96 well culture plate  (Thermo Fisher)


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    Structured Review

    Thermo Fisher 96 well culture plate
    96 Well Culture Plate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/96 well culture plate/product/Thermo Fisher
    Average 99 stars, based on 22 article reviews
    Price from $9.99 to $1999.99
    96 well culture plate - by Bioz Stars, 2020-03
    99/100 stars

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    Related Articles

    MTT Assay:

    Article Title: Synthesis and evaluation of pyrazolone compounds as SARS-coronavirus 3C-like protease inhibitors.
    Article Snippet: Cytotoxicity assay Cell viability was determined by MTT 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide, 32 using Vybrant Ò MTT cell proliferation assay kit purchased from Molecular Probes, USA. .. Human embryonic kidney (HEK) 293 cells (2 Â 10 5 /ml) were seeded into a 96-well culture plate containing 0.1 ml of Minimum Essential Medium (MEM) (Gibico, Invitrogen, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibico) and cultured in 5% CO 2 at 37°C.

    In Vitro:

    Article Title: Adverse reproductive effects of S100A9 on bovine sperm and early embryonic development in vitro
    Article Snippet: Paragraph title: Sperm preparation and in vitro culture of sperm ... To investigate the effect of S100A9 on sperm viability, semen at a concentration of 2 × 104 cells/well, 100 μl/well, was seeded in a 96-well culture plate (Thermo Fisher Scientific Inc.) and treated with S100A9 (4 and 8 μg/ml) or same volume of distilled water as the control for 5 h at 38.5 °C in a humidified atmosphere with 5% CO2 .

    Fluorescence:

    Article Title: Adverse reproductive effects of S100A9 on bovine sperm and early embryonic development in vitro
    Article Snippet: To investigate the effect of S100A9 on sperm viability, semen at a concentration of 2 × 104 cells/well, 100 μl/well, was seeded in a 96-well culture plate (Thermo Fisher Scientific Inc.) and treated with S100A9 (4 and 8 μg/ml) or same volume of distilled water as the control for 5 h at 38.5 °C in a humidified atmosphere with 5% CO2 . .. Living sperm were showed bright green fluorescence, whereas dead and dying sperm produced red and yellow fluorescence, respectively.

    Cell Culture:

    Article Title: Synthesis and evaluation of pyrazolone compounds as SARS-coronavirus 3C-like protease inhibitors.
    Article Snippet: .. Human embryonic kidney (HEK) 293 cells (2 Â 10 5 /ml) were seeded into a 96-well culture plate containing 0.1 ml of Minimum Essential Medium (MEM) (Gibico, Invitrogen, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibico) and cultured in 5% CO 2 at 37°C. .. Cytotoxicity assay After the incubation, 10 lL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) stock solution was added into each well.

    Cytotoxicity Assay:

    Article Title: Synthesis and evaluation of pyrazolone compounds as SARS-coronavirus 3C-like protease inhibitors.
    Article Snippet: Paragraph title: Cytotoxicity assay ... Human embryonic kidney (HEK) 293 cells (2 Â 10 5 /ml) were seeded into a 96-well culture plate containing 0.1 ml of Minimum Essential Medium (MEM) (Gibico, Invitrogen, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibico) and cultured in 5% CO 2 at 37°C.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Synthesis and evaluation of pyrazolone compounds as SARS-coronavirus 3C-like protease inhibitors.
    Article Snippet: Human embryonic kidney (HEK) 293 cells (2 Â 10 5 /ml) were seeded into a 96-well culture plate containing 0.1 ml of Minimum Essential Medium (MEM) (Gibico, Invitrogen, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibico) and cultured in 5% CO 2 at 37°C. .. The levels of formazan were determined by optical density at 540 nm using an ELISA reader and represented as cell viability.

