ethylenediaminetetraacetic acid  (Thermo Fisher)


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    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 ethylenediaminetetraacetic acid
    The preparation of B6 epithelium (EPI) p35 + B6 stroma-endothelium (S-E) composite graft and transplantation model. Harvested B6 corneas were incubated with pHAGE-CMV-p35-IZsGreenW lentiviral vector (3 × 10 5 IU/mL) in the presence of hexadimethrine bromide for 1 h. Transduced corneal layers were treated with <t>ethylenediaminetetraacetic</t> acid for 45 min, and subsequently the intact sheet of epithelium was peeled off and transplanted on untreated S-E of B6 (a) . The photos illustrate the separation of the epithelial layer and the replacement of p35 -treated epithelium on S-E of the same strain (b) . Transduction efficacy in the epithelium was confirmed by detection of IZsGreenW in confocal fluorescence microscopy. Murine corneas were successfully transduced with p35 (visible IZsGreenW expressing cells in green and nuclei in blue ) and untreated negative control (visible nuclei) (c) . The composite grafts were transplanted in Balb/c (B/c) mice (day 0) (d) . B/c mice as recipients were transplanted with B/c EPI + B/c S-E ( n = 5), B6 EPI + B6 S-E ( n = 5), B6 EPI empty + B6 S-E ( n = 4), and B6 EPI p35 + B6 S-E ( n = 4) composite grafts (e)
    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
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    Average 97 stars, based on 1 article reviews
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    Images

    1) Product Images from "Gene Therapy for Modulation of T-Cell-Mediated Immune Response Provoked by Corneal Transplantation"

    Article Title: Gene Therapy for Modulation of T-Cell-Mediated Immune Response Provoked by Corneal Transplantation

    Journal: Human Gene Therapy

    doi: 10.1089/hum.2017.044

    The preparation of B6 epithelium (EPI) p35 + B6 stroma-endothelium (S-E) composite graft and transplantation model. Harvested B6 corneas were incubated with pHAGE-CMV-p35-IZsGreenW lentiviral vector (3 × 10 5 IU/mL) in the presence of hexadimethrine bromide for 1 h. Transduced corneal layers were treated with ethylenediaminetetraacetic acid for 45 min, and subsequently the intact sheet of epithelium was peeled off and transplanted on untreated S-E of B6 (a) . The photos illustrate the separation of the epithelial layer and the replacement of p35 -treated epithelium on S-E of the same strain (b) . Transduction efficacy in the epithelium was confirmed by detection of IZsGreenW in confocal fluorescence microscopy. Murine corneas were successfully transduced with p35 (visible IZsGreenW expressing cells in green and nuclei in blue ) and untreated negative control (visible nuclei) (c) . The composite grafts were transplanted in Balb/c (B/c) mice (day 0) (d) . B/c mice as recipients were transplanted with B/c EPI + B/c S-E ( n = 5), B6 EPI + B6 S-E ( n = 5), B6 EPI empty + B6 S-E ( n = 4), and B6 EPI p35 + B6 S-E ( n = 4) composite grafts (e)
    Figure Legend Snippet: The preparation of B6 epithelium (EPI) p35 + B6 stroma-endothelium (S-E) composite graft and transplantation model. Harvested B6 corneas were incubated with pHAGE-CMV-p35-IZsGreenW lentiviral vector (3 × 10 5 IU/mL) in the presence of hexadimethrine bromide for 1 h. Transduced corneal layers were treated with ethylenediaminetetraacetic acid for 45 min, and subsequently the intact sheet of epithelium was peeled off and transplanted on untreated S-E of B6 (a) . The photos illustrate the separation of the epithelial layer and the replacement of p35 -treated epithelium on S-E of the same strain (b) . Transduction efficacy in the epithelium was confirmed by detection of IZsGreenW in confocal fluorescence microscopy. Murine corneas were successfully transduced with p35 (visible IZsGreenW expressing cells in green and nuclei in blue ) and untreated negative control (visible nuclei) (c) . The composite grafts were transplanted in Balb/c (B/c) mice (day 0) (d) . B/c mice as recipients were transplanted with B/c EPI + B/c S-E ( n = 5), B6 EPI + B6 S-E ( n = 5), B6 EPI empty + B6 S-E ( n = 4), and B6 EPI p35 + B6 S-E ( n = 4) composite grafts (e)

    Techniques Used: Transplantation Assay, Incubation, Plasmid Preparation, Transduction, Fluorescence, Microscopy, Expressing, Negative Control, Mouse Assay

