical properties of intercalated DNA. a Resonance mode ratio evaluated on bare DNA resonators and DNA intercalated with YOYO-1, GelRed, and CisPt. Dashed lines represent the theoretical value of the ratio between the resonance modes for a clamped–clamped beam without stress. b Frequency of the fundamental resonance mode as a function of the ratio of the bundle radius over the square of the length. The squares correspond to the experimental data and the lines to the linear fit. c Effective Young’s modulus of the four different types of DNA resonators. Error bars were computed from the standard deviation of 25 measurements. d Effective Young’s modulus of DNA bundles prepared with different concentrations of CisPt. The 1× concentration is the sample reported in the other panels. e Splitting of the first flexural vibration mode observed in vacuum, due to the asymmetry in the bundle section geometry. The spectra are centered with respect to the average of the two flexural modes. f Relative frequency splitting of the flexural vibration mode of the four different types of DNA resonators. Error bars were computed from the standard deviation of 10 measurements. Source data are provided as a Source Data file

    Journal: Nature Communications

    Article Title: Nanomechanical DNA resonators for sensing and structural analysis of DNA-ligand complexes

    doi: 10.1038/s41467-019-09612-0

    Figure Lengend Snippet: Mechanical properties of intercalated DNA. a Resonance mode ratio evaluated on bare DNA resonators and DNA intercalated with YOYO-1, GelRed, and CisPt. Dashed lines represent the theoretical value of the ratio between the resonance modes for a clamped–clamped beam without stress. b Frequency of the fundamental resonance mode as a function of the ratio of the bundle radius over the square of the length. The squares correspond to the experimental data and the lines to the linear fit. c Effective Young’s modulus of the four different types of DNA resonators. Error bars were computed from the standard deviation of 25 measurements. d Effective Young’s modulus of DNA bundles prepared with different concentrations of CisPt. The 1× concentration is the sample reported in the other panels. e Splitting of the first flexural vibration mode observed in vacuum, due to the asymmetry in the bundle section geometry. The spectra are centered with respect to the average of the two flexural modes. f Relative frequency splitting of the flexural vibration mode of the four different types of DNA resonators. Error bars were computed from the standard deviation of 10 measurements. Source data are provided as a Source Data file

    Article Snippet: λ-DNA (48502 bp sequence available at a producer’s website, New England Biolabs, Ipswich, MA, USA) was preheated at 65 ˚C for 10 min before the incubation with a saturating amount of GelRed (Biotium, Hayward, CA, USA) or YOYO-1 (ThermoFisher, Waltham, MA, USA), MW: 1.271 g/mol, for 2 h at the constant temperature of 30 ˚C.

    Techniques: Standard Deviation, Concentration Assay

    SEM and fluorescence microscope images of intercalated DNA. After the complete evaporation of the solvent ( a ), DNA bundles can be imaged by SEM: they are homogeneously suspended and distributed between silicon micro-pillars. In ( b ), a particular of a suspended DNA bundle, covering the pillar–pillar distance of 12 μm. DNA/intercalator optical images acquired using an excitation of ( c ) 540/25 nm (DM 565, BA 605/55) for the GelRed evaluation and of ( d ) 465–495 nm as in the case of YOYO-1. The scale bars correspond to 10 μm, except for ( b ) which corresponds to 1 μm

    Journal: Nature Communications

    Article Title: Nanomechanical DNA resonators for sensing and structural analysis of DNA-ligand complexes

    doi: 10.1038/s41467-019-09612-0

    Figure Lengend Snippet: SEM and fluorescence microscope images of intercalated DNA. After the complete evaporation of the solvent ( a ), DNA bundles can be imaged by SEM: they are homogeneously suspended and distributed between silicon micro-pillars. In ( b ), a particular of a suspended DNA bundle, covering the pillar–pillar distance of 12 μm. DNA/intercalator optical images acquired using an excitation of ( c ) 540/25 nm (DM 565, BA 605/55) for the GelRed evaluation and of ( d ) 465–495 nm as in the case of YOYO-1. The scale bars correspond to 10 μm, except for ( b ) which corresponds to 1 μm

    Article Snippet: λ-DNA (48502 bp sequence available at a producer’s website, New England Biolabs, Ipswich, MA, USA) was preheated at 65 ˚C for 10 min before the incubation with a saturating amount of GelRed (Biotium, Hayward, CA, USA) or YOYO-1 (ThermoFisher, Waltham, MA, USA), MW: 1.271 g/mol, for 2 h at the constant temperature of 30 ˚C.

    Techniques: Fluorescence, Microscopy, Evaporation

    SMD simulations of pristine DNA and DNA intercalated with YOYO-1 and CisPt. a Simulated conformational structures of the bare DNA (top), DNA intercalated with YOYO-1 (center), and CisPt (bottom) under uniaxial stretching deformation. In each figure, the first corresponds to the initial structure, while the second represents the final conformation reached at the maximum simulated strain. b Stress–strain curve of the unidirectional traction applied to the DNA (blue), DNA/YOYO-1 (green), and DNA/CisPt (orange) systems. The Young’s moduli ( E SMD ) for each complex are calculated from the slope of the linear fitting. The strain at each force has been averaged over three simulations and the corresponding standard errors are reported. c Calculated Young’s modulus ( E SMD ) values for DNA (blue), DNA/YOYO-1 (green), and DNA–CisPt (orange) complexes. d Change in the number of hydrogen bonds of DNA (blue), DNA/YOYO-1 (green), and DNA/CisPt (orange) systems during the simulation time applying the maximum stress value. Source data are provided as a Source Data file

    Journal: Nature Communications

    Article Title: Nanomechanical DNA resonators for sensing and structural analysis of DNA-ligand complexes

    doi: 10.1038/s41467-019-09612-0

    Figure Lengend Snippet: SMD simulations of pristine DNA and DNA intercalated with YOYO-1 and CisPt. a Simulated conformational structures of the bare DNA (top), DNA intercalated with YOYO-1 (center), and CisPt (bottom) under uniaxial stretching deformation. In each figure, the first corresponds to the initial structure, while the second represents the final conformation reached at the maximum simulated strain. b Stress–strain curve of the unidirectional traction applied to the DNA (blue), DNA/YOYO-1 (green), and DNA/CisPt (orange) systems. The Young’s moduli ( E SMD ) for each complex are calculated from the slope of the linear fitting. The strain at each force has been averaged over three simulations and the corresponding standard errors are reported. c Calculated Young’s modulus ( E SMD ) values for DNA (blue), DNA/YOYO-1 (green), and DNA–CisPt (orange) complexes. d Change in the number of hydrogen bonds of DNA (blue), DNA/YOYO-1 (green), and DNA/CisPt (orange) systems during the simulation time applying the maximum stress value. Source data are provided as a Source Data file

    Article Snippet: λ-DNA (48502 bp sequence available at a producer’s website, New England Biolabs, Ipswich, MA, USA) was preheated at 65 ˚C for 10 min before the incubation with a saturating amount of GelRed (Biotium, Hayward, CA, USA) or YOYO-1 (ThermoFisher, Waltham, MA, USA), MW: 1.271 g/mol, for 2 h at the constant temperature of 30 ˚C.

    Techniques: