nunc maxisorb 96 well plates  (Thermo Fisher)


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    Structured Review

    Thermo Fisher nunc maxisorb 96 well plates
    Nunc Maxisorb 96 Well Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nunc maxisorb 96 well plates/product/Thermo Fisher
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nunc maxisorb 96 well plates - by Bioz Stars, 2020-04
    85/100 stars

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    Related Articles

    Transfection:

    Article Title: Increased Immunogenicity and Protective Efficacy of Influenza M2e Fused to a Tetramerizing Protein
    Article Snippet: HeLa-M2 ELISA HeLa enzyme-linked immunosorbent assay (ELISA) plates were prepared as follows: HeLa cells transfected with M2-expressing plasmid (HeLa-M2) encoding a tetracycline response element (Tet) and HeLa cells with an empty plasmid (HeLa-C10) were obtained from Robert B. Couch . .. The cells were grown to confluency in Nunc Maxisorb™ 96-well plates by seeding wells with 1×105 HeLa cells in 150 µL of Iscove’s Modified Dulbecco’s medium (Invitrogen) supplemented with 2-mercaptoethanol at 0.05 mM, transferrin (Sigma-Aldrich) at 0.005 mg/mL, gentamicin (Riedel-de Haën, Seelze, Germany) at 0.05 mg/mL, and 10% fetal bovine serum (FBS) (Lonza, Copenhagen, Denmark).

    Avidin-Biotin Assay:

    Article Title: Regulation of dendritic cell interleukin-12 secretion by tumour cell necrosis
    Article Snippet: Briefly, NUNC-maxisorb 96-well plates (G ibco Life Technologies, Paisley, UK) were coated with purified antihuman IL-12p40 (clone C8·3) or purified antihuman IL-12p70 (clone 20C2) or rat antihuman IL-10 monoclonal antibody (clone JES3–9D7) and incubated overnight at 4°C. .. Cytokine binding was then detected with biotinylated-mouse monoclonal antihuman IL-12 (p40/p70) detection antibody (clone C8·6) or rat antihuman IL-10 clone JES3–12G8) or mouse antihuman IFN-γ (clone 4S.B3), followed by horseradish peroxidase-conjugated avidin (Vector Laboratories, Peterborough, UK) and a substrate solution containing 150 mg 2,2-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid, pH 4·5, mixed with H2 O2 at a final concentration of 0·03%).

    Purification:

    Article Title: Regulation of dendritic cell interleukin-12 secretion by tumour cell necrosis
    Article Snippet: .. Briefly, NUNC-maxisorb 96-well plates (G ibco Life Technologies, Paisley, UK) were coated with purified antihuman IL-12p40 (clone C8·3) or purified antihuman IL-12p70 (clone 20C2) or rat antihuman IL-10 monoclonal antibody (clone JES3–9D7) and incubated overnight at 4°C. ..

    Enzyme-linked Immunosorbent Assay:

    Article Title: Increased Immunogenicity and Protective Efficacy of Influenza M2e Fused to a Tetramerizing Protein
    Article Snippet: Paragraph title: HeLa-M2 ELISA ... The cells were grown to confluency in Nunc Maxisorb™ 96-well plates by seeding wells with 1×105 HeLa cells in 150 µL of Iscove’s Modified Dulbecco’s medium (Invitrogen) supplemented with 2-mercaptoethanol at 0.05 mM, transferrin (Sigma-Aldrich) at 0.005 mg/mL, gentamicin (Riedel-de Haën, Seelze, Germany) at 0.05 mg/mL, and 10% fetal bovine serum (FBS) (Lonza, Copenhagen, Denmark).

    Article Title: Regulation of dendritic cell interleukin-12 secretion by tumour cell necrosis
    Article Snippet: Cytokine production from the stimulated and the unstimulated cells was measured in culture supernatants collected after 48 h by ELISA. .. Briefly, NUNC-maxisorb 96-well plates (G ibco Life Technologies, Paisley, UK) were coated with purified antihuman IL-12p40 (clone C8·3) or purified antihuman IL-12p70 (clone 20C2) or rat antihuman IL-10 monoclonal antibody (clone JES3–9D7) and incubated overnight at 4°C.

