edta  (Thermo Fisher)


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  • 97
    Name:
    EDTA
    Description:
    Thermo Scientific Pierce EDTA is useful as a chelator of alkaline earth metals as well as iron copper and zinc in a variety of laboratory methods
    Catalog Number:
    17892
    Price:
    None
    Category:
    Lab Reagents and Chemicals
    Applications:
    DNA & RNA Purification & Analysis
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    Structured Review

    Thermo Fisher edta
    Conductance measurements through dipeptide-treated nanopores as a function of pH change (DOPA-Lys: purple, DOPA-His: orange, and DOPA-Glu: green). Measurements were performed in 0.14 M <t>KCl,</t> 10 mM Tris-HCl/succinic acid, 1 mM <t>EDTA</t> (pH 4.5, 6, 7.5, and 9) at 100 mV. In some of the points, the error bars are smaller than the marking.
    Thermo Scientific Pierce EDTA is useful as a chelator of alkaline earth metals as well as iron copper and zinc in a variety of laboratory methods
    https://www.bioz.com/result/edta/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
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    Images

    1) Product Images from "Durable, Stable, and Functional Nanopores Decorated by Self-Assembled Dipeptides"

    Article Title: Durable, Stable, and Functional Nanopores Decorated by Self-Assembled Dipeptides

    Journal: ACS Applied Materials & Interfaces

    doi: 10.1021/acsami.0c00062

    Conductance measurements through dipeptide-treated nanopores as a function of pH change (DOPA-Lys: purple, DOPA-His: orange, and DOPA-Glu: green). Measurements were performed in 0.14 M KCl, 10 mM Tris-HCl/succinic acid, 1 mM EDTA (pH 4.5, 6, 7.5, and 9) at 100 mV. In some of the points, the error bars are smaller than the marking.
    Figure Legend Snippet: Conductance measurements through dipeptide-treated nanopores as a function of pH change (DOPA-Lys: purple, DOPA-His: orange, and DOPA-Glu: green). Measurements were performed in 0.14 M KCl, 10 mM Tris-HCl/succinic acid, 1 mM EDTA (pH 4.5, 6, 7.5, and 9) at 100 mV. In some of the points, the error bars are smaller than the marking.

    Techniques Used:

    (a) Currents through representative uncoated (red) and DOPA-His dipeptide-coated (blue) 12 nm diameter nanopores at 100 mV. Currents through each nanopore were measured repeatedly five times in 1 M KCl, 10 mM Tris-HCl, 1 mM EDTA (pH 7.5). After each measurement, the chambers were cleaned with water and the solution was refilled. The graph presents the average of these five measurements. Note the much larger error bar for the uncoated nanopore. (b) Similar currents were measured [under the same conditions as in (a)] through a single nanopore along several months without any further treatment.
    Figure Legend Snippet: (a) Currents through representative uncoated (red) and DOPA-His dipeptide-coated (blue) 12 nm diameter nanopores at 100 mV. Currents through each nanopore were measured repeatedly five times in 1 M KCl, 10 mM Tris-HCl, 1 mM EDTA (pH 7.5). After each measurement, the chambers were cleaned with water and the solution was refilled. The graph presents the average of these five measurements. Note the much larger error bar for the uncoated nanopore. (b) Similar currents were measured [under the same conditions as in (a)] through a single nanopore along several months without any further treatment.

    Techniques Used:

    dsDNA translocation time through 10 nm untreated and DOPA-His-coated nanopores. The measurements were done at 1 M KCl, 10 mM Tris-HCl, 1 mM EDTA, and pH 7.5, 200 mV, pore size 10 nm. (a) Translocation dwell time histogram for 2 kbp DNA (left) and an example of 2 kbp DNA translocation events (right), through DOPA-His-coated and uncoated nanopores. (b) Translocation dwell time histogram for 48 kbp DNA (left) and an example of 48 kbp DNA translocation events (right) through DOPA-His-coated and uncoated nanopore.
    Figure Legend Snippet: dsDNA translocation time through 10 nm untreated and DOPA-His-coated nanopores. The measurements were done at 1 M KCl, 10 mM Tris-HCl, 1 mM EDTA, and pH 7.5, 200 mV, pore size 10 nm. (a) Translocation dwell time histogram for 2 kbp DNA (left) and an example of 2 kbp DNA translocation events (right), through DOPA-His-coated and uncoated nanopores. (b) Translocation dwell time histogram for 48 kbp DNA (left) and an example of 48 kbp DNA translocation events (right) through DOPA-His-coated and uncoated nanopore.

    Techniques Used: Translocation Assay

    Related Articles

    Incubation:

    Article Title: Alteration of a Single Hydrogen Bond between Class II Molecules and Peptide Results in Rapid Degradation of Class II Molecules after Invariant Chain Removal
    Article Snippet: .. CHO cells were treated similarly, but before protease treatment they were maintained adherent in tissue culture dishes, and harvested immediately before enzyme treatment by brief incubation with EDTA (Versene; GIBCO BRL ). ..

    Titration:

    Article Title: Drawbacks of Dialysis Procedures for Removal of EDTA
    Article Snippet: .. A calibration curve was obtained by titration of samples containing 100 μM PAR and 10 μM Zn with known concentrations of EDTA and measuring the absorbance at 492 nm, using a Varioskan Flash (Thermo) microplate reader. .. The amount of EDTA in the protein preparations was determined from their absorbance at 492 nm after incubation with 100 μM PAR and 10 μM Zn, and extrapolation using the calibration curve.

    Confocal Microscopy:

    Article Title: Mesenchymal Stromal Cells Express GARP/LRRC32 on Their Surface: Effects on Their Biology and Immunomodulatory Capacity
    Article Snippet: Protein was detected using an Odyssey Image scanner system (LI-COR Biosciences). .. Confocal Microscopy For LAP/GARP costaining, mASCs were harvested with EDTA as described above and stained with GARP-PE (YGIC86; eBioscience) and purified anti-mouse LAP/TGF-β1 (TW7–16B4) followed by a donkey anti-mouse IgG-Alexa488 (Molecular Probes). .. For phospho-Smad2/3 staining, NT, CTRL-LV, LV#3, and LV#6 mASCs were plated in Lab-Tek II cc2 eight-well chamber slides (Nalgene, Thermo Fisher Scientific, Waltham, MA, http://www.thermofisher.com ) at 5,000 cells/well and cultured in MesenCult supplemented with 0.2% FCS for 2 days.

    Staining:

    Article Title: Mesenchymal Stromal Cells Express GARP/LRRC32 on Their Surface: Effects on Their Biology and Immunomodulatory Capacity
    Article Snippet: Protein was detected using an Odyssey Image scanner system (LI-COR Biosciences). .. Confocal Microscopy For LAP/GARP costaining, mASCs were harvested with EDTA as described above and stained with GARP-PE (YGIC86; eBioscience) and purified anti-mouse LAP/TGF-β1 (TW7–16B4) followed by a donkey anti-mouse IgG-Alexa488 (Molecular Probes). .. For phospho-Smad2/3 staining, NT, CTRL-LV, LV#3, and LV#6 mASCs were plated in Lab-Tek II cc2 eight-well chamber slides (Nalgene, Thermo Fisher Scientific, Waltham, MA, http://www.thermofisher.com ) at 5,000 cells/well and cultured in MesenCult supplemented with 0.2% FCS for 2 days.

    Purification:

    Article Title: Mesenchymal Stromal Cells Express GARP/LRRC32 on Their Surface: Effects on Their Biology and Immunomodulatory Capacity
    Article Snippet: Protein was detected using an Odyssey Image scanner system (LI-COR Biosciences). .. Confocal Microscopy For LAP/GARP costaining, mASCs were harvested with EDTA as described above and stained with GARP-PE (YGIC86; eBioscience) and purified anti-mouse LAP/TGF-β1 (TW7–16B4) followed by a donkey anti-mouse IgG-Alexa488 (Molecular Probes). .. For phospho-Smad2/3 staining, NT, CTRL-LV, LV#3, and LV#6 mASCs were plated in Lab-Tek II cc2 eight-well chamber slides (Nalgene, Thermo Fisher Scientific, Waltham, MA, http://www.thermofisher.com ) at 5,000 cells/well and cultured in MesenCult supplemented with 0.2% FCS for 2 days.

    Article Title: A type IV modification-dependent restriction enzyme SauUSI from Staphylococcus aureus subsp. aureus USA300
    Article Snippet: The protein was diluted in NEB Diluent A buffer and stored at −20°C. .. To remove the divalent metal ions bound by the purified SauUSI, the enzyme was supplemented with 20 mM EDTA and then dialyzed against a buffer (50 mM NaCl, 20 mM Tris–HCl, pH 8, 1 mM DTT) for 48 h at 4°C using a dialysis cassette (10 000 Da MWCO; Thermo Scientific Pierce). .. For batch purification of wt SauUSI and variants using chitin beads, 10 ml of cells were cultured to late log phase at 37°C.

    Lambda DNA Preparation:

    Article Title: Durable, Stable, and Functional Nanopores Decorated by Self-Assembled Dipeptides
    Article Snippet: .. Linear double-stranded DNA (2 kb NoLimits DNA Fragment and 48 kb Lambda DNA (Thermo Fisher Scientific)) translocation experiments were done with 1 M KCl, 10 mM Tris-HCl, 1 mM EDTA, 10% glycerol (pH 7.5). .. A pair of Ag/AgCl pellet electrodes were immersed in the two reservoirs and connected to an Axopatch 200B amplifier (Molecular Devices, Inc.) to record ionic current flow through the nanopore.

    Translocation Assay:

    Article Title: Durable, Stable, and Functional Nanopores Decorated by Self-Assembled Dipeptides
    Article Snippet: .. Linear double-stranded DNA (2 kb NoLimits DNA Fragment and 48 kb Lambda DNA (Thermo Fisher Scientific)) translocation experiments were done with 1 M KCl, 10 mM Tris-HCl, 1 mM EDTA, 10% glycerol (pH 7.5). .. A pair of Ag/AgCl pellet electrodes were immersed in the two reservoirs and connected to an Axopatch 200B amplifier (Molecular Devices, Inc.) to record ionic current flow through the nanopore.

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    Thermo Fisher cell impermeant
    Quantification of tumor spheroid acidification. A–C : > 1-wk-old U251 human glioma spheroids were preincubated and then placed for confocal imaging in pH 6.0, 7.4, and 8.8 baths with 20 μM <t>cell-impermeant</t> seminaphtharhodafluor-5F
    Cell Impermeant, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher u251
    Projection electrophoresis permits the simultaneous analysis of hundreds of single cells by concurrent separation after simultaneous lysis. ( a ) Maximum intensity projection 3D renderings of example separation lanes read out by tiled light-sheet microscopy. ( b ) xz ( y -summed) contour plots of background-subtracted actinin and GAPDH protein signal within the lanes depicted in ( a ). ( c ) Revolved z -intensity profiles (arbitrary fluorescence units, AFU, vs. z ) for the four separation lanes depicted in ( a , b ). Each plot depicts z -directional intensity profiles (summed fluorescence within each xy ROI vs. z ) for GAPDH (magenta) and actinin (cyan), revolved around the z -axis to generate 3D rendering of fluorescence distribution. ( d ) Histogram quantification shows 86% of <t>U251</t> cells lyse within 5 s of initiating lysis. ( e ) Quantified fluorescence intensity data for n = 159 separation lanes passing quality control for both the GAPDH and actinin channels. Each plot depicts revolved z -intensity profiles. ( f ) Full-gel wide-field fluorescence image of calcein-stained live BT474 breast tumor cells before analysis. ( g ) Subset of the live cells from ( f ), within a 1.75 × 1.75 mm light-sheet microscopy field of view (scale bar depicts 200 μm). ( h ) Post-separation wide-field fluorescence image of probed GAPDH signal within the same field of view as ( g ). ( f – h ) are representative of n > 3 separation gels. ( i ) Maximum intensity projection 3D rendering (representative of duplicate separation gels) of a light-sheet microscopy image (same field of view as ( g , h )), showing 3D separations of GAPDH and actinin from tens of separation lanes, each corresponding to signal from the settled cells in microwells depicted in ( f , g ). ( j ) Overlay image of segmented spots corresponding to live BT474 cells in microwells (green) and probed GAPDH bands after separation (magenta), for the same separation gel depicted in ( f , i ). ( k ) Quantification of correspondence between the segmented live cells and bands (via intensity thresholding) within the same separation lanes as ( j ). ( l ) Quantified migration distances from a total of n = 507 (GAPDH) and n = 303 (actinin) lanes passing quality control in two projection electrophoresis gels. ( m ) Quantified z -direction peak widths for the same bands analyzed in ( l ). (n-o) Map of the variation in GAPDH ( n ) and actinin ( o ) electromigration distances across the xy gel area. Source data are provided as a Source Data file.
    U251, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher pc14 pe6 pgf1 br4 cells
    Nintedanib and anti-VEGF/Ang2 nanobody prevent brain metastases formation. 9.4 T MRI after Gadolinium contrast administration was performed on day 26 after intracardial tumor cell injection. a Bar chart illustrating the reduced percentage of mice with metastases, detectable in cMRI. b Representative T1-w cMRI images depicting larger intracranial metastases in the control groups, indicated by arrowheads. scale bar = 2 mm. c Scatter plots showing the reduction of number and volume of cranial metastases in cMRI. d Scatter plots depicting the number and volume of meningeal metastases in cMRI e Quantification demonstrating a higher histological tumor–tissue ratio in control mice at their time of death. f Representative histological slices with higher number and size of <t>PC14-PE6</t> <t>pGF1</t> <t>Br4</t> metastases in the control group. Fluorescent staining: DAPI (blue) = nucleus, GFP (green) = PC14-PE6 tumor cells, Alexa Flour 546 (orange) = collagen-IV positive vascular basement membrane. Arrows indicate GFP-positive metastatic lesions. Scale bar = 1000 µm. Mean values with standard errors of the mean are shown. *p
    Pc14 Pe6 Pgf1 Br4 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher trypsin edta
    Membrane binding of the key PNN component aggrecan is biochemically altered in <t>Ptprz1</t> KO and Tnr KO mice brains. Brain homogenates were treated with ChABC to remove the hyaluronan backbone of PNNs or chondroitinase in the presence of <t>EDTA</t> (ChABC EDTA) and centrifuged to obtain soluble release ( R ) and insoluble pellet ( P ) fractions. A , Western blotting image showing release of PNN marker aggrecan into soluble phase from brain homogenates of WT, Ptprz1 KO, and Tnr KO mice by ChABC treatment alone and ChABC EDTA treatment. The release of aggrecan into the soluble fraction required ChABC treatment in addition with EDTA in WT brain homogenates. Aggrecan was released more readily with just ChABC treatment in Ptprz1 KO and Tnr KO animals. B , quantification showing ratio of the soluble release fraction ( R ) to total aggrecan levels (soluble release (R) + insoluble pellet (P)) in WT, Ptprz1 KO, and Tnr KO mice. There was a statistically significant difference in the release of aggrecan among the three genotypes as determined by one-way ANOVA (F(2,7) = 14.94, p = 0.0030). A Tukey's post hoc test showed that aggrecan was released much more readily in Ptprz1 KO mice (42 ± 0.04%, p = 0.0340) as well as in Tnr KO mice (58 ± 13%, p = 0.0024) compared with WT mice (15 ± 9%) when treated with just ChABC. There was no significant difference in aggrecan release between Ptprz1 KO and Tnr KO brains. Treatment with ChABC alongside EDTA led to almost complete release of aggrecan into the soluble release fraction in all genotypes. The ratio of release to total in Ptprz1 KO, Tnr KO and WT mice was not significantly different among the genotypes in the ChABC with EDTA treatment group. B , bars in graphs represent percentage release ± S.D.
    Trypsin Edta, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Quantification of tumor spheroid acidification. A–C : > 1-wk-old U251 human glioma spheroids were preincubated and then placed for confocal imaging in pH 6.0, 7.4, and 8.8 baths with 20 μM cell-impermeant seminaphtharhodafluor-5F

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Autocrine regulation of glioma cell proliferation via pHe-sensitive K+ channels

    doi: 10.1152/ajpcell.00097.2013

    Figure Lengend Snippet: Quantification of tumor spheroid acidification. A–C : > 1-wk-old U251 human glioma spheroids were preincubated and then placed for confocal imaging in pH 6.0, 7.4, and 8.8 baths with 20 μM cell-impermeant seminaphtharhodafluor-5F

    Article Snippet: Spheroids > 1 wk old were preincubated in sulfate- and phosphate-free (SPF) pH 6.0, 7.4, and 8.8 baths (for composition, see Electrophysiology ) for 2 h at 37°C and then placed in a solution with the cell-impermeant pH indicator dye seminaphtharhodafluor-5F 5-(and 6-)carboxylic acid (SNARF-5F, 20 μM; catalog no. S-23922, Invitrogen) on the stage of a confocal microscope (model FV1000, Olympus) with 405-, 473-, and 559-nm diode lasers.

    Techniques: Imaging

    Projection electrophoresis permits the simultaneous analysis of hundreds of single cells by concurrent separation after simultaneous lysis. ( a ) Maximum intensity projection 3D renderings of example separation lanes read out by tiled light-sheet microscopy. ( b ) xz ( y -summed) contour plots of background-subtracted actinin and GAPDH protein signal within the lanes depicted in ( a ). ( c ) Revolved z -intensity profiles (arbitrary fluorescence units, AFU, vs. z ) for the four separation lanes depicted in ( a , b ). Each plot depicts z -directional intensity profiles (summed fluorescence within each xy ROI vs. z ) for GAPDH (magenta) and actinin (cyan), revolved around the z -axis to generate 3D rendering of fluorescence distribution. ( d ) Histogram quantification shows 86% of U251 cells lyse within 5 s of initiating lysis. ( e ) Quantified fluorescence intensity data for n = 159 separation lanes passing quality control for both the GAPDH and actinin channels. Each plot depicts revolved z -intensity profiles. ( f ) Full-gel wide-field fluorescence image of calcein-stained live BT474 breast tumor cells before analysis. ( g ) Subset of the live cells from ( f ), within a 1.75 × 1.75 mm light-sheet microscopy field of view (scale bar depicts 200 μm). ( h ) Post-separation wide-field fluorescence image of probed GAPDH signal within the same field of view as ( g ). ( f – h ) are representative of n > 3 separation gels. ( i ) Maximum intensity projection 3D rendering (representative of duplicate separation gels) of a light-sheet microscopy image (same field of view as ( g , h )), showing 3D separations of GAPDH and actinin from tens of separation lanes, each corresponding to signal from the settled cells in microwells depicted in ( f , g ). ( j ) Overlay image of segmented spots corresponding to live BT474 cells in microwells (green) and probed GAPDH bands after separation (magenta), for the same separation gel depicted in ( f , i ). ( k ) Quantification of correspondence between the segmented live cells and bands (via intensity thresholding) within the same separation lanes as ( j ). ( l ) Quantified migration distances from a total of n = 507 (GAPDH) and n = 303 (actinin) lanes passing quality control in two projection electrophoresis gels. ( m ) Quantified z -direction peak widths for the same bands analyzed in ( l ). (n-o) Map of the variation in GAPDH ( n ) and actinin ( o ) electromigration distances across the xy gel area. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: 3D projection electrophoresis for single-cell immunoblotting

    doi: 10.1038/s41467-020-19738-1

    Figure Lengend Snippet: Projection electrophoresis permits the simultaneous analysis of hundreds of single cells by concurrent separation after simultaneous lysis. ( a ) Maximum intensity projection 3D renderings of example separation lanes read out by tiled light-sheet microscopy. ( b ) xz ( y -summed) contour plots of background-subtracted actinin and GAPDH protein signal within the lanes depicted in ( a ). ( c ) Revolved z -intensity profiles (arbitrary fluorescence units, AFU, vs. z ) for the four separation lanes depicted in ( a , b ). Each plot depicts z -directional intensity profiles (summed fluorescence within each xy ROI vs. z ) for GAPDH (magenta) and actinin (cyan), revolved around the z -axis to generate 3D rendering of fluorescence distribution. ( d ) Histogram quantification shows 86% of U251 cells lyse within 5 s of initiating lysis. ( e ) Quantified fluorescence intensity data for n = 159 separation lanes passing quality control for both the GAPDH and actinin channels. Each plot depicts revolved z -intensity profiles. ( f ) Full-gel wide-field fluorescence image of calcein-stained live BT474 breast tumor cells before analysis. ( g ) Subset of the live cells from ( f ), within a 1.75 × 1.75 mm light-sheet microscopy field of view (scale bar depicts 200 μm). ( h ) Post-separation wide-field fluorescence image of probed GAPDH signal within the same field of view as ( g ). ( f – h ) are representative of n > 3 separation gels. ( i ) Maximum intensity projection 3D rendering (representative of duplicate separation gels) of a light-sheet microscopy image (same field of view as ( g , h )), showing 3D separations of GAPDH and actinin from tens of separation lanes, each corresponding to signal from the settled cells in microwells depicted in ( f , g ). ( j ) Overlay image of segmented spots corresponding to live BT474 cells in microwells (green) and probed GAPDH bands after separation (magenta), for the same separation gel depicted in ( f , i ). ( k ) Quantification of correspondence between the segmented live cells and bands (via intensity thresholding) within the same separation lanes as ( j ). ( l ) Quantified migration distances from a total of n = 507 (GAPDH) and n = 303 (actinin) lanes passing quality control in two projection electrophoresis gels. ( m ) Quantified z -direction peak widths for the same bands analyzed in ( l ). (n-o) Map of the variation in GAPDH ( n ) and actinin ( o ) electromigration distances across the xy gel area. Source data are provided as a Source Data file.

    Article Snippet: Single-cell separations Adherent U251 glioblastoma and BT474 breast tumor cells were detached from culture flasks with 0.05% Trypsin-EDTA (Life Technologies 25300120) (U251) or 5 mM EDTA (Invitrogen 15575-038) in PBS (BT474) and resuspended in cold PBS (Life Technologies 10010049) at a concentration of 1.5 × 106 cells/mL.

    Techniques: Electrophoresis, Lysis, Microscopy, Fluorescence, Staining, Migration

    Design and verification of sample preparation for projection electrophoresis of single mammalian cells. ( a ) High-density endogenous protein bands (ii) correspond to single-cell settling in microwells (i). Scale bars represent 1 mm (left full-gel images) and 200 μm (right zoom images). ( b ) Illustration of top-view and side-view geometries shown in protein dilution studies ( c , d ). ( c ) Modeling and experimental quantification of diffusional dilution during lysis. Simulated and experimental top-view images of diffusional protein dilution during lysis, and side-view simulated results are shown. The simulated initial TurboGFP concentration was 2 μM. Experimental image is representative of 12 monitored cells across 3 independent lysis experiments. Scale bars represent 50 μm. ( d ) Modeling the impact of diffusion during electrophoresis on detectable in-gel protein concentration. Side and top view TurboGFP concentration profiles are shown at different times during electrophoresis. Simulated initial TurboGFP concentration (before lysis and electrophoresis) was 2 μM. Scale bars represent 30 μm. ( e ) Quantification of the percent of protein remaining in the microwell region during lysis (experiment plots mean and standard deviation of n = 12 cells across 3 lysis experiments). ( f ) Quantification of the change in the spatial maximum protein concentration as a function of time after protein solubilization (experiment plots mean and standard deviation of n = 12 cells across 3 lysis experiments). ( g ) Simulated maximum protein concentration vs. electrophoresis time, for 3 model proteins. ( h ) Representative β-tubulin separations from U251 glioblastoma cells lysed with different buffer formulations (2× RIPA lysis buffer and 2× RIPA including 8 M urea), both after 10 s electrophoresis. Lysis/EP buffer requires 8 M urea for fast protein solubilization and electromigration. ( i ) Maximum intensity projection 3D renderings and z -intensity profiles of probed GAPDH bands from single BT474 breast tumor cells. ( j ) Microwell packing density (impacting assay throughput) is dependent on protein band diffusion before photocapture. Protein diffusion profiles confirm that a microwell pitch of 200 μm is sufficient to resolve bands from neighboring microwells. After 10 s EP, the mean peak width ( σ xy ) of the xy GAPDH spots is 32 ± 11 μm (mean ± standard deviation of n = 47 single-cell separation lanes across 5 replicate separation gels). The depicted confocal slice micrograph is representative of 20 confocal image stacks, across different regions of 5 replicate separation gels. At a microwell pitch of 192 μm (6 σ xy ),

