96 well elisa plates  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99

    Structured Review

    Thermo Fisher 96 well elisa plates
    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, 2020-02
    99/100 stars

    Images

    Related Articles

    Cytometry:

    Article Title: CCR7-deficiency allows Accelerated Clearance of Chlamydia from the Female Reproductive Tract
    Article Snippet: After 72 h of incubation at 37°C, supernatants were collected and added to 96-well ELISA plates (Costar, Corning, NY) that had been pre-coated with purified anti-IFN-γ (R4-6A2), anti-IL-2, anti-IL-4, anti-IL-6, anti-IL-17A, anti-IL-23 (eBiosciences) or anti-IL-10 (R & D). .. In other experiments, stimulated mononuclear cells recovered from the FRT were stimulated with HKEBs for 48 hours and stained using antibodies specific for CD4, CD44, IFN-γ, and IL-17A and examined by flow cytometry, as described above.

    Blocking Assay:

    Article Title: Identification of a mimotope for circulating anti-cytokeratin 8/18 antibody and its usage for the diagnosis of breast cancer
    Article Snippet: .. After complex formation between CK8 and CK18 or truncated CK18, mixtures were coated onto 96-well ELISA plates at a concentration of about 1 μ g/well (Maxisorp; NUNC, Thermo Scientific, Rochester, NY) and incubated at 4°C for 16 h. The plates were blocked with 5% skim milk in TBST at RT for 1 h. Diluted K94 auto antibodies, from 0.06 to 0.48 μ g per well in blocking buffer, were then incubated in these wells for 90 min. After washing with TBST, the wells were incubated with HRP-linked anti-mouse IgGAM antibody (1:2,500, diluted in blocking buffer, Abcam). .. Visualization was performed with 3, 3′, 5, 5′-tetramethylbenzidine (TMB, Pierce) at 100 μ l per well.

    Article Title: Haploinsufficiency of cathepsin D leads to lysosomal dysfunction and promotes cell-to-cell transmission of α-synuclein aggregates
    Article Snippet: Briefly, 96-well enzyme-linked immunosorbent assay plates (Nalge Nunc International, Rochester, NY, USA) were coated with 1 μ g/ml of capture antibody (Ab62) in 50 mM carbonate buffer (pH 9.6) at 4 °C overnight. .. After washing with PBS with 0.05% Tween 20 (PBST), SuperBlock T20 (PBS) Blocking Buffer (Thermo Scientific, Rockford, IL, USA) was added to each well.

    Article Title: RGMb is a novel binding partner for PD-L2 and its engagement with PD-L2 promotes respiratory tolerance
    Article Snippet: .. To test the capacities of mRGMb antibodies and mPD-L2 fusion proteins to block RGMb binding to BMP-2/4, 96-well ELISA plates were coated with 1 µg/ml of recombinant mouse BMP-2 (Invitrogen) or BMP-4 (R & D Systems). mRGMb antibodies, isotype controls, mPD-L2-hIgG1/IgA, mPD-L2-mIgG2a/IgA, or control Ig fusion proteins at the indicated concentrations were preincubated with 20 µg/ml mRGMb-HIS (R & D Systems) for 45 min at 4°C, then added to the plates and incubated for 1 h at 37°C. .. Anti–penta-HIS-HRP (QIAGEN) at 1:1,000 was used for detection.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Identification of a mimotope for circulating anti-cytokeratin 8/18 antibody and its usage for the diagnosis of breast cancer
    Article Snippet: .. After complex formation between CK8 and CK18 or truncated CK18, mixtures were coated onto 96-well ELISA plates at a concentration of about 1 μ g/well (Maxisorp; NUNC, Thermo Scientific, Rochester, NY) and incubated at 4°C for 16 h. The plates were blocked with 5% skim milk in TBST at RT for 1 h. Diluted K94 auto antibodies, from 0.06 to 0.48 μ g per well in blocking buffer, were then incubated in these wells for 90 min. After washing with TBST, the wells were incubated with HRP-linked anti-mouse IgGAM antibody (1:2,500, diluted in blocking buffer, Abcam). .. Visualization was performed with 3, 3′, 5, 5′-tetramethylbenzidine (TMB, Pierce) at 100 μ l per well.

    Article Title: Longevity of adenovirus vector immunity in mice and its implications for vaccine efficacy
    Article Snippet: .. Briefly, 96-well ELISA plates (Thermo Fisher Scientific Clear Flat-Bottom Immuno Nonsterile 96-Well Plates) were coated with 0.5 μg/ml of purified GFP protein (Upstate, Temecula, CA) or HA protein of HK/156 (MyBioSource, Inc., San Diego, CA), incubated overnight at 4°C, and blocked with 1% bovine serum albumin (BSA) in PBS. ..

    Article Title: Repression of GSK3 restores NK cell cytotoxicity in AML patients
    Article Snippet: .. ELISA ELISA assays were performed in 96-well ELISA plates using TNF-α, IFN-γ, IL-10 and TGF-β ELISA kits (eBioscience, San Diego, CA, USA) according to the manufacturer's instructions. .. Lentiviral transductions 293 T cells (ATCC, Manassas, VA, USA) were co-transfected with one of the following plasmids: shGSK3β, shGSK3α, pLKO (Sigma-Aldrich, St Louis, MO, USA), GSK3β and GSK3α (both full-length human sequences were cloned into pLVX-EF1alpha-IRES-mCherry using the EcorR1/Not1 sites; Clontech, Mountain View, CA, USA) GSK3β contains a serine to alanine mutation at position 9 and GSK3α contains a serine to alanine mutation at position 21 to prevent the inactivation of these kinases by inhibitory phosphorylation.

    Article Title: HIV-1 subtype C superinfected individuals mount low autologous neutralizing antibody responses prior to intrasubtype superinfection
    Article Snippet: .. Briefly, 96-well ELISA plates were coated overnight with 100 μl (2 μg/ml) purified gp120 protein (GeneART) from the Zambian subtype C seroconverter ZM205F [ , ] at 4°C. ..

    Article Title: In vivo rescue of recombinant Zika virus from an infectious cDNA clone and its implications in vaccine development
    Article Snippet: .. Briefly, 96-well ELISA plates (ThermoFisher) were coated with extracts from ZIKV-infected Vero cells, and incubated overnight at 4 °C. .. Coated wells were blocked with 1% BSA in PBS, and incubated with 2-fold serial dilutions of mice sera (starting dilution of 1:100) for 1 h at 37 °C, following by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG for 1 h at 37 °C.

    Article Title: Epitope Mapping of the Immunodominant Invariable Region of Borrelia burgdorferi VlsE in Three Host Species
    Article Snippet: .. Briefly, 96-well ELISA plates were coated with streptavidin (Pierce Chemical Company, Rockford, Ill.) in coating buffer (0.1 M carbonate buffer, pH 9.2), followed by incubation with biotinylated peptides. ..

    Article Title: Increased Susceptibility to Salmonella Infection in Signal Regulatory Protein alpha (SIRP?) Deficient Mice
    Article Snippet: .. After 24-48 h incubation at 37°C, the supernatants were collected and added to 96-well ELISA plates (Costar, Corning, NY) pre-coated with purified anti-IFNγ, anti-IL-2 or anti-IL-4 (eBiosciences, San Diego, CA). .. Cytokine production was detected using biotinylated anti-IFNγ, anti-IL-2 and anti-IL-4 (eBiosciences, San Diego, CA), respectively, followed by ExtrAvidin peroxidase substrate (Sigma-Aldrich, St. Louis, MO).

    Article Title: Haploinsufficiency of cathepsin D leads to lysosomal dysfunction and promotes cell-to-cell transmission of α-synuclein aggregates
    Article Snippet: .. Briefly, 96-well enzyme-linked immunosorbent assay plates (Nalge Nunc International, Rochester, NY, USA) were coated with 1 μ g/ml of capture antibody (Ab62) in 50 mM carbonate buffer (pH 9.6) at 4 °C overnight. .. After washing with PBS with 0.05% Tween 20 (PBST), SuperBlock T20 (PBS) Blocking Buffer (Thermo Scientific, Rockford, IL, USA) was added to each well.