    Concentration Assay:

    Article Title: Regulation of dendritic cell interleukin-12 secretion by tumour cell necrosis
    Article Snippet: Briefly, NUNC-maxisorb 96-well plates (G ibco Life Technologies, Paisley, UK) were coated with purified antihuman IL-12p40 (clone C8·3) or purified antihuman IL-12p70 (clone 20C2) or rat antihuman IL-10 monoclonal antibody (clone JES3–9D7) and incubated overnight at 4°C. .. Cytokine binding was then detected with biotinylated-mouse monoclonal antihuman IL-12 (p40/p70) detection antibody (clone C8·6) or rat antihuman IL-10 clone JES3–12G8) or mouse antihuman IFN-γ (clone 4S.B3), followed by horseradish peroxidase-conjugated avidin (Vector Laboratories, Peterborough, UK) and a substrate solution containing 150 mg 2,2-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid, pH 4·5, mixed with H2 O2 at a final concentration of 0·03%).

    Incubation:

    Article Title: Bacterial Polysaccharide Specificity of the Pattern Recognition Receptor Langerin Is Highly Species-dependent *
    Article Snippet: .. For mannan plate assays, NUNC Maxisorb 96-well plates (Thermo Fisher Scientific) were coated with 10 μg/ml mannan in 50 m m carbonate buffer, pH 9.6, blocked with 2% BSA in TBS-T for 1 h. Wells were washed three times with TBS-T + 5 m m CaCl2 , incubated with protein in the same buffer for at least 90 min at room temperature, washed, and incubated with StrepTactin-HRP conjugate (IBA) in 2% BSA in TBS-T + 5 m m CaCl2 for 1 h. Plates were developed by adding TMB solution (tebu-bio). ..

    Article Title: Increased Immunogenicity and Protective Efficacy of Influenza M2e Fused to a Tetramerizing Protein
    Article Snippet: The cells were grown to confluency in Nunc Maxisorb™ 96-well plates by seeding wells with 1×105 HeLa cells in 150 µL of Iscove’s Modified Dulbecco’s medium (Invitrogen) supplemented with 2-mercaptoethanol at 0.05 mM, transferrin (Sigma-Aldrich) at 0.005 mg/mL, gentamicin (Riedel-de Haën, Seelze, Germany) at 0.05 mg/mL, and 10% fetal bovine serum (FBS) (Lonza, Copenhagen, Denmark). .. After two days of incubation at 37°C in air/CO2 , the media was flicked out and the cells were fixed with 0.05% glutaraldehyde (Sigma-Aldrich) for 20 min at RT.

    Article Title: Regulation of dendritic cell interleukin-12 secretion by tumour cell necrosis
    Article Snippet: .. Briefly, NUNC-maxisorb 96-well plates (G ibco Life Technologies, Paisley, UK) were coated with purified antihuman IL-12p40 (clone C8·3) or purified antihuman IL-12p70 (clone 20C2) or rat antihuman IL-10 monoclonal antibody (clone JES3–9D7) and incubated overnight at 4°C. ..

    Modification:

    Article Title: Increased Immunogenicity and Protective Efficacy of Influenza M2e Fused to a Tetramerizing Protein
    Article Snippet: .. The cells were grown to confluency in Nunc Maxisorb™ 96-well plates by seeding wells with 1×105 HeLa cells in 150 µL of Iscove’s Modified Dulbecco’s medium (Invitrogen) supplemented with 2-mercaptoethanol at 0.05 mM, transferrin (Sigma-Aldrich) at 0.005 mg/mL, gentamicin (Riedel-de Haën, Seelze, Germany) at 0.05 mg/mL, and 10% fetal bovine serum (FBS) (Lonza, Copenhagen, Denmark). .. The media was further supplemented with 1 µg/mL doxycycline (Sigma-Aldrich) for induction of Tet.