    Concentration Assay:

    Article Title: Adverse reproductive effects of S100A9 on bovine sperm and early embryonic development in vitro
    Article Snippet: .. To investigate the effect of S100A9 on sperm viability, semen at a concentration of 2 × 104 cells/well, 100 μl/well, was seeded in a 96-well culture plate (Thermo Fisher Scientific Inc.) and treated with S100A9 (4 and 8 μg/ml) or same volume of distilled water as the control for 5 h at 38.5 °C in a humidified atmosphere with 5% CO2 . .. In the present study, IVF was performed for 5 h due to suppress polyspermy fertilization, so the same time was set for sperm exposure of S100A9.

    Incubation:

    Article Title: Synthesis and evaluation of pyrazolone compounds as SARS-coronavirus 3C-like protease inhibitors.
    Article Snippet: Human embryonic kidney (HEK) 293 cells (2 Â 10 5 /ml) were seeded into a 96-well culture plate containing 0.1 ml of Minimum Essential Medium (MEM) (Gibico, Invitrogen, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibico) and cultured in 5% CO 2 at 37°C. .. Cytotoxicity assay After the incubation, 10 lL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) stock solution was added into each well.

    Proliferation Assay:

    Article Title: Synthesis and evaluation of pyrazolone compounds as SARS-coronavirus 3C-like protease inhibitors.
    Article Snippet: Cytotoxicity assay Cell viability was determined by MTT 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide, 32 using Vybrant Ò MTT cell proliferation assay kit purchased from Molecular Probes, USA. .. Human embryonic kidney (HEK) 293 cells (2 Â 10 5 /ml) were seeded into a 96-well culture plate containing 0.1 ml of Minimum Essential Medium (MEM) (Gibico, Invitrogen, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibico) and cultured in 5% CO 2 at 37°C.

    Produced:

    Article Title: Adverse reproductive effects of S100A9 on bovine sperm and early embryonic development in vitro
    Article Snippet: To investigate the effect of S100A9 on sperm viability, semen at a concentration of 2 × 104 cells/well, 100 μl/well, was seeded in a 96-well culture plate (Thermo Fisher Scientific Inc.) and treated with S100A9 (4 and 8 μg/ml) or same volume of distilled water as the control for 5 h at 38.5 °C in a humidified atmosphere with 5% CO2 . .. Living sperm were showed bright green fluorescence, whereas dead and dying sperm produced red and yellow fluorescence, respectively.

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    Thermo Fisher 96 well culture plate
    Spheroids growth using different techniques, a comparison between spheroids that were prepared using the following formats: 96 well plate with agarose; Rounded bottom 96 well plate; 3D petri dish with 35 wells; Hanging drop method. Spheroids were made of 5,000 cells per spheroid. Assembly time was 3 days for all methods except the hanging drop method which was 5 days. Bar = 100 µm.
    96 Well Culture Plate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/96 well culture plate/product/Thermo Fisher
    Average 99 stars, based on 22 article reviews
    Price from $9.99 to $1999.99
    96 well culture plate - by Bioz Stars, 2020-03
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher viral genomic rna
    ABT-263 induces the premature death of cells <t>transfected</t> with IAV genomic <t>RNA</t> (vRNA) or plasmid DNA (pDNA). ( A ) Fluorescent microscopy images showing that ABT-263 kills vRNA-transfected (160 ng) but not mock-transfected RPE cells at 8 h post transfection. Asymmetric cyanine dye stains the dsDNA of dead cells. Hoechst stains DNA in living cells; ( B ) CTxG plot showing that ABT-263 (3 µM) induces that premature death of RPE cells transfected with increasing concentrations of vRNA. Mean ± SD, n = 3; ( C ) Fluorescent and bright field microscopy of RPE cells showing that ABT-263 kills eGFP-expressing plasmid transfected (300 ng) but not mock-transfected RPE cells at 6 h post transfection; ( D ) CTG graph showing that the viability of ABT-263-treated (3 µM) cells decreases with increasing concentrations of transfected plasmid DNA. Mean ± SD, n = 3.
    Viral Genomic Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/viral genomic rna/product/Thermo Fisher
    Average 99 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    viral genomic rna - by Bioz Stars, 2020-03
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    99
    Thermo Fisher elisa plates
    Description and characterization of the chimeric human <t>FasL-derived</t> constructs Panel A: Schematic representation of soluble FasL (sFasL), Flag-tagged sFasL (sfFasL), polymeric Flag-tagged soluble FasL (pfFasL), polymeric TCR γ4 and δ5 Flag-tagged soluble FasL generating the TCR-pfFasL upon cotransfection, and beta2-microglobulin-fused HLA-A*02: 01 Flag-tagged soluble FasL (HLA-pfFasL). The f and p symbols represent the flag epitope and the LIF receptor-derived domain triggering the polymerisation of the FasL oligomers, respectively. Panel B: direct immunoblot of the supernatants from COS cells transfected with the empty vector (control) or the FasL constructs sFasL, sfFasL and pfFasL. Panel C: immunoprecipitation of the TCR-pfFasL chimera from transfected HEK cells, using an irrelevant IgG1 antibody, the anti-Flag (clone M2), the anti-FasL (clone 10F2), the anti-TCRγδ (clone IMU-510) or the anti-TCRδ5 (clone 12C7) antibodies. Panel D: immunoprecipitation of the HLA-pfFasL chimera from the supernatant of COS cells, with anti-Flag, anti-FasL or anti-β2microglobulin antibodies. As controls, the same experiment was performed with irrelevant IgG1 and IgG2 antibodies. Panel E: cytotoxic effect of the FasL chimeras. The indicated chimeras, as supernatants from transfected cells and quantitated using the <t>ELISA</t> for FasL, were incubated at the indicated concentrations with Jurkat cells. After 18 h, the MTT cell viability assay was performed. The anti-Flag M2 antibody at 0.5 µg/ml was added to sfFasL to render it cytotoxic.
    Elisa Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 430 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/elisa plates/product/Thermo Fisher
    Average 99 stars, based on 430 article reviews
    Price from $9.99 to $1999.99
    elisa plates - by Bioz Stars, 2020-03
    99/100 stars
      Buy from Supplier

    Image Search Results


    Spheroids growth using different techniques, a comparison between spheroids that were prepared using the following formats: 96 well plate with agarose; Rounded bottom 96 well plate; 3D petri dish with 35 wells; Hanging drop method. Spheroids were made of 5,000 cells per spheroid. Assembly time was 3 days for all methods except the hanging drop method which was 5 days. Bar = 100 µm.

    Journal: Scientific Reports

    Article Title: Tumor cells and their crosstalk with endothelial cells in 3D spheroids

    doi: 10.1038/s41598-017-10699-y

    Figure Lengend Snippet: Spheroids growth using different techniques, a comparison between spheroids that were prepared using the following formats: 96 well plate with agarose; Rounded bottom 96 well plate; 3D petri dish with 35 wells; Hanging drop method. Spheroids were made of 5,000 cells per spheroid. Assembly time was 3 days for all methods except the hanging drop method which was 5 days. Bar = 100 µm.

    Article Snippet: Spheroids formation using multi-well agarose-coated plates Agarose hydrogel 1.5% (100 µl) was added to each well of a 96-well culture plate (Thermo Fisher Scientific, Denmark), and incubated at 37 °C for 2 hours.

    Techniques:

    ABT-263 induces the premature death of cells transfected with IAV genomic RNA (vRNA) or plasmid DNA (pDNA). ( A ) Fluorescent microscopy images showing that ABT-263 kills vRNA-transfected (160 ng) but not mock-transfected RPE cells at 8 h post transfection. Asymmetric cyanine dye stains the dsDNA of dead cells. Hoechst stains DNA in living cells; ( B ) CTxG plot showing that ABT-263 (3 µM) induces that premature death of RPE cells transfected with increasing concentrations of vRNA. Mean ± SD, n = 3; ( C ) Fluorescent and bright field microscopy of RPE cells showing that ABT-263 kills eGFP-expressing plasmid transfected (300 ng) but not mock-transfected RPE cells at 6 h post transfection; ( D ) CTG graph showing that the viability of ABT-263-treated (3 µM) cells decreases with increasing concentrations of transfected plasmid DNA. Mean ± SD, n = 3.

    Journal: Viruses

    Article Title: Antiviral Properties of Chemical Inhibitors of Cellular Anti-Apoptotic Bcl-2 Proteins

    doi: 10.3390/v9100271

    Figure Lengend Snippet: ABT-263 induces the premature death of cells transfected with IAV genomic RNA (vRNA) or plasmid DNA (pDNA). ( A ) Fluorescent microscopy images showing that ABT-263 kills vRNA-transfected (160 ng) but not mock-transfected RPE cells at 8 h post transfection. Asymmetric cyanine dye stains the dsDNA of dead cells. Hoechst stains DNA in living cells; ( B ) CTxG plot showing that ABT-263 (3 µM) induces that premature death of RPE cells transfected with increasing concentrations of vRNA. Mean ± SD, n = 3; ( C ) Fluorescent and bright field microscopy of RPE cells showing that ABT-263 kills eGFP-expressing plasmid transfected (300 ng) but not mock-transfected RPE cells at 6 h post transfection; ( D ) CTG graph showing that the viability of ABT-263-treated (3 µM) cells decreases with increasing concentrations of transfected plasmid DNA. Mean ± SD, n = 3.

    Article Snippet: Transfections of RPE Cells with vRNA or Plasmid DNA RPE cells were cultured to 80% confluence in 96 well plates and transfected with 160 ng viral genomic RNA using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA) or with 30, 100, or 300 ng of plasmid DNA (pEGFP) using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Transfection, Plasmid Preparation, Microscopy, Expressing, CTG Assay

    Description and characterization of the chimeric human FasL-derived constructs Panel A: Schematic representation of soluble FasL (sFasL), Flag-tagged sFasL (sfFasL), polymeric Flag-tagged soluble FasL (pfFasL), polymeric TCR γ4 and δ5 Flag-tagged soluble FasL generating the TCR-pfFasL upon cotransfection, and beta2-microglobulin-fused HLA-A*02: 01 Flag-tagged soluble FasL (HLA-pfFasL). The f and p symbols represent the flag epitope and the LIF receptor-derived domain triggering the polymerisation of the FasL oligomers, respectively. Panel B: direct immunoblot of the supernatants from COS cells transfected with the empty vector (control) or the FasL constructs sFasL, sfFasL and pfFasL. Panel C: immunoprecipitation of the TCR-pfFasL chimera from transfected HEK cells, using an irrelevant IgG1 antibody, the anti-Flag (clone M2), the anti-FasL (clone 10F2), the anti-TCRγδ (clone IMU-510) or the anti-TCRδ5 (clone 12C7) antibodies. Panel D: immunoprecipitation of the HLA-pfFasL chimera from the supernatant of COS cells, with anti-Flag, anti-FasL or anti-β2microglobulin antibodies. As controls, the same experiment was performed with irrelevant IgG1 and IgG2 antibodies. Panel E: cytotoxic effect of the FasL chimeras. The indicated chimeras, as supernatants from transfected cells and quantitated using the ELISA for FasL, were incubated at the indicated concentrations with Jurkat cells. After 18 h, the MTT cell viability assay was performed. The anti-Flag M2 antibody at 0.5 µg/ml was added to sfFasL to render it cytotoxic.

    Journal: PLoS ONE

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL

    doi: 10.1371/journal.pone.0073375

    Figure Lengend Snippet: Description and characterization of the chimeric human FasL-derived constructs Panel A: Schematic representation of soluble FasL (sFasL), Flag-tagged sFasL (sfFasL), polymeric Flag-tagged soluble FasL (pfFasL), polymeric TCR γ4 and δ5 Flag-tagged soluble FasL generating the TCR-pfFasL upon cotransfection, and beta2-microglobulin-fused HLA-A*02: 01 Flag-tagged soluble FasL (HLA-pfFasL). The f and p symbols represent the flag epitope and the LIF receptor-derived domain triggering the polymerisation of the FasL oligomers, respectively. Panel B: direct immunoblot of the supernatants from COS cells transfected with the empty vector (control) or the FasL constructs sFasL, sfFasL and pfFasL. Panel C: immunoprecipitation of the TCR-pfFasL chimera from transfected HEK cells, using an irrelevant IgG1 antibody, the anti-Flag (clone M2), the anti-FasL (clone 10F2), the anti-TCRγδ (clone IMU-510) or the anti-TCRδ5 (clone 12C7) antibodies. Panel D: immunoprecipitation of the HLA-pfFasL chimera from the supernatant of COS cells, with anti-Flag, anti-FasL or anti-β2microglobulin antibodies. As controls, the same experiment was performed with irrelevant IgG1 and IgG2 antibodies. Panel E: cytotoxic effect of the FasL chimeras. The indicated chimeras, as supernatants from transfected cells and quantitated using the ELISA for FasL, were incubated at the indicated concentrations with Jurkat cells. After 18 h, the MTT cell viability assay was performed. The anti-Flag M2 antibody at 0.5 µg/ml was added to sfFasL to render it cytotoxic.

    Article Snippet: The anti-FasL 14C2 or the anti-Flag mAbs were pre-coated overnight onto 96 well ELISA plates (Maxisorp Nunc, Thermo Scientific, Rochester, USA) respectively at 1 µg or 0.25 µg/well in hydrogenocarbonate coating buffer (pH = 9.6).

    Techniques: Derivative Assay, Construct, Cotransfection, FLAG-tag, Transfection, Plasmid Preparation, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Incubation, MTT Assay, Viability Assay

    Direct association of sFasL to the pfFasL-containing chimeric proteins during co-expression. Panel A: Identical amounts of pfFasL (1 µg, according to the Flag ELISA) produced in the presence of the indicated ratios of added sFasL plasmid (left panels) was immunoprecipitated with the anti-FasL (upper panel) or anti-Flag (lower panel) antibodies, followed by a SDS-PAGE under reducing conditions and immunoblotting with an anti-FasL antibody. As a control, the same experiment was performed for the sFasL molecule (3 µg according to the FasL ELISA, right panel). Panel B: Densitometric detection and quantification of the pfFasL (grey bars) and the sFasL (black bars) fractions, following transfection of the pfFasL plasmid in the presence of the indicated proportion of the sFasL plasmid. The measures were normalized to the condition lacking sFasL. Mean+/- sd from three experiments. Panel C: The TCR-pfFasL chimera (2 µg, according to an ELISA specific for the TCR-pFasL molecule using anti-TCRδ5 (clone 12C7) and anti-FasL (clone 10F2) as capture and tracing antibodies, respectively), produced in the absence or the presence of the sFasL plasmid at the indicated ratio, was immunoprecipitated with the anti-TCRδ5 antibody, then separated by 10% SDS-PAGE under reducing conditions and revealed by immunoblotting with the anti-FasL antibody. As a control, the immunoprecipitation experiment was performed with 2 µg of sFasL protein. Panel D: COS supernatants containing pfFasL (4 µg/ml according to the Flag ELISA) produced alone, was mixed with culture medium or sFasL (15 µg/ml) produced separately in a total volume of 1 ml, and incubated for 24 h at 37°C. Then the recombinant proteins were immunoprecipitated (left panels) with the anti-FasL (upper panel) or anti-Flag (lower panel) antibodies, followed by a SDS-PAGE under reducing conditions and immunoblotting with an anti-FasL antibody. As a control, the same experiment was performed for the sFasL molecule (15 µg according to the FasL ELISA, right panel).