    2) Product Images from "The ?2 Integrin Subunit-Deficient Mouse"

    Article Title: The ?2 Integrin Subunit-Deficient Mouse

    Journal: The American Journal of Pathology

    doi:

    Platelet adhesion and aggregation. A: Platelets purified from wild-type (+/+), heterozygous (+/−), and α 2 -deficient (−/−) animals were assayed for adhesion to type I collagen. The assays were performed for 1 hour in phosphate-buffered saline with 2 mmol/L of Mg 2+ or ethylenediaminetetraacetic acid. B: Platelet adhesion to type I collagen at shear rates of 150 seconds −1 was assayed in a parallel-plate flow chamber apparatus. Micrographs of the collagen substrate and the deposition of platelets from wild-type (+/+), heterozygous (+/−), and α 2 -deficient (−/−) mice are depicted. C: The surface area covered by adherent wild-type, heterozygous, or α 2 -deficient platelets was quantitated and expressed as a percentage of wild-type platelet adhesion. D: Platelet aggregation of heterozygous (+/−) or α 2 -deficient (−/−) platelets was initiated by either the addition of 2.5 or 5 μg/ml of soluble equine tendon collagen or 0.5 U/ml of thrombin.
    Figure Legend Snippet: Platelet adhesion and aggregation. A: Platelets purified from wild-type (+/+), heterozygous (+/−), and α 2 -deficient (−/−) animals were assayed for adhesion to type I collagen. The assays were performed for 1 hour in phosphate-buffered saline with 2 mmol/L of Mg 2+ or ethylenediaminetetraacetic acid. B: Platelet adhesion to type I collagen at shear rates of 150 seconds −1 was assayed in a parallel-plate flow chamber apparatus. Micrographs of the collagen substrate and the deposition of platelets from wild-type (+/+), heterozygous (+/−), and α 2 -deficient (−/−) mice are depicted. C: The surface area covered by adherent wild-type, heterozygous, or α 2 -deficient platelets was quantitated and expressed as a percentage of wild-type platelet adhesion. D: Platelet aggregation of heterozygous (+/−) or α 2 -deficient (−/−) platelets was initiated by either the addition of 2.5 or 5 μg/ml of soluble equine tendon collagen or 0.5 U/ml of thrombin.

