versene solution  (Thermo Fisher)


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    Name:
    Versene Solution
    Description:
    Versene is an EDTA solution for use as a gentle non enzymatic cell dissociation reagent Gibco Versene Solution 0 48 mM is formulated as 0 2 g EDTA Na4 per liter of Phosphate Buffered Saline PBS Product UseFor Research Use Only Not for use in diagnostic procedures Dual site cGMP Manufacturing and Quality SystemGibco Versene Solution is manufactured at a cGMP compliant facility located in Paisley Scotland UK The facility is registered with the FDA as a medical device manufacturer and is certified to the ISO 13485 standard For supply chain continuity we offers an identical Gibco Versene Solution product made in our Grand Island facility 15040 066 This facility is registered with the FDA as a medical device manufacturer and is certified to the ISO 13485 standards
    Catalog Number:
    15040033
    Price:
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    Category:
    Cell Culture Transfection Reagents
    Applications:
    Cell Culture|Mammalian Cell Culture
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    Structured Review

    Thermo Fisher versene solution
    TMEM16F is required for annexin A2 and A5 cell surface localisation. (A) TMEM16F-knockout (KO) cells have severely reduced annexin A2 and A5 on their surface. Wild-type (WT), matched controls and TMEM-knockout HeLa cells were incubated in <t>versene</t> (EDTA solution) or not (SFM) for 10 min at 37°C before the eluate was collected and analysed for annexin A2 and A5 by western blotting. A representative western blot is shown ( n =4). (B) Expression of mCherry–mTMEM16F rescues lipid movement in TMEM16F-knockout cells. Wild-type and TMEM16F-knockout HeLa cells alone or expressing mCherry–mTMEM16F were treated with ionomycin and analysed for recombinant annexin-A5–Cy5 binding by flow cytometry. A representative experiment is shown ( n =3). (C) Expression of mCherry–mTMEM16F rescues annexin A2 and A5 expression at the cell surface. Annexin A2 and A5 on the cell surface were evaluated through treatment with EDTA and western blotting as described in A. A representative experiment is shown ( n =4). A quantification of cell surface annexin A2 in HeLa transfected or not with mTMEM16F is presented (fold change measured as band intensity [transfected(eluate/lysate)/untransfected(eluate/lysate)]) Results are mean±s.e.m. from n =5 biological replicates; * P
    Versene is an EDTA solution for use as a gentle non enzymatic cell dissociation reagent Gibco Versene Solution 0 48 mM is formulated as 0 2 g EDTA Na4 per liter of Phosphate Buffered Saline PBS Product UseFor Research Use Only Not for use in diagnostic procedures Dual site cGMP Manufacturing and Quality SystemGibco Versene Solution is manufactured at a cGMP compliant facility located in Paisley Scotland UK The facility is registered with the FDA as a medical device manufacturer and is certified to the ISO 13485 standard For supply chain continuity we offers an identical Gibco Versene Solution product made in our Grand Island facility 15040 066 This facility is registered with the FDA as a medical device manufacturer and is certified to the ISO 13485 standards
    https://www.bioz.com/result/versene solution/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    versene solution - by Bioz Stars, 2021-04
    97/100 stars

    Images

    1) Product Images from "Transbilayer phospholipid movement facilitates the translocation of annexin across membranes"

    Article Title: Transbilayer phospholipid movement facilitates the translocation of annexin across membranes