    Journal: PLoS ONE

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL

    doi: 10.1371/journal.pone.0073375

    Figure Lengend Snippet: Direct association of sFasL to the pfFasL-containing chimeric proteins during co-expression. Panel A: Identical amounts of pfFasL (1 µg, according to the Flag ELISA) produced in the presence of the indicated ratios of added sFasL plasmid (left panels) was immunoprecipitated with the anti-FasL (upper panel) or anti-Flag (lower panel) antibodies, followed by a SDS-PAGE under reducing conditions and immunoblotting with an anti-FasL antibody. As a control, the same experiment was performed for the sFasL molecule (3 µg according to the FasL ELISA, right panel). Panel B: Densitometric detection and quantification of the pfFasL (grey bars) and the sFasL (black bars) fractions, following transfection of the pfFasL plasmid in the presence of the indicated proportion of the sFasL plasmid. The measures were normalized to the condition lacking sFasL. Mean+/- sd from three experiments. Panel C: The TCR-pfFasL chimera (2 µg, according to an ELISA specific for the TCR-pFasL molecule using anti-TCRδ5 (clone 12C7) and anti-FasL (clone 10F2) as capture and tracing antibodies, respectively), produced in the absence or the presence of the sFasL plasmid at the indicated ratio, was immunoprecipitated with the anti-TCRδ5 antibody, then separated by 10% SDS-PAGE under reducing conditions and revealed by immunoblotting with the anti-FasL antibody. As a control, the immunoprecipitation experiment was performed with 2 µg of sFasL protein. Panel D: COS supernatants containing pfFasL (4 µg/ml according to the Flag ELISA) produced alone, was mixed with culture medium or sFasL (15 µg/ml) produced separately in a total volume of 1 ml, and incubated for 24 h at 37°C. Then the recombinant proteins were immunoprecipitated (left panels) with the anti-FasL (upper panel) or anti-Flag (lower panel) antibodies, followed by a SDS-PAGE under reducing conditions and immunoblotting with an anti-FasL antibody. As a control, the same experiment was performed for the sFasL molecule (15 µg according to the FasL ELISA, right panel).

    Article Snippet: The anti-FasL 14C2 or the anti-Flag mAbs were pre-coated overnight onto 96 well ELISA plates (Maxisorp Nunc, Thermo Scientific, Rochester, USA) respectively at 1 µg or 0.25 µg/well in hydrogenocarbonate coating buffer (pH = 9.6).

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Produced, Plasmid Preparation, Immunoprecipitation, SDS Page, Transfection, Incubation, Recombinant

    Effect of sFasL on the supernatant production of the Flag-tagged FasL constructs. Panels A to D : An increasing amount expressed in percentage, of the sFasL encoding plasmid, was co-transfected with a fixed amount of the plasmids encoding sfFasL (Panel A), pfFasL (Panel B), TCR-pfFasL (Panel C) and HLA-pfFasL (Panel D). The secreted proteins were quantified in culture supernatants using an ELISA specific for FasL (shaded histograms, right-hand scale) and for Flag-tagged FasL (curves, left-hand scale). For the Flag ELISA, the measured concentrations were normalized according to the condition lacking sFasL. Are presented the mean +/- sd of four independent transfection experiments. * 0.02≤p≤0.05; ** p≤0.02. Panel E : direct anti-FasL immunoblot analysis of identical volumes of the cell culture supernatant containing pfFasL produced alone and with 50% of the sFasL plasmid, after SDS-PAGE separation under reducing conditions.