    Techniques Used: Purification, Flow Cytometry, Mouse 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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  • 97
    Thermo Fisher immunoblot buffer
    Time course for DNA damage and p53 network response in HT1080 cells following exposure to etoposide, quercetin, or methyl methanesulfonate. Representative Western blots from three separate experiments are shown. HT1080 cells were exposed to 0.1% DMSO (control cells; Ctrl), 1-μM etoposide (ETP), 30-μM quercetin (QUE), or 200-μM methyl methanesulfonate (MMS) for 3, 8, or 24 h. Total cellular protein was isolated and subjected to <t>immunoblot</t> analysis. GAPDH was used as an internal control.
    Immunoblot Buffer, 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
    Signalling pathways in <t>TL2i</t> cells. ( a ) Phase-contrast microphotographs, AP activity and immunofluorescence labelling of OCT4, SSEA4 and TRA-1–60 in TL2i cells. ( b ) A colony-forming assay with TL2i-OS3–10 and TL2i-H9S3-2 cells (representative experiment). Cell clumps were plated in TL2i medium supplemented with pharmacological inhibitors of JAK2 (SD1029 at 10 μM), FGFR (SU5402 at 10 μM) and SMADs (SB431542 at 10 μM), and cultivated for 5 days. Upper panels: staining to reveal AP activity; bottom panels: histograms showing the percentage of undifferentiated, mixed and differentiated colonies. ( n =3; error bars indicates the mean±s.e.m.). ( c ) Histogram representation of the mRNA level (ΔCt) of pluripotency genes in TL2i-OS3–10 cells before and treatment with FGFR inhibitor SU5402 for 5 days after normalization to GAPDH (ΔCt=1). ( n =3, mean±s.d.). ( d ) Characteristics of TL2i-OS3–10 cells after propagation on Matrigel without MEF; AP, alkaline phosphatase activity. ( e ) Histograms showing the percentage of undifferentiated, mixed and differentiated colonies ( n =3; error bars indicate the mean±s.e.m.) in a colony-forming assay with TL2i-OS3–10 cells (representative experiment). Cell clumps were plated in a medium supplemented with pharmacological inhibitors of FGFR (SU5402 at 10 μM) and SMADs (SB431542 at 10 μM), and cultivated for 5 days with LIF+4′-OHT. ( f ) Histogram representation of the mRNA level (ΔCt) of LIFR , GP130 , JAK and STAT3 genes in TL2i-H9S3-2 cells, after normalization to GAPDH (ΔCt=1), then to TL-H9S3-2 (blue bars) and F-H9S3-2 cells (red bars). ( n =3, mean±s. d.). ( g ) Western blot analysis of STAT3 and STAT3-ER T2 expression in OSCAR, F-H9S3-2, TL-H9S3-2 and TL2i-H9S3-2 cells, analysed with antibodies to total STAT3, phospho-(Tyr705)-STAT3 and phospho-(Ser720)-STAT3. One representative experiment of three repeats is shown. ( h ) Phase-contrast microphotographs (PC) and immunofluorescence labelling of OCT4, NANOG, SSEA4 and TRA-1–81 in TL2i-OS3–10 cells after culturing in 2i/LIF medium without 4′-OHT for 30 passages. ( i ) Western blot analysis of STAT3 and STAT3-ER T2 expression in TL-OS3–10 cells and TL2i-OS3–10 cells (+/−4′-OHT), analysed with antibodies to total STAT3. One representative experiment of two repeats is shown. ( j ) Phase-contrast microphotographs (PC), AP detection and immunofluorescence labelling of OCT4, NANOG, TRA-1–81 and SSEA4 in TL2i-OS3–10 cells after culturing in N2B27+2i/LIF basal medium for eight passages. ( k ) Histogram representation of the cloning efficiency of F, TL and TL2i cells (OS3–10, H9S3–2 and H9S3–14 lines) after single-cell dissociation with 0.05% <t>trypsin-EDTA</t> and re-plating on feeders in the presence of 10 μM ROCK inhibitor Y-27632 for 24 h post dissociation. Tukey's test; n =3; error bars indicate the mean±s. e. Scale bar, 50 μm ( a , d , h , i ).
    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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    Thermo Fisher plasmid dna
    Rescue efficiency in clonal cell lines. HeLa cells were infected with wild-type AAV at specific MOIs and plated out at 24 h to make single-cell clones. Three weeks postinfection, individual cell lines were examined for the presence of latent AAV by a rescue assay. Briefly, cells were seeded in 96-well dishes and infected with wild-type adenovirus at an MOI of 10. At full cytopathic effect (48 to 72 h), cells were lysed by addition of NaOH and <t>EDTA</t> and denatured for 15 min at 68°C. Lysates were filtered onto nylon membranes with a slot blot apparatus and probed for the presence of wild-type AAV <t>DNA.</t> A representative blot from a wild-type AAV MOI 100 infection is shown in panel A. Panel B graphs MOIs from 0 to 1,000. Each point represents a minimum of 36 cell lines screened.
    Plasmid Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dm nitrophen
    DDI in physiological ACSF (1.3 mM Mg 2+ ) evoked by voltage steps and photolysis of caged Ca 2+ in mitral cells. ( A ) Single sweeps showing that photolysis of <t>DM-Nitrophen</t> evokes DDI that is similar to that evoked by using a voltage step. ( Inset ) A 30-ms section of the trace, illustrating the unitary IPSCs that constitute DDI. ( B ) APV (100 μM) blocks responses to voltage steps and Ca 2+ uncaging in the same cell. ( C ) Summary plot ( n = 6) of the action of cadmium (150 μM) on DDI evoked by voltage steps (open circles) and photolysis of DM-Nitrophen (filled circles) in the same mitral cells. ( D ) Cumulative frequency histogram summarizing unitary IPSCs evoked by using voltage steps (open circles, n = 1,958), flash photolysis (filled circles, n = 799), and spontaneous events in cadmium (solid line, n = 438). ( Inset ) Average of ≈25 aligned IPSCs from one cell evoked by voltage steps (V step ), Ca 2+ uncaging (Flash), and occurring in cadmium (Cd).
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    Image Search Results


    Time course for DNA damage and p53 network response in HT1080 cells following exposure to etoposide, quercetin, or methyl methanesulfonate. Representative Western blots from three separate experiments are shown. HT1080 cells were exposed to 0.1% DMSO (control cells; Ctrl), 1-μM etoposide (ETP), 30-μM quercetin (QUE), or 200-μM methyl methanesulfonate (MMS) for 3, 8, or 24 h. Total cellular protein was isolated and subjected to immunoblot analysis. GAPDH was used as an internal control.