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.217034

    TMEM16F is required for annexin A2 and A5 cell surface localisation. (A) TMEM16F-knockout (KO) cells have severely reduced annexin A2 and A5 on their surface. Wild-type (WT), matched controls and TMEM-knockout HeLa cells were incubated in versene (EDTA solution) or not (SFM) for 10 min at 37°C before the eluate was collected and analysed for annexin A2 and A5 by western blotting. A representative western blot is shown ( n =4). (B) Expression of mCherry–mTMEM16F rescues lipid movement in TMEM16F-knockout cells. Wild-type and TMEM16F-knockout HeLa cells alone or expressing mCherry–mTMEM16F were treated with ionomycin and analysed for recombinant annexin-A5–Cy5 binding by flow cytometry. A representative experiment is shown ( n =3). (C) Expression of mCherry–mTMEM16F rescues annexin A2 and A5 expression at the cell surface. Annexin A2 and A5 on the cell surface were evaluated through treatment with EDTA and western blotting as described in A. A representative experiment is shown ( n =4). A quantification of cell surface annexin A2 in HeLa transfected or not with mTMEM16F is presented (fold change measured as band intensity [transfected(eluate/lysate)/untransfected(eluate/lysate)]) Results are mean±s.e.m. from n =5 biological replicates; * P
    Figure Legend Snippet: TMEM16F is required for annexin A2 and A5 cell surface localisation. (A) TMEM16F-knockout (KO) cells have severely reduced annexin A2 and A5 on their surface. Wild-type (WT), matched controls and TMEM-knockout HeLa cells were incubated in versene (EDTA solution) or not (SFM) for 10 min at 37°C before the eluate was collected and analysed for annexin A2 and A5 by western blotting. A representative western blot is shown ( n =4). (B) Expression of mCherry–mTMEM16F rescues lipid movement in TMEM16F-knockout cells. Wild-type and TMEM16F-knockout HeLa cells alone or expressing mCherry–mTMEM16F were treated with ionomycin and analysed for recombinant annexin-A5–Cy5 binding by flow cytometry. A representative experiment is shown ( n =3). (C) Expression of mCherry–mTMEM16F rescues annexin A2 and A5 expression at the cell surface. Annexin A2 and A5 on the cell surface were evaluated through treatment with EDTA and western blotting as described in A. A representative experiment is shown ( n =4). A quantification of cell surface annexin A2 in HeLa transfected or not with mTMEM16F is presented (fold change measured as band intensity [transfected(eluate/lysate)/untransfected(eluate/lysate)]) Results are mean±s.e.m. from n =5 biological replicates; * P

    Techniques Used: Knock-Out, Incubation, Western Blot, Expressing, Recombinant, Binding Assay, Flow Cytometry, Cytometry, Transfection

    Cinnamycin facilitates annexin translocation across membranes in cells. (A) Cinnamycin lipid movement activity. HeLa cells were treated with 1 µM cinnamycin for 50 min at 37°C. Then, recombinant annexin-A5–Cy5 (as a probe for PS) and propidium iodide (PI) (to exclude PI-containing dead cells) were added, and cells were incubated for a further 10 min at 37°C. Annexin-A5–Cy5 binding and PI accumulation were analysed by flow cytometry. Representative histograms of annexin-A5–Cy5 binding to live cells are shown ( n =3). (B) Western blotting analysis of cell lysates and eluates of HeLa cells treated with cinnamycin (30 min at 37°C) and then with EDTA (10 min at 37°C) as indicated. Quantification of cell surface annexin A2 and annexin A5 {fold change measured as band intensity [cinnamycin(eluate/lysate)/DMSO(eluate/lysate)]} is shown. Results are mean±s.e.m. from n =3 biological replicates; * P
    Figure Legend Snippet: Cinnamycin facilitates annexin translocation across membranes in cells. (A) Cinnamycin lipid movement activity. HeLa cells were treated with 1 µM cinnamycin for 50 min at 37°C. Then, recombinant annexin-A5–Cy5 (as a probe for PS) and propidium iodide (PI) (to exclude PI-containing dead cells) were added, and cells were incubated for a further 10 min at 37°C. Annexin-A5–Cy5 binding and PI accumulation were analysed by flow cytometry. Representative histograms of annexin-A5–Cy5 binding to live cells are shown ( n =3). (B) Western blotting analysis of cell lysates and eluates of HeLa cells treated with cinnamycin (30 min at 37°C) and then with EDTA (10 min at 37°C) as indicated. Quantification of cell surface annexin A2 and annexin A5 {fold change measured as band intensity [cinnamycin(eluate/lysate)/DMSO(eluate/lysate)]} is shown. Results are mean±s.e.m. from n =3 biological replicates; * P

    Techniques Used: Translocation Assay, Activity Assay, Recombinant, Incubation, Binding Assay, Flow Cytometry, Cytometry, Western Blot

    Related Articles

    Migration:

    Article Title: Interleukin‐6 drives melanoma cell motility through p38α‐MAPK‐dependent up‐regulation of WNT5A expression), Interleukin‐6 drives melanoma cell motility through p38α‐MAPK‐dependent up‐regulation of WNT5A expression
    Article Snippet: The final concentration of DMSO never exceeded 0.4% (v/v). .. 2.2 Cell migration Following serum starvation, the cells were detached with Versene and resuspended in cell media supplemented with 1% FBS; the cell concentration was then calculated using an automated cell counter (Countess™, Invitrogen). .. At the start of each experiment, 50,000 cells were suspended in 0.5 ml low serum media (only supplemented with 1% FBS) and added to cell culture inserts (BD Bioscience) with 8‐μm pore‐size membranes in the absence or presence of recombinant IL‐6 (Peprotech; 20 ng/ml) and Box5 (Calbiochem; 100 or 500 μM).

    Concentration Assay:

    Article Title: Interleukin‐6 drives melanoma cell motility through p38α‐MAPK‐dependent up‐regulation of WNT5A expression), Interleukin‐6 drives melanoma cell motility through p38α‐MAPK‐dependent up‐regulation of WNT5A expression
    Article Snippet: The final concentration of DMSO never exceeded 0.4% (v/v). .. 2.2 Cell migration Following serum starvation, the cells were detached with Versene and resuspended in cell media supplemented with 1% FBS; the cell concentration was then calculated using an automated cell counter (Countess™, Invitrogen). .. At the start of each experiment, 50,000 cells were suspended in 0.5 ml low serum media (only supplemented with 1% FBS) and added to cell culture inserts (BD Bioscience) with 8‐μm pore‐size membranes in the absence or presence of recombinant IL‐6 (Peprotech; 20 ng/ml) and Box5 (Calbiochem; 100 or 500 μM).

    Incubation:

    Article Title: A New Group B Adenovirus Receptor Is Expressed at High Levels on Human Stem and Tumor Cells ▿
    Article Snippet: .. MES cells were detached from culture dishes by incubation with Versene (Gibco), and 1 × 104 cells/chamber were plated on eight-well Tissue-Tek chamber slides (Nalge Nunc International) and incubated at 37°C overnight. .. The next day, cells were incubated with Ad3 or Ad35 labeled with Cy3 at an MOI of 8,000 VP/cell at 4°C for 45 min in 200 μl adhesion buffer. hSF6 and CD34+ cells were incubated in suspension with 8,000 VP/cell Cy-3 labeled Ad3 or Ad35 at 4°C for 45 min in 100 μl adhesion buffer.

    Recombinant:

    Article Title: Transbilayer phospholipid movement facilitates the translocation of annexin across membranes
    Article Snippet: Antibodies Antibodies used were: mouse monoclonal anti-annexin A2 (BD Biosciences; 610071; 1:1000), mouse monoclonal anti-annexin A5 (Abcam; ab54775, 1:1000), mouse monoclonal anti-transferrin receptor (Zymed; H68.4; 1:1000), rabbit polyclonal anti-actin (Sigma; A2066; 1:2000), mouse monoclonal anti-tubulin (Sigma; T9026; 1:4000), mouse monoclonal anti-FLAG (Sigma-Aldrich; clone M2; 1:4000), mouse monoclonal anti-Arf1 (Santa Cruz Biotechnology; sc-53168, 1:1000), rat anti-galectin-3 (Biolegend; 125401; western blotting, 1:2000) mouse monoclonal anti-Arf6 (Santa Cruz Biotechnology; clone 3A-1; 1:500), mouse monoclonal anti-LAMP2 (Biolegend; 354302; western blotting, 1:1000), rat polyclonal anti-galectin-3 conjugated to Alexa Fluor 647 (Biolegend; 125408; flow cytometry, 1:100) and rabbit polyclonal anti-mCherry antibody (Genetex; GTX128508-S). .. Reagents Reagents used were: cinnamycin (Santa Cruz Biotechnology; sc-391464), mastoparan X (Alfa Aesar; J61173), recombinant annexin-A5–FITC (Abcam; ab14085), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Sigma-Aldrich; 54008), 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC, Sigma-Aldrich; P3017), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE, Avanti Polar Lipids, 810145) and proteinase K (Molecular Biology; BP1700-100), EGTA (Sigma-Aldrich; E3889), sodium dithionite (Sigma-Aldrich; 71699), versene solution containing ethylenediaminetetraacetic acid (EDTA) (Gibco; 15040-033), propidium iodide solution (Biolegend; 421301), QuickExtract DNA extraction solution (Epicenter; QE0905T), Herculase II fusion DNA polymerase (Agilent; 600675), annexin-V–FITC and annexin-V–Cy5 Apoptosis Staining/Detection Kit (ab14085, ab14150), annexin-V conjugated to Alexa Fluor 647 (Biolegend; 640912) and ionomycin (Cayman Chemical Company; 10004974), recombinant galectin-3 (Biolegend; 599706) and recombinant anneaxin A5 (Novus NBP1-30265). .. Oligonucleotides for TMEM16F CRISPR targeting and sequencing were synthesised from Sigma-Aldrich ( Table S1 ).