    Journal: PLoS ONE

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL

    doi: 10.1371/journal.pone.0073375

    Figure Lengend Snippet: Effect of sFasL on the supernatant production of the Flag-tagged FasL constructs. Panels A to D : An increasing amount expressed in percentage, of the sFasL encoding plasmid, was co-transfected with a fixed amount of the plasmids encoding sfFasL (Panel A), pfFasL (Panel B), TCR-pfFasL (Panel C) and HLA-pfFasL (Panel D). The secreted proteins were quantified in culture supernatants using an ELISA specific for FasL (shaded histograms, right-hand scale) and for Flag-tagged FasL (curves, left-hand scale). For the Flag ELISA, the measured concentrations were normalized according to the condition lacking sFasL. Are presented the mean +/- sd of four independent transfection experiments. * 0.02≤p≤0.05; ** p≤0.02. Panel E : direct anti-FasL immunoblot analysis of identical volumes of the cell culture supernatant containing pfFasL produced alone and with 50% of the sFasL plasmid, after SDS-PAGE separation under reducing conditions.

    Article Snippet: The anti-FasL 14C2 or the anti-Flag mAbs were pre-coated overnight onto 96 well ELISA plates (Maxisorp Nunc, Thermo Scientific, Rochester, USA) respectively at 1 µg or 0.25 µg/well in hydrogenocarbonate coating buffer (pH = 9.6).

    Techniques: Construct, Plasmid Preparation, Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture, Produced, SDS Page

    Effect of sFasL on cell targeting of the FasL-containing chimeras. Panel A : Schematic description of the experimental model used. The chimera is enriched at the surface of the CD32-expressing L-cells via its HLA targeting module and an anti-HLA monoclonal antibody. Panel B: murine Fas (continuous line), human CD32 (dashed line) and IgG1 isotype-matched control (shaded histogram) staining of the CD32+ L-cell transfectant. Living cells were gated on the basis of the morphological parameters. Panel C : Fas sensitivity of the CD32+ L-cell transfectant to the indicated concentrations of the anti-Fas JO-2 antibody (circles), the HLA-pfFasL chimera expressed alone (triangle) or in the presence of 25% of the sFasL plasmid (squares), in the MTT viability assay. Panel D : The CD32+ L-cells were incubated with the HLA-pfFasL chimera produced in the presence (black bars) or in the absence (white bars) of 25% of the sFasL plasmid, together with the indicated irrelevant IgG1 isotype-matched, anti-beta-2 microglobulin or anti-Flag antibodies. The concentrations of the chimera that triggered 15% of cell death and were at 15 and 0.3 ng/ml in the absence and presence of sFasL, as estimated using the ELISA specific for the Flag-tagged FasL. Cytotoxicity was measured with the propidium iodide assay and normalized to the effect of the chimera in the absence of antibody. Are presented the mean +/- sd of three independent experiments. Panel E: reversal in the presence of the blocking anti-FasL and anti-CD32 antibodies, of the cytotoxic effect of the immune complexes between the anti-Flag antibody and HLA-pfFasL co-expressed with sFasL. Are presented the mean +/- sd of three independent experiments. ns : non significant ; ** p≤0.02.

    Journal: PLoS ONE

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL

    doi: 10.1371/journal.pone.0073375

    Figure Lengend Snippet: Effect of sFasL on cell targeting of the FasL-containing chimeras. Panel A : Schematic description of the experimental model used. The chimera is enriched at the surface of the CD32-expressing L-cells via its HLA targeting module and an anti-HLA monoclonal antibody. Panel B: murine Fas (continuous line), human CD32 (dashed line) and IgG1 isotype-matched control (shaded histogram) staining of the CD32+ L-cell transfectant. Living cells were gated on the basis of the morphological parameters. Panel C : Fas sensitivity of the CD32+ L-cell transfectant to the indicated concentrations of the anti-Fas JO-2 antibody (circles), the HLA-pfFasL chimera expressed alone (triangle) or in the presence of 25% of the sFasL plasmid (squares), in the MTT viability assay. Panel D : The CD32+ L-cells were incubated with the HLA-pfFasL chimera produced in the presence (black bars) or in the absence (white bars) of 25% of the sFasL plasmid, together with the indicated irrelevant IgG1 isotype-matched, anti-beta-2 microglobulin or anti-Flag antibodies. The concentrations of the chimera that triggered 15% of cell death and were at 15 and 0.3 ng/ml in the absence and presence of sFasL, as estimated using the ELISA specific for the Flag-tagged FasL. Cytotoxicity was measured with the propidium iodide assay and normalized to the effect of the chimera in the absence of antibody. Are presented the mean +/- sd of three independent experiments. Panel E: reversal in the presence of the blocking anti-FasL and anti-CD32 antibodies, of the cytotoxic effect of the immune complexes between the anti-Flag antibody and HLA-pfFasL co-expressed with sFasL. Are presented the mean +/- sd of three independent experiments. ns : non significant ; ** p≤0.02.