    Binding Assay:

    Article Title: Regulation of dendritic cell interleukin-12 secretion by tumour cell necrosis
    Article Snippet: Briefly, NUNC-maxisorb 96-well plates (G ibco Life Technologies, Paisley, UK) were coated with purified antihuman IL-12p40 (clone C8·3) or purified antihuman IL-12p70 (clone 20C2) or rat antihuman IL-10 monoclonal antibody (clone JES3–9D7) and incubated overnight at 4°C. .. Cytokine binding was then detected with biotinylated-mouse monoclonal antihuman IL-12 (p40/p70) detection antibody (clone C8·6) or rat antihuman IL-10 clone JES3–12G8) or mouse antihuman IFN-γ (clone 4S.B3), followed by horseradish peroxidase-conjugated avidin (Vector Laboratories, Peterborough, UK) and a substrate solution containing 150 mg 2,2-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid, pH 4·5, mixed with H2 O2 at a final concentration of 0·03%).

    Plasmid Preparation:

    Article Title: Increased Immunogenicity and Protective Efficacy of Influenza M2e Fused to a Tetramerizing Protein
    Article Snippet: HeLa-M2 ELISA HeLa enzyme-linked immunosorbent assay (ELISA) plates were prepared as follows: HeLa cells transfected with M2-expressing plasmid (HeLa-M2) encoding a tetracycline response element (Tet) and HeLa cells with an empty plasmid (HeLa-C10) were obtained from Robert B. Couch . .. The cells were grown to confluency in Nunc Maxisorb™ 96-well plates by seeding wells with 1×105 HeLa cells in 150 µL of Iscove’s Modified Dulbecco’s medium (Invitrogen) supplemented with 2-mercaptoethanol at 0.05 mM, transferrin (Sigma-Aldrich) at 0.005 mg/mL, gentamicin (Riedel-de Haën, Seelze, Germany) at 0.05 mg/mL, and 10% fetal bovine serum (FBS) (Lonza, Copenhagen, Denmark).

    Article Title: Regulation of dendritic cell interleukin-12 secretion by tumour cell necrosis
    Article Snippet: Briefly, NUNC-maxisorb 96-well plates (G ibco Life Technologies, Paisley, UK) were coated with purified antihuman IL-12p40 (clone C8·3) or purified antihuman IL-12p70 (clone 20C2) or rat antihuman IL-10 monoclonal antibody (clone JES3–9D7) and incubated overnight at 4°C. .. Cytokine binding was then detected with biotinylated-mouse monoclonal antihuman IL-12 (p40/p70) detection antibody (clone C8·6) or rat antihuman IL-10 clone JES3–12G8) or mouse antihuman IFN-γ (clone 4S.B3), followed by horseradish peroxidase-conjugated avidin (Vector Laboratories, Peterborough, UK) and a substrate solution containing 150 mg 2,2-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid, pH 4·5, mixed with H2 O2 at a final concentration of 0·03%).

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  • 99
    Thermo Fisher 96 well elisa plates
    Specificity of <t>ELISA</t> and RT–PCR assays The specificity of the PTX3 ELISA was tested using different dilutions of 2C3 antibody to measure immobilized murine and human PTX3. Purified recombinant murine and human PTX3 were immobilized in <t>96‐well</t> ELISA plates, and then, different dilutions of 2C3 were added. The graph shows dose–response of 2C3 on immobilized murine or human PTX3. Human PTX3 was not detected by 2C3 antibody. Evaluation of the amplification efficiency of real‐time RT–PCR assay designed for PTX3 expression in astrocyte cell cultures. (B, C) Melting curve and amplification plot of PTX3 RT–qPCR assay. (D) Standard curves of PTX3 and GAPDH, used as reference mRNA, obtained using fivefold serial dilutions of the cDNA (420, 84, 16.8, 3.36 ng). The threshold cycle ( C t ) values ( y ‐axis) are plotted against log 10 values of cDNA input amounts ( x ‐axis). The graphs are parallel lines and the calculated efficiencies (E) are, respectively, of 1.13 and 1.12 from a y‐slope of −3.04 and −3.07 and a correlation coefficient ( R 2 ) > 0.9.
    96 Well Elisa Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 101 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/96 well elisa plates/product/Thermo Fisher
    Average 99 stars, based on 101 article reviews
    Price from $9.99 to $1999.99
    96 well elisa plates - by Bioz Stars, 2020-04
    99/100 stars
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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, 2020-04
    99/100 stars
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    85
    Thermo Fisher grp78 elisa microtiter 96 well plates
    Analysis of <t>GRP78–HTJ1</t> and OxPL–GRP78 interactions. (A) GRP78 and HTJ1 expression in HPAEC and human lung microvascular endothelial cells was detected by Western Blot. (B, C) GRP78 interactions were analyzed in coimmunoprecipitation assays using lysates from control or DMPC- (10 μg/ml, 15 min) or OxPAPC-stimulated (10 μg/ml) cells with antibody to GRP78 (B, top), HTJ1 (B, bottom), or EO6 antibody recognizing OxPL (C). (D) Human recombinant GRP78 was incubated with OxPAPC, OxPAPS, or their oxidation-resistant analogues DMPC or DMPS. Left, native gel electrophoresis, followed by Western blot with anti-GRP78 antibody. Shift in electrophoretic mobility of GRP78 incubated with OxPAPS, but not DMPS, indicates formation of GRP78–OxPAPS complex. Right, SDS–PAGE, followed by Western blot with EO6 antibody and reprobing with anti-GRP78 antibody. Positive EO6 immunoreactivity of GRP78 preincubated with OxPAPC indicates formation of GRP78–OxPAPC complex. (E) <t>ELISA</t> plates coated with OxPAPS or DMPS or control uncoated plates incubated with PBS incubated with human recombinant GRP78 (left) or HPAEC lysates (middle and right). The bound GRP78 was detected using anti-GRP78 antibody. * p
    Grp78 Elisa Microtiter 96 Well Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/grp78 elisa microtiter 96 well plates/product/Thermo Fisher
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    grp78 elisa microtiter 96 well plates - by Bioz Stars, 2020-04
    85/100 stars
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    Specificity of ELISA and RT–PCR assays The specificity of the PTX3 ELISA was tested using different dilutions of 2C3 antibody to measure immobilized murine and human PTX3. Purified recombinant murine and human PTX3 were immobilized in 96‐well ELISA plates, and then, different dilutions of 2C3 were added. The graph shows dose–response of 2C3 on immobilized murine or human PTX3. Human PTX3 was not detected by 2C3 antibody. Evaluation of the amplification efficiency of real‐time RT–PCR assay designed for PTX3 expression in astrocyte cell cultures. (B, C) Melting curve and amplification plot of PTX3 RT–qPCR assay. (D) Standard curves of PTX3 and GAPDH, used as reference mRNA, obtained using fivefold serial dilutions of the cDNA (420, 84, 16.8, 3.36 ng). The threshold cycle ( C t ) values ( y ‐axis) are plotted against log 10 values of cDNA input amounts ( x ‐axis). The graphs are parallel lines and the calculated efficiencies (E) are, respectively, of 1.13 and 1.12 from a y‐slope of −3.04 and −3.07 and a correlation coefficient ( R 2 ) > 0.9.