    DNA Extraction:

    Article Title: Transbilayer phospholipid movement facilitates the translocation of annexin across membranes
    Article Snippet: Antibodies Antibodies used were: mouse monoclonal anti-annexin A2 (BD Biosciences; 610071; 1:1000), mouse monoclonal anti-annexin A5 (Abcam; ab54775, 1:1000), mouse monoclonal anti-transferrin receptor (Zymed; H68.4; 1:1000), rabbit polyclonal anti-actin (Sigma; A2066; 1:2000), mouse monoclonal anti-tubulin (Sigma; T9026; 1:4000), mouse monoclonal anti-FLAG (Sigma-Aldrich; clone M2; 1:4000), mouse monoclonal anti-Arf1 (Santa Cruz Biotechnology; sc-53168, 1:1000), rat anti-galectin-3 (Biolegend; 125401; western blotting, 1:2000) mouse monoclonal anti-Arf6 (Santa Cruz Biotechnology; clone 3A-1; 1:500), mouse monoclonal anti-LAMP2 (Biolegend; 354302; western blotting, 1:1000), rat polyclonal anti-galectin-3 conjugated to Alexa Fluor 647 (Biolegend; 125408; flow cytometry, 1:100) and rabbit polyclonal anti-mCherry antibody (Genetex; GTX128508-S). .. Reagents Reagents used were: cinnamycin (Santa Cruz Biotechnology; sc-391464), mastoparan X (Alfa Aesar; J61173), recombinant annexin-A5–FITC (Abcam; ab14085), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Sigma-Aldrich; 54008), 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC, Sigma-Aldrich; P3017), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE, Avanti Polar Lipids, 810145) and proteinase K (Molecular Biology; BP1700-100), EGTA (Sigma-Aldrich; E3889), sodium dithionite (Sigma-Aldrich; 71699), versene solution containing ethylenediaminetetraacetic acid (EDTA) (Gibco; 15040-033), propidium iodide solution (Biolegend; 421301), QuickExtract DNA extraction solution (Epicenter; QE0905T), Herculase II fusion DNA polymerase (Agilent; 600675), annexin-V–FITC and annexin-V–Cy5 Apoptosis Staining/Detection Kit (ab14085, ab14150), annexin-V conjugated to Alexa Fluor 647 (Biolegend; 640912) and ionomycin (Cayman Chemical Company; 10004974), recombinant galectin-3 (Biolegend; 599706) and recombinant anneaxin A5 (Novus NBP1-30265). .. Oligonucleotides for TMEM16F CRISPR targeting and sequencing were synthesised from Sigma-Aldrich ( Table S1 ).

    Staining:

    Article Title: Transbilayer phospholipid movement facilitates the translocation of annexin across membranes
    Article Snippet: Antibodies Antibodies used were: mouse monoclonal anti-annexin A2 (BD Biosciences; 610071; 1:1000), mouse monoclonal anti-annexin A5 (Abcam; ab54775, 1:1000), mouse monoclonal anti-transferrin receptor (Zymed; H68.4; 1:1000), rabbit polyclonal anti-actin (Sigma; A2066; 1:2000), mouse monoclonal anti-tubulin (Sigma; T9026; 1:4000), mouse monoclonal anti-FLAG (Sigma-Aldrich; clone M2; 1:4000), mouse monoclonal anti-Arf1 (Santa Cruz Biotechnology; sc-53168, 1:1000), rat anti-galectin-3 (Biolegend; 125401; western blotting, 1:2000) mouse monoclonal anti-Arf6 (Santa Cruz Biotechnology; clone 3A-1; 1:500), mouse monoclonal anti-LAMP2 (Biolegend; 354302; western blotting, 1:1000), rat polyclonal anti-galectin-3 conjugated to Alexa Fluor 647 (Biolegend; 125408; flow cytometry, 1:100) and rabbit polyclonal anti-mCherry antibody (Genetex; GTX128508-S). .. Reagents Reagents used were: cinnamycin (Santa Cruz Biotechnology; sc-391464), mastoparan X (Alfa Aesar; J61173), recombinant annexin-A5–FITC (Abcam; ab14085), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Sigma-Aldrich; 54008), 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC, Sigma-Aldrich; P3017), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE, Avanti Polar Lipids, 810145) and proteinase K (Molecular Biology; BP1700-100), EGTA (Sigma-Aldrich; E3889), sodium dithionite (Sigma-Aldrich; 71699), versene solution containing ethylenediaminetetraacetic acid (EDTA) (Gibco; 15040-033), propidium iodide solution (Biolegend; 421301), QuickExtract DNA extraction solution (Epicenter; QE0905T), Herculase II fusion DNA polymerase (Agilent; 600675), annexin-V–FITC and annexin-V–Cy5 Apoptosis Staining/Detection Kit (ab14085, ab14150), annexin-V conjugated to Alexa Fluor 647 (Biolegend; 640912) and ionomycin (Cayman Chemical Company; 10004974), recombinant galectin-3 (Biolegend; 599706) and recombinant anneaxin A5 (Novus NBP1-30265). .. Oligonucleotides for TMEM16F CRISPR targeting and sequencing were synthesised from Sigma-Aldrich ( Table S1 ).

    Transfection:

    Article Title: The Ebola-Glycoprotein Modulates the Function of Natural Killer Cells
    Article Snippet: To obtain GP or GP-GFP expressing cells, HEK293T cells were plated in 10-cm plates 24 h prior to transfection and transiently transfected using a calcium-phosphate-based reagent with 15 µg DNA per 10-cm plate. .. In all experiments floating cells were collected and pooled with Adherent Cells, harvested using a gentle non-enzymatic cell dissociation reagent (Versene, Gibco™, 15040-033), at 24 h post transfection. .. Antibodies, Fusion-Ig Proteins, and Reagents The following antibodies (Abs) and other materials were used: phycoerythrin (PE)-conjugated anti-human HLA-A, B, C (W6/32, BioLegend), PE-conjugated anti-human MICA (159227, R & D Systems), PE-conjugated anti-CD107a (H4A3, SouthernBiotech), PE-conjugated anti-human IgG (polyclonal, Jackson ImmunoResearch), FITC-conjugated anti-KIR2DL2 (CH-L, BD Biosciences), FITC-conjugated anti-human IgG (polyclonal, Jackson ImmunoResearch), Alexa Fluor 647-conjugated streptavidin (Jackson ImmunoResearch), allophycocyanin (APC)-conjugated anti-mouse IgG (polyclonal, Jackson ImmunoResearch), Pacific Blue-conjugated anti-human CD16, purified/biotinylated anti-SUDV-GP (3C10), purified anti-B7H6, Capture: purified anti-human IFN-γ (NIB42, BioLegend), Detection: biotin anti-human IFN-γ (4S.B3, BioLegend), 7-aminoactinomycin D (7AAD) (BioLegend), anti-phosphotyrosine 4G10, anti-PLCγ1 (Upstate), anti-SHP-1 (Santa Cruz), anti-GAPDH (Biodesign), and p -nitrophenyl phosphate (pNPP; New England BioLabs).

    Flow Cytometry:

    Article Title: Macromolecular Uptake of Alkyl Chain-Modified Guanidinoglycoside Molecular Transporters
    Article Snippet: For binding studies, cells were treated with the diluted conjugate solutions (300 μL) after removal of the medium and incubated for 30 min at 4°C. .. Cells were washed with ice-chilled phosphate buffered saline (PBS, 300 μL) twice, detached with Versene (EDTA, 100 μL, Life Technologies), diluted with PBS containing 5% BSA and analyzed by flow cytometry. ..

    Cytometry:

    Article Title: Macromolecular Uptake of Alkyl Chain-Modified Guanidinoglycoside Molecular Transporters
    Article Snippet: For binding studies, cells were treated with the diluted conjugate solutions (300 μL) after removal of the medium and incubated for 30 min at 4°C. .. Cells were washed with ice-chilled phosphate buffered saline (PBS, 300 μL) twice, detached with Versene (EDTA, 100 μL, Life Technologies), diluted with PBS containing 5% BSA and analyzed by flow cytometry. ..