    Article Snippet: The anti-FasL 14C2 or the anti-Flag mAbs were pre-coated overnight onto 96 well ELISA plates (Maxisorp Nunc, Thermo Scientific, Rochester, USA) respectively at 1 µg or 0.25 µg/well in hydrogenocarbonate coating buffer (pH = 9.6).

    Techniques: Expressing, Staining, Transfection, Plasmid Preparation, MTT Assay, Viability Assay, Incubation, Produced, Enzyme-linked Immunosorbent Assay, Blocking Assay

    Effect of sFasL on the cytotoxic activity of the Flag-tagged FasL chimeras. The FasL-derived proteins sfFasL (Panel A), pfFasL (Panel B), TCR-pfFasL (Panel C) and HLA-pfFasL (Panel D) were expressed alone or upon co-transfection with the indicated percentage of the plasmid encoding sFasL. A fixed concentration triggering 25 to 40% of cell death (1.9 ng/ml for sfFasL, 0.6 ng/ml for pfFasL, 0.7 ng/ml for HLA-pfFasL and 2.2 ng/ml for TCR-pfFasL), for the FasL-derived protein quantitated with the ELISA specific for Flag-tagged FasL, was incubated with the Fas-sensitive Jurkat cells. For the sfFasL construct, the filled squares and the empty squares depict the cytotoxicity of sfFasL in the presence and absence of the cross-linking anti-Flag antibody at 0.5 µg/ml), respectively. Cytotoxicity was estimated by a measure of the remaining viable cells using the MTT assay. Are presented the mean +/- sd of four independent transfection experiments. * 0.01≤p≤0.05; ** p≤0.01.

    Journal: PLoS ONE

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL

    doi: 10.1371/journal.pone.0073375

    Figure Lengend Snippet: Effect of sFasL on the cytotoxic activity of the Flag-tagged FasL chimeras. The FasL-derived proteins sfFasL (Panel A), pfFasL (Panel B), TCR-pfFasL (Panel C) and HLA-pfFasL (Panel D) were expressed alone or upon co-transfection with the indicated percentage of the plasmid encoding sFasL. A fixed concentration triggering 25 to 40% of cell death (1.9 ng/ml for sfFasL, 0.6 ng/ml for pfFasL, 0.7 ng/ml for HLA-pfFasL and 2.2 ng/ml for TCR-pfFasL), for the FasL-derived protein quantitated with the ELISA specific for Flag-tagged FasL, was incubated with the Fas-sensitive Jurkat cells. For the sfFasL construct, the filled squares and the empty squares depict the cytotoxicity of sfFasL in the presence and absence of the cross-linking anti-Flag antibody at 0.5 µg/ml), respectively. Cytotoxicity was estimated by a measure of the remaining viable cells using the MTT assay. Are presented the mean +/- sd of four independent transfection experiments. * 0.01≤p≤0.05; ** p≤0.01.

    Article Snippet: The anti-FasL 14C2 or the anti-Flag mAbs were pre-coated overnight onto 96 well ELISA plates (Maxisorp Nunc, Thermo Scientific, Rochester, USA) respectively at 1 µg or 0.25 µg/well in hydrogenocarbonate coating buffer (pH = 9.6).

    Techniques: Activity Assay, Derivative Assay, Cotransfection, Plasmid Preparation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Construct, MTT Assay, Transfection