    Journal: Toxicological Sciences

    Article Title: Profiling Dose-Dependent Activation of p53-Mediated Signaling Pathways by Chemicals with Distinct Mechanisms of DNA Damage

    doi: 10.1093/toxsci/kfu153

    Figure Lengend Snippet: Time course for DNA damage and p53 network response in HT1080 cells following exposure to etoposide, quercetin, or methyl methanesulfonate. Representative Western blots from three separate experiments are shown. HT1080 cells were exposed to 0.1% DMSO (control cells; Ctrl), 1-μM etoposide (ETP), 30-μM quercetin (QUE), or 200-μM methyl methanesulfonate (MMS) for 3, 8, or 24 h. Total cellular protein was isolated and subjected to immunoblot analysis. GAPDH was used as an internal control.

    Article Snippet: 24 h after plating, cells were exposed to 1-μM etoposide, 30-μM quercetin, or 200-μM methyl methanesulfonate for 3, 8, or 24 h. After chemical treatment, cells were washed twice with PBS, homogenized in immunoblot buffer (0.1-M Tris-HCl pH8.0; 0.05-M ethylenediaminetetraacetic acid, pH 8.0; 0.5 M NaCl; 1% sodium dodecyl sulfate (SDS) and 1% sarkosyl) plus 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail (Pierce).

    Techniques: Western Blot, Isolation

    Signalling pathways in TL2i cells. ( a ) Phase-contrast microphotographs, AP activity and immunofluorescence labelling of OCT4, SSEA4 and TRA-1–60 in TL2i cells. ( b ) A colony-forming assay with TL2i-OS3–10 and TL2i-H9S3-2 cells (representative experiment). Cell clumps were plated in TL2i medium supplemented with pharmacological inhibitors of JAK2 (SD1029 at 10 μM), FGFR (SU5402 at 10 μM) and SMADs (SB431542 at 10 μM), and cultivated for 5 days. Upper panels: staining to reveal AP activity; bottom panels: histograms showing the percentage of undifferentiated, mixed and differentiated colonies. ( n =3; error bars indicates the mean±s.e.m.). ( c ) Histogram representation of the mRNA level (ΔCt) of pluripotency genes in TL2i-OS3–10 cells before and treatment with FGFR inhibitor SU5402 for 5 days after normalization to GAPDH (ΔCt=1). ( n =3, mean±s.d.). ( d ) Characteristics of TL2i-OS3–10 cells after propagation on Matrigel without MEF; AP, alkaline phosphatase activity. ( e ) Histograms showing the percentage of undifferentiated, mixed and differentiated colonies ( n =3; error bars indicate the mean±s.e.m.) in a colony-forming assay with TL2i-OS3–10 cells (representative experiment). Cell clumps were plated in a medium supplemented with pharmacological inhibitors of FGFR (SU5402 at 10 μM) and SMADs (SB431542 at 10 μM), and cultivated for 5 days with LIF+4′-OHT. ( f ) Histogram representation of the mRNA level (ΔCt) of LIFR , GP130 , JAK and STAT3 genes in TL2i-H9S3-2 cells, after normalization to GAPDH (ΔCt=1), then to TL-H9S3-2 (blue bars) and F-H9S3-2 cells (red bars). ( n =3, mean±s. d.). ( g ) Western blot analysis of STAT3 and STAT3-ER T2 expression in OSCAR, F-H9S3-2, TL-H9S3-2 and TL2i-H9S3-2 cells, analysed with antibodies to total STAT3, phospho-(Tyr705)-STAT3 and phospho-(Ser720)-STAT3. One representative experiment of three repeats is shown. ( h ) Phase-contrast microphotographs (PC) and immunofluorescence labelling of OCT4, NANOG, SSEA4 and TRA-1–81 in TL2i-OS3–10 cells after culturing in 2i/LIF medium without 4′-OHT for 30 passages. ( i ) Western blot analysis of STAT3 and STAT3-ER T2 expression in TL-OS3–10 cells and TL2i-OS3–10 cells (+/−4′-OHT), analysed with antibodies to total STAT3. One representative experiment of two repeats is shown. ( j ) Phase-contrast microphotographs (PC), AP detection and immunofluorescence labelling of OCT4, NANOG, TRA-1–81 and SSEA4 in TL2i-OS3–10 cells after culturing in N2B27+2i/LIF basal medium for eight passages. ( k ) Histogram representation of the cloning efficiency of F, TL and TL2i cells (OS3–10, H9S3–2 and H9S3–14 lines) after single-cell dissociation with 0.05% trypsin-EDTA and re-plating on feeders in the presence of 10 μM ROCK inhibitor Y-27632 for 24 h post dissociation. Tukey's test; n =3; error bars indicate the mean±s. e. Scale bar, 50 μm ( a , d , h , i ).