    Article Title: RGMb is a novel binding partner for PD-L2 and its engagement with PD-L2 promotes respiratory tolerance
    Article Snippet: .. To test the capacities of mRGMb antibodies and mPD-L2 fusion proteins to block RGMb binding to BMP-2/4, 96-well ELISA plates were coated with 1 µg/ml of recombinant mouse BMP-2 (Invitrogen) or BMP-4 (R & D Systems). mRGMb antibodies, isotype controls, mPD-L2-hIgG1/IgA, mPD-L2-mIgG2a/IgA, or control Ig fusion proteins at the indicated concentrations were preincubated with 20 µg/ml mRGMb-HIS (R & D Systems) for 45 min at 4°C, then added to the plates and incubated for 1 h at 37°C. .. Anti–penta-HIS-HRP (QIAGEN) at 1:1,000 was used for detection.

    Article Title: CCR7-deficiency allows Accelerated Clearance of Chlamydia from the Female Reproductive Tract
    Article Snippet: .. After 72 h of incubation at 37°C, supernatants were collected and added to 96-well ELISA plates (Costar, Corning, NY) that had been pre-coated with purified anti-IFN-γ (R4-6A2), anti-IL-2, anti-IL-4, anti-IL-6, anti-IL-17A, anti-IL-23 (eBiosciences) or anti-IL-10 (R & D). .. Cytokine production was detected using biotinylated antibodies specific for each cytokine (eBiosciences), followed by the addition of ExtrAvidin-Peroxidase and TMB substrate (Sigma-Aldrich and BD Biosciences).

    Article Title: Biochemical Engineering of Cell Surface Sialic Acids Stimulates Axonal Growth
    Article Snippet: .. Protein was determined in 96-well ELISA plates using 200 μl of bicinchonic acid protein reagent (Pierce, Rockford, IL) and a 50 μl sample. .. Plates were evaluated in a 96-well ELISA reader (Spectra) at 570 nm.

    Incubation:

    Article Title: Identification of a mimotope for circulating anti-cytokeratin 8/18 antibody and its usage for the diagnosis of breast cancer
    Article Snippet: .. After complex formation between CK8 and CK18 or truncated CK18, mixtures were coated onto 96-well ELISA plates at a concentration of about 1 μ g/well (Maxisorp; NUNC, Thermo Scientific, Rochester, NY) and incubated at 4°C for 16 h. The plates were blocked with 5% skim milk in TBST at RT for 1 h. Diluted K94 auto antibodies, from 0.06 to 0.48 μ g per well in blocking buffer, were then incubated in these wells for 90 min. After washing with TBST, the wells were incubated with HRP-linked anti-mouse IgGAM antibody (1:2,500, diluted in blocking buffer, Abcam). .. Visualization was performed with 3, 3′, 5, 5′-tetramethylbenzidine (TMB, Pierce) at 100 μ l per well.

    Article Title: Longevity of adenovirus vector immunity in mice and its implications for vaccine efficacy
    Article Snippet: .. Briefly, 96-well ELISA plates (Thermo Fisher Scientific Clear Flat-Bottom Immuno Nonsterile 96-Well Plates) were coated with 0.5 μg/ml of purified GFP protein (Upstate, Temecula, CA) or HA protein of HK/156 (MyBioSource, Inc., San Diego, CA), incubated overnight at 4°C, and blocked with 1% bovine serum albumin (BSA) in PBS. ..

    Article Title: HIV-1 subtype C superinfected individuals mount low autologous neutralizing antibody responses prior to intrasubtype superinfection
    Article Snippet: Briefly, 96-well ELISA plates were coated overnight with 100 μl (2 μg/ml) purified gp120 protein (GeneART) from the Zambian subtype C seroconverter ZM205F [ , ] at 4°C. .. Plates were washed again, and 100 μl/well of five-fold serially diluted heat-inactivated plasma was incubated for 1 hour at 37°C.