    Journal: Nature Communications

    Article Title: 3D projection electrophoresis for single-cell immunoblotting

    doi: 10.1038/s41467-020-19738-1

    Figure Lengend Snippet: Design and verification of sample preparation for projection electrophoresis of single mammalian cells. ( a ) High-density endogenous protein bands (ii) correspond to single-cell settling in microwells (i). Scale bars represent 1 mm (left full-gel images) and 200 μm (right zoom images). ( b ) Illustration of top-view and side-view geometries shown in protein dilution studies ( c , d ). ( c ) Modeling and experimental quantification of diffusional dilution during lysis. Simulated and experimental top-view images of diffusional protein dilution during lysis, and side-view simulated results are shown. The simulated initial TurboGFP concentration was 2 μM. Experimental image is representative of 12 monitored cells across 3 independent lysis experiments. Scale bars represent 50 μm. ( d ) Modeling the impact of diffusion during electrophoresis on detectable in-gel protein concentration. Side and top view TurboGFP concentration profiles are shown at different times during electrophoresis. Simulated initial TurboGFP concentration (before lysis and electrophoresis) was 2 μM. Scale bars represent 30 μm. ( e ) Quantification of the percent of protein remaining in the microwell region during lysis (experiment plots mean and standard deviation of n = 12 cells across 3 lysis experiments). ( f ) Quantification of the change in the spatial maximum protein concentration as a function of time after protein solubilization (experiment plots mean and standard deviation of n = 12 cells across 3 lysis experiments). ( g ) Simulated maximum protein concentration vs. electrophoresis time, for 3 model proteins. ( h ) Representative β-tubulin separations from U251 glioblastoma cells lysed with different buffer formulations (2× RIPA lysis buffer and 2× RIPA including 8 M urea), both after 10 s electrophoresis. Lysis/EP buffer requires 8 M urea for fast protein solubilization and electromigration. ( i ) Maximum intensity projection 3D renderings and z -intensity profiles of probed GAPDH bands from single BT474 breast tumor cells. ( j ) Microwell packing density (impacting assay throughput) is dependent on protein band diffusion before photocapture. Protein diffusion profiles confirm that a microwell pitch of 200 μm is sufficient to resolve bands from neighboring microwells. After 10 s EP, the mean peak width ( σ xy ) of the xy GAPDH spots is 32 ± 11 μm (mean ± standard deviation of n = 47 single-cell separation lanes across 5 replicate separation gels). The depicted confocal slice micrograph is representative of 20 confocal image stacks, across different regions of 5 replicate separation gels. At a microwell pitch of 192 μm (6 σ xy ),

    Article Snippet: Single-cell separations Adherent U251 glioblastoma and BT474 breast tumor cells were detached from culture flasks with 0.05% Trypsin-EDTA (Life Technologies 25300120) (U251) or 5 mM EDTA (Invitrogen 15575-038) in PBS (BT474) and resuspended in cold PBS (Life Technologies 10010049) at a concentration of 1.5 × 106 cells/mL.

    Techniques: Sample Prep, Electrophoresis, Lysis, Concentration Assay, Diffusion-based Assay, Protein Concentration, Standard Deviation

    Nintedanib and anti-VEGF/Ang2 nanobody prevent brain metastases formation. 9.4 T MRI after Gadolinium contrast administration was performed on day 26 after intracardial tumor cell injection. a Bar chart illustrating the reduced percentage of mice with metastases, detectable in cMRI. b Representative T1-w cMRI images depicting larger intracranial metastases in the control groups, indicated by arrowheads. scale bar = 2 mm. c Scatter plots showing the reduction of number and volume of cranial metastases in cMRI. d Scatter plots depicting the number and volume of meningeal metastases in cMRI e Quantification demonstrating a higher histological tumor–tissue ratio in control mice at their time of death. f Representative histological slices with higher number and size of PC14-PE6 pGF1 Br4 metastases in the control group. Fluorescent staining: DAPI (blue) = nucleus, GFP (green) = PC14-PE6 tumor cells, Alexa Flour 546 (orange) = collagen-IV positive vascular basement membrane. Arrows indicate GFP-positive metastatic lesions. Scale bar = 1000 µm. Mean values with standard errors of the mean are shown. *p

    Journal: Clinical & Experimental Metastasis

    Article Title: Nintedanib and a bi-specific anti-VEGF/Ang2 nanobody selectively prevent brain metastases of lung adenocarcinoma cells

    doi: 10.1007/s10585-020-10055-x

    Figure Lengend Snippet: Nintedanib and anti-VEGF/Ang2 nanobody prevent brain metastases formation. 9.4 T MRI after Gadolinium contrast administration was performed on day 26 after intracardial tumor cell injection. a Bar chart illustrating the reduced percentage of mice with metastases, detectable in cMRI. b Representative T1-w cMRI images depicting larger intracranial metastases in the control groups, indicated by arrowheads. scale bar = 2 mm. c Scatter plots showing the reduction of number and volume of cranial metastases in cMRI. d Scatter plots depicting the number and volume of meningeal metastases in cMRI e Quantification demonstrating a higher histological tumor–tissue ratio in control mice at their time of death. f Representative histological slices with higher number and size of PC14-PE6 pGF1 Br4 metastases in the control group. Fluorescent staining: DAPI (blue) = nucleus, GFP (green) = PC14-PE6 tumor cells, Alexa Flour 546 (orange) = collagen-IV positive vascular basement membrane. Arrows indicate GFP-positive metastatic lesions. Scale bar = 1000 µm. Mean values with standard errors of the mean are shown. *p

    Article Snippet: Briefly, PC14-PE6 pGF1 Br4 cells were trypsinized (Gibco, Life Science Technologies, cat. no.: 25200-056), washed, counted and resuspended in PBS (cat. no: D8537, Sigma Life Sciences) in a final concentration of 5 × 106 cells/mL.