    Journal: The EMBO Journal

    Article Title: Pentraxin 3 regulates synaptic function by inducing AMPA receptor clustering via ECM remodeling and β1‐integrin

    doi: 10.15252/embj.201899529

    Figure Lengend Snippet: Specificity of ELISA and RT–PCR assays The specificity of the PTX3 ELISA was tested using different dilutions of 2C3 antibody to measure immobilized murine and human PTX3. Purified recombinant murine and human PTX3 were immobilized in 96‐well ELISA plates, and then, different dilutions of 2C3 were added. The graph shows dose–response of 2C3 on immobilized murine or human PTX3. Human PTX3 was not detected by 2C3 antibody. Evaluation of the amplification efficiency of real‐time RT–PCR assay designed for PTX3 expression in astrocyte cell cultures. (B, C) Melting curve and amplification plot of PTX3 RT–qPCR assay. (D) Standard curves of PTX3 and GAPDH, used as reference mRNA, obtained using fivefold serial dilutions of the cDNA (420, 84, 16.8, 3.36 ng). The threshold cycle ( C t ) values ( y ‐axis) are plotted against log 10 values of cDNA input amounts ( x ‐axis). The graphs are parallel lines and the calculated efficiencies (E) are, respectively, of 1.13 and 1.12 from a y‐slope of −3.04 and −3.07 and a correlation coefficient ( R 2 ) > 0.9.

    Article Snippet: Briefly, 96‐well ELISA plates (Nunc MaxiSorp, Thermo Fischer Scientific, Roskilde, Denmark) were coated with monoclonal antibody 2C3 anti‐mouse PTX3 in coating buffer (15 mM carbonate buffer pH 9.6) and incubated overnight at 4°C.

    Techniques: Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Purification, Recombinant, Amplification, Quantitative RT-PCR, Expressing

    Specificity of ELISA and RT–PCR assays A The specificity of the PTX3 ELISA was tested using different dilutions of 2C3 antibody to measure immobilized murine and human PTX3. Purified recombinant murine and human PTX3 were immobilized in 96‐well ELISA plates, and then, different dilutions of 2C3 were added. The graph shows dose–response of 2C3 on immobilized murine or human PTX3. Human PTX3 was not detected by 2C3 antibody. B–D Evaluation of the amplification efficiency of real‐time RT–PCR assay designed for PTX3 expression in astrocyte cell cultures. (B, C) Melting curve and amplification plot of PTX3 RT–qPCR assay. (D) Standard curves of PTX3 and GAPDH, used as reference mRNA, obtained using fivefold serial dilutions of the cDNA (420, 84, 16.8, 3.36 ng). The threshold cycle ( C t ) values ( y ‐axis) are plotted against log 10 values of cDNA input amounts ( x ‐axis). The graphs are parallel lines and the calculated efficiencies (E) are, respectively, of 1.13 and 1.12 from a y‐slope of −3.04 and −3.07 and a correlation coefficient ( R 2 ) > 0.9.

    Journal: The EMBO Journal

    Article Title: Pentraxin 3 regulates synaptic function by inducing AMPA receptor clustering via ECM remodeling and β1‐integrin

    doi: 10.15252/embj.201899529

    Figure Lengend Snippet: Specificity of ELISA and RT–PCR assays A The specificity of the PTX3 ELISA was tested using different dilutions of 2C3 antibody to measure immobilized murine and human PTX3. Purified recombinant murine and human PTX3 were immobilized in 96‐well ELISA plates, and then, different dilutions of 2C3 were added. The graph shows dose–response of 2C3 on immobilized murine or human PTX3. Human PTX3 was not detected by 2C3 antibody. B–D Evaluation of the amplification efficiency of real‐time RT–PCR assay designed for PTX3 expression in astrocyte cell cultures. (B, C) Melting curve and amplification plot of PTX3 RT–qPCR assay. (D) Standard curves of PTX3 and GAPDH, used as reference mRNA, obtained using fivefold serial dilutions of the cDNA (420, 84, 16.8, 3.36 ng). The threshold cycle ( C t ) values ( y ‐axis) are plotted against log 10 values of cDNA input amounts ( x ‐axis). The graphs are parallel lines and the calculated efficiencies (E) are, respectively, of 1.13 and 1.12 from a y‐slope of −3.04 and −3.07 and a correlation coefficient ( R 2 ) > 0.9.