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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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    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

    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

    Anti-rotaviral activity of RV6-26. (A) Inhibition of in vitro transcription: EDTA-activated DLP were incubated with 200 nM combining sites of different antibodies and mRNA was synthesized in vitro using selected components of the Riboprobe SP6 system (transcription was mediated by the viral RNA-dependent RNA polymerase not the SP6 DNA-dependent RNA polymerase). First-strand cDNA was synthesized by reverse transcription using a VP6-specific primer. Amplification of cDNA with VP6-specific primers was monitored in a real-time PCR using SYBR Green; the concentrations of RNA estimated from a standard curve constructed using reference RNA extracted from RRV are plotted. (B) Inhibition of rotavirus replication by IgA: polarized monolayers of Caco-2 cells grown on Transwell inserts were treated with polymeric IgA in the basal compartment and inoculated apically with trypsin-activated RRV (MOI = 5) at ambient temperature for 1 h and then cultured for 16 in medium containing trypsin. Amount of rotavirus in the inserts was titrated by inoculating MA104 cells and culturing for 16 h, followed by acetone-fixation and staining with anti-rotavirus polyclonal antibodies conjugated to either Alexa568 or IRDye 800. Detection was done either by scanning on Licor or by counting fluorescent foci.

    Journal: PLoS ONE

    Article Title: Human Rotavirus VP6-Specific Antibodies Mediate Intracellular Neutralization by Binding to a Quaternary Structure in the Transcriptional Pore

    doi: 10.1371/journal.pone.0061101

    Figure Lengend Snippet: Anti-rotaviral activity of RV6-26. (A) Inhibition of in vitro transcription: EDTA-activated DLP were incubated with 200 nM combining sites of different antibodies and mRNA was synthesized in vitro using selected components of the Riboprobe SP6 system (transcription was mediated by the viral RNA-dependent RNA polymerase not the SP6 DNA-dependent RNA polymerase). First-strand cDNA was synthesized by reverse transcription using a VP6-specific primer. Amplification of cDNA with VP6-specific primers was monitored in a real-time PCR using SYBR Green; the concentrations of RNA estimated from a standard curve constructed using reference RNA extracted from RRV are plotted. (B) Inhibition of rotavirus replication by IgA: polarized monolayers of Caco-2 cells grown on Transwell inserts were treated with polymeric IgA in the basal compartment and inoculated apically with trypsin-activated RRV (MOI = 5) at ambient temperature for 1 h and then cultured for 16 in medium containing trypsin. Amount of rotavirus in the inserts was titrated by inoculating MA104 cells and culturing for 16 h, followed by acetone-fixation and staining with anti-rotavirus polyclonal antibodies conjugated to either Alexa568 or IRDye 800. Detection was done either by scanning on Licor or by counting fluorescent foci.

    Article Snippet: Infection medium (IM), used when cells were inoculated with rotavirus and cultured, contained all of the above supplements except serum, and trypsin-EDTA (Invitrogen) was added to a final concentration of 1 µg/mL.

    Techniques: Activity Assay, Inhibition, In Vitro, Incubation, Synthesized, Amplification, Real-time Polymerase Chain Reaction, SYBR Green Assay, Construct, Cell Culture, Staining

    The effect of trypsinization on receptor expression. Lu1205 cells were detached from culture dishes by 0.05% trypsin-EDTA. Cells were incubated for 60 min in medium before being treated with trypsin for 5min. ICAM-1 (A) and α v β 3 (B) expressions

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

    Article Title: Sequential binding of ?v?3 and ICAM-1 determines fibrin-mediated melanoma capture and stable adhesion to CD11b/CD18 on neutrophils 1

    doi: 10.4049/jimmunol.1000494

    Figure Lengend Snippet: The effect of trypsinization on receptor expression. Lu1205 cells were detached from culture dishes by 0.05% trypsin-EDTA. Cells were incubated for 60 min in medium before being treated with trypsin for 5min. ICAM-1 (A) and α v β 3 (B) expressions

    Article Snippet: Prior to each experiment, Lu1205 cells were detached with 0.05% trypsin/EDTA (Invitrogen) and washed twice with fresh medium.

    Techniques: Expressing, Incubation