nunc maxisorp 96 well plate  (Thermo Fisher)


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    Structured Review

    Thermo Fisher nunc maxisorp 96 well plate
    Nunc Maxisorp 96 Well Plate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nunc maxisorp 96 well plate/product/Thermo Fisher
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nunc maxisorp 96 well plate - by Bioz Stars, 2020-04
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    Related Articles

    Enzyme-linked Immunosorbent Assay:

    Article Title: Use of Heterologous Vesiculovirus G Proteins Circumvents the Humoral Anti-envelope Immunity in Lentivector-Based In Vivo Gene Delivery
    Article Snippet: Paragraph title: Ig-Isotyping ELISA ... Wells in a Nunc Maxisorp 96-well plate (Thermo Scientific) were coated overnight at 4°C with 50 ng/well in PBS recombinant VSVind.G protein (Source Bioscience).

    Article Title: Interactions between the Aggregatibacter actinomycetemcomitans secretin HofQ and host cytokines indicate a link between natural competence and interleukin-8 uptake
    Article Snippet: .. Anti-HofQ antibodies in A. actinomycetemcomitans-positive patient sera examined by ELISA Recombinant emHofQ (500 ng) or BSA as a control in PBSN were incubated at RT overnight in a Nunc Maxisorp 96-well plate. ..

    Article Title: Identification of new EphA4 inhibitors by virtual screening of FDA-approved drugs
    Article Snippet: Paragraph title: ELISA ... We immobilized recombinant mouse EphA4 ectodomain Fc chimera (R & D Systems) in buffer (25 mM sodium carbonate and 25 mM sodium bicarbonate, pH 9.7) on a Nunc MaxiSorp 96-well plate (eBioscience).

    Article Title: De novo protein sequencing, humanization and in vitro effects of an antihuman CD34 mouse monoclonal antibody
    Article Snippet: Paragraph title: ELISA ... In brief, a Nunc™ MaxiSorp 96-well plate (Thermo Fisher Scientific) was coated with the human CD34 protein (Fc tag; Sino Biological Incorporation, Beijing, China) in a volume of 50 μL at a concentration of 5 µg/mL and incubated at 4 °C for 18 h. After blocking with the StartingBlock™ blocking buffer (Thermo Fisher Scientific) and being washing with PBS containing 0.01% Tween-20 (PBST) three times, the samples were added to the plates and incubated for 1 h at 37 °C.

    Article Title: Identification of new EphA4 inhibitors by virtual screening of FDA-approved drugs
    Article Snippet: .. ELISA We immobilized recombinant mouse EphA4 ectodomain Fc chimera (R & D Systems) in buffer (25 mM sodium carbonate and 25 mM sodium bicarbonate, pH 9.7) on a Nunc MaxiSorp 96-well plate (eBioscience). .. We subsequently incubated the plate with biotinylated recombinant mouse ephrin-A1 in TBST (20 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.01% [v/v] Tween-20) at 30 °C for 1 h, followed by streptavidin-conjugated HRP (Thermo Scientific) for 1 h. We then incubated the plate with the substrate, TMB One Solution (Promega), until a blue color developed.

    Negative Control:

    Article Title: Interactions between the Aggregatibacter actinomycetemcomitans secretin HofQ and host cytokines indicate a link between natural competence and interleukin-8 uptake
    Article Snippet: Wells of a Nunc Maxisorp 96-well plate (Thermo Fisher Scientific) were coated with 6 pmol of IL-1β or IL-8 in PBS at 4°C overnight. .. BSA was used as a negative control.

    Article Title: Interactions between the Aggregatibacter actinomycetemcomitans secretin HofQ and host cytokines indicate a link between natural competence and interleukin-8 uptake
    Article Snippet: Time-resolved fluorometric immunoassay (TRFIA) Wells of a Nunc Maxisorp 96-well plate (Thermo Fisher Scientific) were coated with 6 pmol of IL-1β or IL-8 in PBS at 4°C overnight. .. BSA was used as a negative control.

    Amplification:

    Article Title: Theranostic Application of a Novel G-Quadruplex-Forming DNA Aptamer Targeting Malate Synthase of Mycobacterium tuberculosis
    Article Snippet: Monitoring the Rounds of SELEX After 10 iterative rounds of SELEX, the archived aptamer populations from rounds R2, R4, R6, R7, R8, R9, and R10 were amplified through PCR using 5′ biotinylated forward primer and rA-containing reverse primer, followed by strand separation using denaturing urea-PAGE as described previously. .. For this, 500 ng of purified MS protein was coated onto a Nunc MaxiSorp 96-well plate (Thermo Scientific) overnight at 4°C followed by blocking with 5% BSA at RT.

    Concentration Assay:

    Article Title: De novo protein sequencing, humanization and in vitro effects of an antihuman CD34 mouse monoclonal antibody
    Article Snippet: .. In brief, a Nunc™ MaxiSorp 96-well plate (Thermo Fisher Scientific) was coated with the human CD34 protein (Fc tag; Sino Biological Incorporation, Beijing, China) in a volume of 50 μL at a concentration of 5 µg/mL and incubated at 4 °C for 18 h. After blocking with the StartingBlock™ blocking buffer (Thermo Fisher Scientific) and being washing with PBS containing 0.01% Tween-20 (PBST) three times, the samples were added to the plates and incubated for 1 h at 37 °C. .. After washing, the plates were incubated with horseradish peroxidase-conjugated antihuman lambda light chain antibody (Bethyl Laboratories, Inc., Montgomery, TX, USA) for 1 h at RT, followed by washing with PBST.

    Mutagenesis:

    Article Title: A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8
    Article Snippet: Paragraph title: Binding of bilRI− mutant cells on collagen and fibrinogen ... A total of 1 μg of collagen in sodium bicarbonate buffer (16 mM sodium carbonate, 34 mM sodium bicarbonate, and 0.02% sodium azide, pH 9.6) or 25 ng of fibrinogen in PBSN1 (0.05% sodium azide in PBS1 ) was added to the wells of a Nunc MaxiSorp 96-well plate (Affymetrix).

    Labeling:

    Article Title: Construction of an Immunochromatographic Determination System for N1,N12‐diacetylspermine
    Article Snippet: Briefly, the wells of a NUNC Maxisorp 96‐well plate (Thermo Fisher Scientific Inc., Waltham, MA) were coated with the AcSpm–GMB–peptide conjugate and then blocked with 5% skim milk. .. After thorough washing with phosphate buffered saline (PBS) containing 0.05% Tween 20 (PBS‐T), a horseradish peroxidase labeled antimouse IgG (H+L) antibody (1:6,000; Cappel Laboratories, Westchester, PA) was added to each well, and the plate was incubated for 1 h at room temperature.

    Construct:

    Article Title: A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8
    Article Snippet: Cytokine-binding assay for recombinant BilRI Because BilRI produced unwanted spontaneous dimers when the cysteine at position 20 was included in the recombinant protein, the construct that was used in the cytokine-binding assays contains neither the signal sequence (the first 19 amino acids) nor the C20. .. A total of 100 ng of each cytokine (IL-1β/IL-6/IL-8/IL-10/tumor necrosis factor [TNF]-α/interferon [INF]-γ/transforming growth factor [TGF]-β1) diluted in PBSN buffer (0.05% sodium azide in PBS1, ) was incubated in a Nunc MaxiSorp 96-well plate (Affymetrix, Santa Clara, CA, USA) at RT overnight.

    Purification:

    Article Title: Theranostic Application of a Novel G-Quadruplex-Forming DNA Aptamer Targeting Malate Synthase of Mycobacterium tuberculosis
    Article Snippet: .. For this, 500 ng of purified MS protein was coated onto a Nunc MaxiSorp 96-well plate (Thermo Scientific) overnight at 4°C followed by blocking with 5% BSA at RT. ..

    Produced:

    Article Title: A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8
    Article Snippet: Cytokine-binding assay for recombinant BilRI Because BilRI produced unwanted spontaneous dimers when the cysteine at position 20 was included in the recombinant protein, the construct that was used in the cytokine-binding assays contains neither the signal sequence (the first 19 amino acids) nor the C20. .. A total of 100 ng of each cytokine (IL-1β/IL-6/IL-8/IL-10/tumor necrosis factor [TNF]-α/interferon [INF]-γ/transforming growth factor [TGF]-β1) diluted in PBSN buffer (0.05% sodium azide in PBS1, ) was incubated in a Nunc MaxiSorp 96-well plate (Affymetrix, Santa Clara, CA, USA) at RT overnight.

    Sequencing:

    Article Title: A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8
    Article Snippet: Cytokine-binding assay for recombinant BilRI Because BilRI produced unwanted spontaneous dimers when the cysteine at position 20 was included in the recombinant protein, the construct that was used in the cytokine-binding assays contains neither the signal sequence (the first 19 amino acids) nor the C20. .. A total of 100 ng of each cytokine (IL-1β/IL-6/IL-8/IL-10/tumor necrosis factor [TNF]-α/interferon [INF]-γ/transforming growth factor [TGF]-β1) diluted in PBSN buffer (0.05% sodium azide in PBS1, ) was incubated in a Nunc MaxiSorp 96-well plate (Affymetrix, Santa Clara, CA, USA) at RT overnight.

    Incubation:

    Article Title: Use of Heterologous Vesiculovirus G Proteins Circumvents the Humoral Anti-envelope Immunity in Lentivector-Based In Vivo Gene Delivery
    Article Snippet: Wells in a Nunc Maxisorp 96-well plate (Thermo Scientific) were coated overnight at 4°C with 50 ng/well in PBS recombinant VSVind.G protein (Source Bioscience). .. Following blocking, pooled serum samples were added at 1:100 in the diluent buffer provided and incubated for 1 h at room temperature.

    Article Title: Interactions between the Aggregatibacter actinomycetemcomitans secretin HofQ and host cytokines indicate a link between natural competence and interleukin-8 uptake
    Article Snippet: .. Anti-HofQ antibodies in A. actinomycetemcomitans-positive patient sera examined by ELISA Recombinant emHofQ (500 ng) or BSA as a control in PBSN were incubated at RT overnight in a Nunc Maxisorp 96-well plate. ..

    Article Title: Identification of new EphA4 inhibitors by virtual screening of FDA-approved drugs
    Article Snippet: We immobilized recombinant mouse EphA4 ectodomain Fc chimera (R & D Systems) in buffer (25 mM sodium carbonate and 25 mM sodium bicarbonate, pH 9.7) on a Nunc MaxiSorp 96-well plate (eBioscience). .. We subsequently incubated the plate with biotinylated recombinant mouse ephrin-A1 in TBST (20 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.01% [v/v] Tween-20) at 30 °C for 1 h, followed by streptavidin-conjugated HRP (Thermo Scientific) for 1 h. We then incubated the plate with the substrate, TMB One Solution (Promega), until a blue color developed.

    Article Title: De novo protein sequencing, humanization and in vitro effects of an antihuman CD34 mouse monoclonal antibody
    Article Snippet: .. In brief, a Nunc™ MaxiSorp 96-well plate (Thermo Fisher Scientific) was coated with the human CD34 protein (Fc tag; Sino Biological Incorporation, Beijing, China) in a volume of 50 μL at a concentration of 5 µg/mL and incubated at 4 °C for 18 h. After blocking with the StartingBlock™ blocking buffer (Thermo Fisher Scientific) and being washing with PBS containing 0.01% Tween-20 (PBST) three times, the samples were added to the plates and incubated for 1 h at 37 °C. .. After washing, the plates were incubated with horseradish peroxidase-conjugated antihuman lambda light chain antibody (Bethyl Laboratories, Inc., Montgomery, TX, USA) for 1 h at RT, followed by washing with PBST.

    Article Title: Identification of new EphA4 inhibitors by virtual screening of FDA-approved drugs
    Article Snippet: ELISA We immobilized recombinant mouse EphA4 ectodomain Fc chimera (R & D Systems) in buffer (25 mM sodium carbonate and 25 mM sodium bicarbonate, pH 9.7) on a Nunc MaxiSorp 96-well plate (eBioscience). .. We subsequently incubated the plate with biotinylated recombinant mouse ephrin-A1 in TBST (20 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.01% [v/v] Tween-20) at 30 °C for 1 h, followed by streptavidin-conjugated HRP (Thermo Scientific) for 1 h. We then incubated the plate with the substrate, TMB One Solution (Promega), until a blue color developed.

    Article Title: Interactions between the Aggregatibacter actinomycetemcomitans secretin HofQ and host cytokines indicate a link between natural competence and interleukin-8 uptake
    Article Snippet: .. Recombinant emHofQ (500 ng) or BSA as a control in PBSN were incubated at RT overnight in a Nunc Maxisorp 96-well plate. ..

    Article Title: Construction of an Immunochromatographic Determination System for N1,N12‐diacetylspermine
    Article Snippet: Briefly, the wells of a NUNC Maxisorp 96‐well plate (Thermo Fisher Scientific Inc., Waltham, MA) were coated with the AcSpm–GMB–peptide conjugate and then blocked with 5% skim milk. .. After washing, serial dilutions of DiAcSpm and the anti‐DiAcSpm antibody at appropriate concentrations were added to each well, and the plate was incubated at room temperature for 1 h with constant shaking.

    Article Title: A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8
    Article Snippet: .. A total of 100 ng of each cytokine (IL-1β/IL-6/IL-8/IL-10/tumor necrosis factor [TNF]-α/interferon [INF]-γ/transforming growth factor [TGF]-β1) diluted in PBSN buffer (0.05% sodium azide in PBS1, ) was incubated in a Nunc MaxiSorp 96-well plate (Affymetrix, Santa Clara, CA, USA) at RT overnight. .. Wells were washed 3 times with ion-exchanged water, after which the wells were blocked with blocking buffer (0.25% BSA, 0.02% sodium azide in PBS-T) at 37°C for 3 h. The wells were again washed as above, and 400 ng of C-His-tagged BilRI21-181 was added to the wells and incubated at 4°C overnight.

    Polymerase Chain Reaction:

    Article Title: Theranostic Application of a Novel G-Quadruplex-Forming DNA Aptamer Targeting Malate Synthase of Mycobacterium tuberculosis
    Article Snippet: Monitoring the Rounds of SELEX After 10 iterative rounds of SELEX, the archived aptamer populations from rounds R2, R4, R6, R7, R8, R9, and R10 were amplified through PCR using 5′ biotinylated forward primer and rA-containing reverse primer, followed by strand separation using denaturing urea-PAGE as described previously. .. For this, 500 ng of purified MS protein was coated onto a Nunc MaxiSorp 96-well plate (Thermo Scientific) overnight at 4°C followed by blocking with 5% BSA at RT.

    Competitive ELISA:

    Article Title: Construction of an Immunochromatographic Determination System for N1,N12‐diacetylspermine
    Article Snippet: Paragraph title: Characterization of Anti‐DiAcSpm Monoclonal Antibodies by Competitive ELISA ... Briefly, the wells of a NUNC Maxisorp 96‐well plate (Thermo Fisher Scientific Inc., Waltham, MA) were coated with the AcSpm–GMB–peptide conjugate and then blocked with 5% skim milk.

    Blocking Assay:

    Article Title: Use of Heterologous Vesiculovirus G Proteins Circumvents the Humoral Anti-envelope Immunity in Lentivector-Based In Vivo Gene Delivery
    Article Snippet: Wells in a Nunc Maxisorp 96-well plate (Thermo Scientific) were coated overnight at 4°C with 50 ng/well in PBS recombinant VSVind.G protein (Source Bioscience). .. Following blocking, pooled serum samples were added at 1:100 in the diluent buffer provided and incubated for 1 h at room temperature.

    Article Title: De novo protein sequencing, humanization and in vitro effects of an antihuman CD34 mouse monoclonal antibody
    Article Snippet: .. In brief, a Nunc™ MaxiSorp 96-well plate (Thermo Fisher Scientific) was coated with the human CD34 protein (Fc tag; Sino Biological Incorporation, Beijing, China) in a volume of 50 μL at a concentration of 5 µg/mL and incubated at 4 °C for 18 h. After blocking with the StartingBlock™ blocking buffer (Thermo Fisher Scientific) and being washing with PBS containing 0.01% Tween-20 (PBST) three times, the samples were added to the plates and incubated for 1 h at 37 °C. .. After washing, the plates were incubated with horseradish peroxidase-conjugated antihuman lambda light chain antibody (Bethyl Laboratories, Inc., Montgomery, TX, USA) for 1 h at RT, followed by washing with PBST.

    Article Title: Interactions between the Aggregatibacter actinomycetemcomitans secretin HofQ and host cytokines indicate a link between natural competence and interleukin-8 uptake
    Article Snippet: Wells of a Nunc Maxisorp 96-well plate (Thermo Fisher Scientific) were coated with 6 pmol of IL-1β or IL-8 in PBS at 4°C overnight. .. Empty binding sites were blocked with 200 μl of Alternative Block (BB5, ImmunoChemistry Technologies, #6299) at RT overnight, after which the wells were washed 3 times as described above.