    Article Snippet: Briefly, 96‐well ELISA plates (Nunc MaxiSorp, Thermo Fischer Scientific, Roskilde, Denmark) were coated with monoclonal antibody 2C3 anti‐mouse PTX3 in coating buffer (15 mM carbonate buffer pH 9.6) and incubated overnight at 4°C.

    Techniques: Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Purification, Recombinant, Amplification, Quantitative RT-PCR, Expressing

    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

    Analysis of GRP78–HTJ1 and OxPL–GRP78 interactions. (A) GRP78 and HTJ1 expression in HPAEC and human lung microvascular endothelial cells was detected by Western Blot. (B, C) GRP78 interactions were analyzed in coimmunoprecipitation assays using lysates from control or DMPC- (10 μg/ml, 15 min) or OxPAPC-stimulated (10 μg/ml) cells with antibody to GRP78 (B, top), HTJ1 (B, bottom), or EO6 antibody recognizing OxPL (C). (D) Human recombinant GRP78 was incubated with OxPAPC, OxPAPS, or their oxidation-resistant analogues DMPC or DMPS. Left, native gel electrophoresis, followed by Western blot with anti-GRP78 antibody. Shift in electrophoretic mobility of GRP78 incubated with OxPAPS, but not DMPS, indicates formation of GRP78–OxPAPS complex. Right, SDS–PAGE, followed by Western blot with EO6 antibody and reprobing with anti-GRP78 antibody. Positive EO6 immunoreactivity of GRP78 preincubated with OxPAPC indicates formation of GRP78–OxPAPC complex. (E) ELISA plates coated with OxPAPS or DMPS or control uncoated plates incubated with PBS incubated with human recombinant GRP78 (left) or HPAEC lysates (middle and right). The bound GRP78 was detected using anti-GRP78 antibody. * p

    Journal: Molecular Biology of the Cell

    Article Title: GRP78 is a novel receptor initiating a vascular barrier protective response to oxidized phospholipids

    doi: 10.1091/mbc.E13-12-0743

    Figure Lengend Snippet: Analysis of GRP78–HTJ1 and OxPL–GRP78 interactions. (A) GRP78 and HTJ1 expression in HPAEC and human lung microvascular endothelial cells was detected by Western Blot. (B, C) GRP78 interactions were analyzed in coimmunoprecipitation assays using lysates from control or DMPC- (10 μg/ml, 15 min) or OxPAPC-stimulated (10 μg/ml) cells with antibody to GRP78 (B, top), HTJ1 (B, bottom), or EO6 antibody recognizing OxPL (C). (D) Human recombinant GRP78 was incubated with OxPAPC, OxPAPS, or their oxidation-resistant analogues DMPC or DMPS. Left, native gel electrophoresis, followed by Western blot with anti-GRP78 antibody. Shift in electrophoretic mobility of GRP78 incubated with OxPAPS, but not DMPS, indicates formation of GRP78–OxPAPS complex. Right, SDS–PAGE, followed by Western blot with EO6 antibody and reprobing with anti-GRP78 antibody. Positive EO6 immunoreactivity of GRP78 preincubated with OxPAPC indicates formation of GRP78–OxPAPC complex. (E) ELISA plates coated with OxPAPS or DMPS or control uncoated plates incubated with PBS incubated with human recombinant GRP78 (left) or HPAEC lysates (middle and right). The bound GRP78 was detected using anti-GRP78 antibody. * p

    Article Snippet: GRP78 ELISA Microtiter 96-well plates (MaxiSorp; Nunc, Thermo Scientific, Rochester, NY) were coated with OxPAPS or DMPS (each 100 μg/ml in PBS containing 0.01% BHT) at 4°C overnight.

    Techniques: Expressing, Western Blot, Recombinant, Incubation, Nucleic Acid Electrophoresis, SDS Page, Enzyme-linked Immunosorbent Assay