    Journal: Nature Communications

    Article Title: Reinforcement of STAT3 activity reprogrammes human embryonic stem cells to naive-like pluripotency

    doi: 10.1038/ncomms8095

    Figure Lengend Snippet: Signalling pathways in TL2i cells. ( a ) Phase-contrast microphotographs, AP activity and immunofluorescence labelling of OCT4, SSEA4 and TRA-1–60 in TL2i cells. ( b ) A colony-forming assay with TL2i-OS3–10 and TL2i-H9S3-2 cells (representative experiment). Cell clumps were plated in TL2i medium supplemented with pharmacological inhibitors of JAK2 (SD1029 at 10 μM), FGFR (SU5402 at 10 μM) and SMADs (SB431542 at 10 μM), and cultivated for 5 days. Upper panels: staining to reveal AP activity; bottom panels: histograms showing the percentage of undifferentiated, mixed and differentiated colonies. ( n =3; error bars indicates the mean±s.e.m.). ( c ) Histogram representation of the mRNA level (ΔCt) of pluripotency genes in TL2i-OS3–10 cells before and treatment with FGFR inhibitor SU5402 for 5 days after normalization to GAPDH (ΔCt=1). ( n =3, mean±s.d.). ( d ) Characteristics of TL2i-OS3–10 cells after propagation on Matrigel without MEF; AP, alkaline phosphatase activity. ( e ) Histograms showing the percentage of undifferentiated, mixed and differentiated colonies ( n =3; error bars indicate the mean±s.e.m.) in a colony-forming assay with TL2i-OS3–10 cells (representative experiment). Cell clumps were plated in a medium supplemented with pharmacological inhibitors of FGFR (SU5402 at 10 μM) and SMADs (SB431542 at 10 μM), and cultivated for 5 days with LIF+4′-OHT. ( f ) Histogram representation of the mRNA level (ΔCt) of LIFR , GP130 , JAK and STAT3 genes in TL2i-H9S3-2 cells, after normalization to GAPDH (ΔCt=1), then to TL-H9S3-2 (blue bars) and F-H9S3-2 cells (red bars). ( n =3, mean±s. d.). ( g ) Western blot analysis of STAT3 and STAT3-ER T2 expression in OSCAR, F-H9S3-2, TL-H9S3-2 and TL2i-H9S3-2 cells, analysed with antibodies to total STAT3, phospho-(Tyr705)-STAT3 and phospho-(Ser720)-STAT3. One representative experiment of three repeats is shown. ( h ) Phase-contrast microphotographs (PC) and immunofluorescence labelling of OCT4, NANOG, SSEA4 and TRA-1–81 in TL2i-OS3–10 cells after culturing in 2i/LIF medium without 4′-OHT for 30 passages. ( i ) Western blot analysis of STAT3 and STAT3-ER T2 expression in TL-OS3–10 cells and TL2i-OS3–10 cells (+/−4′-OHT), analysed with antibodies to total STAT3. One representative experiment of two repeats is shown. ( j ) Phase-contrast microphotographs (PC), AP detection and immunofluorescence labelling of OCT4, NANOG, TRA-1–81 and SSEA4 in TL2i-OS3–10 cells after culturing in N2B27+2i/LIF basal medium for eight passages. ( k ) Histogram representation of the cloning efficiency of F, TL and TL2i cells (OS3–10, H9S3–2 and H9S3–14 lines) after single-cell dissociation with 0.05% trypsin-EDTA and re-plating on feeders in the presence of 10 μM ROCK inhibitor Y-27632 for 24 h post dissociation. Tukey's test; n =3; error bars indicate the mean±s. e. Scale bar, 50 μm ( a , d , h , i ).

    Article Snippet: TL2i cells were regularly passaged by single-cell dissociation with 0.05% trypsin-EDTA (Gibco) every 4 days.