    Article Title: Theranostic Application of a Novel G-Quadruplex-Forming DNA Aptamer Targeting Malate Synthase of Mycobacterium tuberculosis
    Article Snippet: .. For this, 500 ng of purified MS protein was coated onto a Nunc MaxiSorp 96-well plate (Thermo Scientific) overnight at 4°C followed by blocking with 5% BSA at RT. ..

    Article Title: Interactions between the Aggregatibacter actinomycetemcomitans secretin HofQ and host cytokines indicate a link between natural competence and interleukin-8 uptake
    Article Snippet: Time-resolved fluorometric immunoassay (TRFIA) Wells of a Nunc Maxisorp 96-well plate (Thermo Fisher Scientific) were coated with 6 pmol of IL-1β or IL-8 in PBS at 4°C overnight. .. Empty binding sites were blocked with 200 μl of Alternative Block (BB5, ImmunoChemistry Technologies, #6299) at RT overnight, after which the wells were washed 3 times as described above.

    Article Title: A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8
    Article Snippet: A total of 100 ng of each cytokine (IL-1β/IL-6/IL-8/IL-10/tumor necrosis factor [TNF]-α/interferon [INF]-γ/transforming growth factor [TGF]-β1) diluted in PBSN buffer (0.05% sodium azide in PBS1, ) was incubated in a Nunc MaxiSorp 96-well plate (Affymetrix, Santa Clara, CA, USA) at RT overnight. .. Wells were washed 3 times with ion-exchanged water, after which the wells were blocked with blocking buffer (0.25% BSA, 0.02% sodium azide in PBS-T) at 37°C for 3 h. The wells were again washed as above, and 400 ng of C-His-tagged BilRI21-181 was added to the wells and incubated at 4°C overnight.

    Mass Spectrometry:

    Article Title: Theranostic Application of a Novel G-Quadruplex-Forming DNA Aptamer Targeting Malate Synthase of Mycobacterium tuberculosis
    Article Snippet: .. For this, 500 ng of purified MS protein was coated onto a Nunc MaxiSorp 96-well plate (Thermo Scientific) overnight at 4°C followed by blocking with 5% BSA at RT. ..

    Modification:

    Article Title: A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8
    Article Snippet: Binding of bilRI− mutant cells on collagen and fibrinogen The binding of A. actinomycetemcomitans to type V collagen and fibrinogen was determined using a microplate assay modified from the methods described by Yu et al and Tang and Mintz. .. A total of 1 μg of collagen in sodium bicarbonate buffer (16 mM sodium carbonate, 34 mM sodium bicarbonate, and 0.02% sodium azide, pH 9.6) or 25 ng of fibrinogen in PBSN1 (0.05% sodium azide in PBS1 ) was added to the wells of a Nunc MaxiSorp 96-well plate (Affymetrix).

    Recombinant:

    Article Title: Use of Heterologous Vesiculovirus G Proteins Circumvents the Humoral Anti-envelope Immunity in Lentivector-Based In Vivo Gene Delivery
    Article Snippet: .. Wells in a Nunc Maxisorp 96-well plate (Thermo Scientific) were coated overnight at 4°C with 50 ng/well in PBS recombinant VSVind.G protein (Source Bioscience). .. Following blocking, pooled serum samples were added at 1:100 in the diluent buffer provided and incubated for 1 h at room temperature.

    Article Title: Interactions between the Aggregatibacter actinomycetemcomitans secretin HofQ and host cytokines indicate a link between natural competence and interleukin-8 uptake
    Article Snippet: .. Anti-HofQ antibodies in A. actinomycetemcomitans-positive patient sera examined by ELISA Recombinant emHofQ (500 ng) or BSA as a control in PBSN were incubated at RT overnight in a Nunc Maxisorp 96-well plate. ..

    Article Title: Identification of new EphA4 inhibitors by virtual screening of FDA-approved drugs
    Article Snippet: .. We immobilized recombinant mouse EphA4 ectodomain Fc chimera (R & D Systems) in buffer (25 mM sodium carbonate and 25 mM sodium bicarbonate, pH 9.7) on a Nunc MaxiSorp 96-well plate (eBioscience). .. We subsequently incubated the plate with biotinylated recombinant mouse ephrin-A1 in TBST (20 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.01% [v/v] Tween-20) at 30 °C for 1 h, followed by streptavidin-conjugated HRP (Thermo Scientific) for 1 h. We then incubated the plate with the substrate, TMB One Solution (Promega), until a blue color developed.