    Techniques: Magnetic Resonance Imaging, Injection, Mouse Assay, Staining

    Membrane binding of the key PNN component aggrecan is biochemically altered in Ptprz1 KO and Tnr KO mice brains. Brain homogenates were treated with ChABC to remove the hyaluronan backbone of PNNs or chondroitinase in the presence of EDTA (ChABC EDTA) and centrifuged to obtain soluble release ( R ) and insoluble pellet ( P ) fractions. A , Western blotting image showing release of PNN marker aggrecan into soluble phase from brain homogenates of WT, Ptprz1 KO, and Tnr KO mice by ChABC treatment alone and ChABC EDTA treatment. The release of aggrecan into the soluble fraction required ChABC treatment in addition with EDTA in WT brain homogenates. Aggrecan was released more readily with just ChABC treatment in Ptprz1 KO and Tnr KO animals. B , quantification showing ratio of the soluble release fraction ( R ) to total aggrecan levels (soluble release (R) + insoluble pellet (P)) in WT, Ptprz1 KO, and Tnr KO mice. There was a statistically significant difference in the release of aggrecan among the three genotypes as determined by one-way ANOVA (F(2,7) = 14.94, p = 0.0030). A Tukey's post hoc test showed that aggrecan was released much more readily in Ptprz1 KO mice (42 ± 0.04%, p = 0.0340) as well as in Tnr KO mice (58 ± 13%, p = 0.0024) compared with WT mice (15 ± 9%) when treated with just ChABC. There was no significant difference in aggrecan release between Ptprz1 KO and Tnr KO brains. Treatment with ChABC alongside EDTA led to almost complete release of aggrecan into the soluble release fraction in all genotypes. The ratio of release to total in Ptprz1 KO, Tnr KO and WT mice was not significantly different among the genotypes in the ChABC with EDTA treatment group. B , bars in graphs represent percentage release ± S.D.

    Journal: The Journal of Biological Chemistry

    Article Title: The protein tyrosine phosphatase RPTPζ/phosphacan is critical for perineuronal net structure

    doi: 10.1074/jbc.RA119.010830

    Figure Lengend Snippet: Membrane binding of the key PNN component aggrecan is biochemically altered in Ptprz1 KO and Tnr KO mice brains. Brain homogenates were treated with ChABC to remove the hyaluronan backbone of PNNs or chondroitinase in the presence of EDTA (ChABC EDTA) and centrifuged to obtain soluble release ( R ) and insoluble pellet ( P ) fractions. A , Western blotting image showing release of PNN marker aggrecan into soluble phase from brain homogenates of WT, Ptprz1 KO, and Tnr KO mice by ChABC treatment alone and ChABC EDTA treatment. The release of aggrecan into the soluble fraction required ChABC treatment in addition with EDTA in WT brain homogenates. Aggrecan was released more readily with just ChABC treatment in Ptprz1 KO and Tnr KO animals. B , quantification showing ratio of the soluble release fraction ( R ) to total aggrecan levels (soluble release (R) + insoluble pellet (P)) in WT, Ptprz1 KO, and Tnr KO mice. There was a statistically significant difference in the release of aggrecan among the three genotypes as determined by one-way ANOVA (F(2,7) = 14.94, p = 0.0030). A Tukey's post hoc test showed that aggrecan was released much more readily in Ptprz1 KO mice (42 ± 0.04%, p = 0.0340) as well as in Tnr KO mice (58 ± 13%, p = 0.0024) compared with WT mice (15 ± 9%) when treated with just ChABC. There was no significant difference in aggrecan release between Ptprz1 KO and Tnr KO brains. Treatment with ChABC alongside EDTA led to almost complete release of aggrecan into the soluble release fraction in all genotypes. The ratio of release to total in Ptprz1 KO, Tnr KO and WT mice was not significantly different among the genotypes in the ChABC with EDTA treatment group. B , bars in graphs represent percentage release ± S.D.

    Article Snippet: Briefly, cortices of embryonic day (E) 16 CD-1 WT or Ptprz1 KO embryos were removed and digested in 0.25% trypsin-EDTA (Thermo Fisher Scientific).

    Techniques: Binding Assay, Mouse Assay, Western Blot, Marker