    Techniques: Activity Assay, Immunofluorescence, Staining, Western Blot, Expressing, Clone Assay

    Rescue efficiency in clonal cell lines. HeLa cells were infected with wild-type AAV at specific MOIs and plated out at 24 h to make single-cell clones. Three weeks postinfection, individual cell lines were examined for the presence of latent AAV by a rescue assay. Briefly, cells were seeded in 96-well dishes and infected with wild-type adenovirus at an MOI of 10. At full cytopathic effect (48 to 72 h), cells were lysed by addition of NaOH and EDTA and denatured for 15 min at 68°C. Lysates were filtered onto nylon membranes with a slot blot apparatus and probed for the presence of wild-type AAV DNA. A representative blot from a wild-type AAV MOI 100 infection is shown in panel A. Panel B graphs MOIs from 0 to 1,000. Each point represents a minimum of 36 cell lines screened.

    Journal: Journal of Virology

    Article Title: Adeno-Associated Virus Site-Specific Integration and AAVS1 Disruption

    doi: 10.1128/JVI.78.15.7874-7882.2004

    Figure Lengend Snippet: Rescue efficiency in clonal cell lines. HeLa cells were infected with wild-type AAV at specific MOIs and plated out at 24 h to make single-cell clones. Three weeks postinfection, individual cell lines were examined for the presence of latent AAV by a rescue assay. Briefly, cells were seeded in 96-well dishes and infected with wild-type adenovirus at an MOI of 10. At full cytopathic effect (48 to 72 h), cells were lysed by addition of NaOH and EDTA and denatured for 15 min at 68°C. Lysates were filtered onto nylon membranes with a slot blot apparatus and probed for the presence of wild-type AAV DNA. A representative blot from a wild-type AAV MOI 100 infection is shown in panel A. Panel B graphs MOIs from 0 to 1,000. Each point represents a minimum of 36 cell lines screened.

    Article Snippet: Cells were electroporated (280 V, 960 μF) with plasmid DNA (30 μg in 30 μl of Tris-EDTA, including 5 μg of pEGFP [Invitrogen]) in a 4-mm gap width electroporation cuvette.

    Techniques: Infection, Clone Assay, Rescue Assay, Dot Blot

    DDI in physiological ACSF (1.3 mM Mg 2+ ) evoked by voltage steps and photolysis of caged Ca 2+ in mitral cells. ( A ) Single sweeps showing that photolysis of DM-Nitrophen evokes DDI that is similar to that evoked by using a voltage step. ( Inset ) A 30-ms section of the trace, illustrating the unitary IPSCs that constitute DDI. ( B ) APV (100 μM) blocks responses to voltage steps and Ca 2+ uncaging in the same cell. ( C ) Summary plot ( n = 6) of the action of cadmium (150 μM) on DDI evoked by voltage steps (open circles) and photolysis of DM-Nitrophen (filled circles) in the same mitral cells. ( D ) Cumulative frequency histogram summarizing unitary IPSCs evoked by using voltage steps (open circles, n = 1,958), flash photolysis (filled circles, n = 799), and spontaneous events in cadmium (solid line, n = 438). ( Inset ) Average of ≈25 aligned IPSCs from one cell evoked by voltage steps (V step ), Ca 2+ uncaging (Flash), and occurring in cadmium (Cd).

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

    Article Title: Mechanisms governing dendritic ?-aminobutyric acid (GABA) release in the rat olfactory bulb

    doi:

    Figure Lengend Snippet: DDI in physiological ACSF (1.3 mM Mg 2+ ) evoked by voltage steps and photolysis of caged Ca 2+ in mitral cells. ( A ) Single sweeps showing that photolysis of DM-Nitrophen evokes DDI that is similar to that evoked by using a voltage step. ( Inset ) A 30-ms section of the trace, illustrating the unitary IPSCs that constitute DDI. ( B ) APV (100 μM) blocks responses to voltage steps and Ca 2+ uncaging in the same cell. ( C ) Summary plot ( n = 6) of the action of cadmium (150 μM) on DDI evoked by voltage steps (open circles) and photolysis of DM-Nitrophen (filled circles) in the same mitral cells. ( D ) Cumulative frequency histogram summarizing unitary IPSCs evoked by using voltage steps (open circles, n = 1,958), flash photolysis (filled circles, n = 799), and spontaneous events in cadmium (solid line, n = 438). ( Inset ) Average of ≈25 aligned IPSCs from one cell evoked by voltage steps (V step ), Ca 2+ uncaging (Flash), and occurring in cadmium (Cd).

    Article Snippet: In experiments using caged Ca2+ , EGTA was replaced with 2 mM DM-Nitrophen (the structurally identical DMNP-EDTA from Molecular Probes was used) loaded 60–70% with CaCl2 and K2 ATP was substituted for MgATP.

    Techniques: Mass Spectrometry