    Article Title: A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8
    Article Snippet: Collagen and fibrinogen solutions were prepared as in the collagen- and fibrinogen-binding assay for recombinant BilRI. .. A total of 1 μg of collagen in sodium bicarbonate buffer (16 mM sodium carbonate, 34 mM sodium bicarbonate, and 0.02% sodium azide, pH 9.6) or 25 ng of fibrinogen in PBSN1 (0.05% sodium azide in PBS1 ) was added to the wells of a Nunc MaxiSorp 96-well plate (Affymetrix).

    Article Title: Identification of new EphA4 inhibitors by virtual screening of FDA-approved drugs
    Article Snippet: .. ELISA We immobilized recombinant mouse EphA4 ectodomain Fc chimera (R & D Systems) in buffer (25 mM sodium carbonate and 25 mM sodium bicarbonate, pH 9.7) on a Nunc MaxiSorp 96-well plate (eBioscience). .. We subsequently incubated the plate with biotinylated recombinant mouse ephrin-A1 in TBST (20 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.01% [v/v] Tween-20) at 30 °C for 1 h, followed by streptavidin-conjugated HRP (Thermo Scientific) for 1 h. We then incubated the plate with the substrate, TMB One Solution (Promega), until a blue color developed.

    Article Title: Interactions between the Aggregatibacter actinomycetemcomitans secretin HofQ and host cytokines indicate a link between natural competence and interleukin-8 uptake
    Article Snippet: .. Recombinant emHofQ (500 ng) or BSA as a control in PBSN were incubated at RT overnight in a Nunc Maxisorp 96-well plate. ..

    Article Title: A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8
    Article Snippet: Paragraph title: Cytokine-binding assay for recombinant BilRI ... A total of 100 ng of each cytokine (IL-1β/IL-6/IL-8/IL-10/tumor necrosis factor [TNF]-α/interferon [INF]-γ/transforming growth factor [TGF]-β1) diluted in PBSN buffer (0.05% sodium azide in PBS1, ) was incubated in a Nunc MaxiSorp 96-well plate (Affymetrix, Santa Clara, CA, USA) at RT overnight.

    Binding Assay:

    Article Title: Interactions between the Aggregatibacter actinomycetemcomitans secretin HofQ and host cytokines indicate a link between natural competence and interleukin-8 uptake
    Article Snippet: Wells of a Nunc Maxisorp 96-well plate (Thermo Fisher Scientific) were coated with 6 pmol of IL-1β or IL-8 in PBS at 4°C overnight. .. Empty binding sites were blocked with 200 μl of Alternative Block (BB5, ImmunoChemistry Technologies, #6299) at RT overnight, after which the wells were washed 3 times as described above.

    Article Title: Theranostic Application of a Novel G-Quadruplex-Forming DNA Aptamer Targeting Malate Synthase of Mycobacterium tuberculosis
    Article Snippet: These ssDNA pools of different rounds were checked for their binding to MS by aptamer-linked immobilized sorbent assay (ALISA). .. For this, 500 ng of purified MS protein was coated onto a Nunc MaxiSorp 96-well plate (Thermo Scientific) overnight at 4°C followed by blocking with 5% BSA at RT.

    Article Title: A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8
    Article Snippet: Paragraph title: Binding of bilRI− mutant cells on collagen and fibrinogen ... A total of 1 μg of collagen in sodium bicarbonate buffer (16 mM sodium carbonate, 34 mM sodium bicarbonate, and 0.02% sodium azide, pH 9.6) or 25 ng of fibrinogen in PBSN1 (0.05% sodium azide in PBS1 ) was added to the wells of a Nunc MaxiSorp 96-well plate (Affymetrix).

    Article Title: Interactions between the Aggregatibacter actinomycetemcomitans secretin HofQ and host cytokines indicate a link between natural competence and interleukin-8 uptake
    Article Snippet: Time-resolved fluorometric immunoassay (TRFIA) Wells of a Nunc Maxisorp 96-well plate (Thermo Fisher Scientific) were coated with 6 pmol of IL-1β or IL-8 in PBS at 4°C overnight. .. Empty binding sites were blocked with 200 μl of Alternative Block (BB5, ImmunoChemistry Technologies, #6299) at RT overnight, after which the wells were washed 3 times as described above.

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    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
      Buy from Supplier

    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
      Buy from Supplier

    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
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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