    Article Title: In vivo rescue of recombinant Zika virus from an infectious cDNA clone and its implications in vaccine development
    Article Snippet: .. Briefly, 96-well ELISA plates (ThermoFisher) were coated with extracts from ZIKV-infected Vero cells, and incubated overnight at 4 °C. .. Coated wells were blocked with 1% BSA in PBS, and incubated with 2-fold serial dilutions of mice sera (starting dilution of 1:100) for 1 h at 37 °C, following by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG for 1 h at 37 °C.

    Article Title: Epitope Mapping of the Immunodominant Invariable Region of Borrelia burgdorferi VlsE in Three Host Species
    Article Snippet: .. Briefly, 96-well ELISA plates were coated with streptavidin (Pierce Chemical Company, Rockford, Ill.) in coating buffer (0.1 M carbonate buffer, pH 9.2), followed by incubation with biotinylated peptides. ..

    Article Title: Increased Susceptibility to Salmonella Infection in Signal Regulatory Protein alpha (SIRP?) Deficient Mice
    Article Snippet: .. After 24-48 h incubation at 37°C, the supernatants were collected and added to 96-well ELISA plates (Costar, Corning, NY) pre-coated with purified anti-IFNγ, anti-IL-2 or anti-IL-4 (eBiosciences, San Diego, CA). .. Cytokine production was detected using biotinylated anti-IFNγ, anti-IL-2 and anti-IL-4 (eBiosciences, San Diego, CA), respectively, followed by ExtrAvidin peroxidase substrate (Sigma-Aldrich, St. Louis, MO).

    Article Title: Haploinsufficiency of cathepsin D leads to lysosomal dysfunction and promotes cell-to-cell transmission of α-synuclein aggregates
    Article Snippet: Briefly, 96-well enzyme-linked immunosorbent assay plates (Nalge Nunc International, Rochester, NY, USA) were coated with 1 μ g/ml of capture antibody (Ab62) in 50 mM carbonate buffer (pH 9.6) at 4 °C overnight. .. After incubation for 1 h at room temperature with shaking, plates were washed five times in PBST.

    Article Title: RGMb is a novel binding partner for PD-L2 and its engagement with PD-L2 promotes respiratory tolerance
    Article Snippet: .. To test the capacities of mRGMb antibodies and mPD-L2 fusion proteins to block RGMb binding to BMP-2/4, 96-well ELISA plates were coated with 1 µg/ml of recombinant mouse BMP-2 (Invitrogen) or BMP-4 (R & D Systems). mRGMb antibodies, isotype controls, mPD-L2-hIgG1/IgA, mPD-L2-mIgG2a/IgA, or control Ig fusion proteins at the indicated concentrations were preincubated with 20 µg/ml mRGMb-HIS (R & D Systems) for 45 min at 4°C, then added to the plates and incubated for 1 h at 37°C. .. Anti–penta-HIS-HRP (QIAGEN) at 1:1,000 was used for detection.

    Article Title: CCR7-deficiency allows Accelerated Clearance of Chlamydia from the Female Reproductive Tract
    Article Snippet: .. After 72 h of incubation at 37°C, supernatants were collected and added to 96-well ELISA plates (Costar, Corning, NY) that had been pre-coated with purified anti-IFN-γ (R4-6A2), anti-IL-2, anti-IL-4, anti-IL-6, anti-IL-17A, anti-IL-23 (eBiosciences) or anti-IL-10 (R & D). .. Cytokine production was detected using biotinylated antibodies specific for each cytokine (eBiosciences), followed by the addition of ExtrAvidin-Peroxidase and TMB substrate (Sigma-Aldrich and BD Biosciences).

    Western Blot:

    Article Title: Identification of a mimotope for circulating anti-cytokeratin 8/18 antibody and its usage for the diagnosis of breast cancer
    Article Snippet: Cytokeratin heterotypic complex formation Western blot analysis of heterotypic CK8/18 complexes were performed following the method of Ditzel et al ( ) with some modifications. .. After complex formation between CK8 and CK18 or truncated CK18, mixtures were coated onto 96-well ELISA plates at a concentration of about 1 μ g/well (Maxisorp; NUNC, Thermo Scientific, Rochester, NY) and incubated at 4°C for 16 h. The plates were blocked with 5% skim milk in TBST at RT for 1 h. Diluted K94 auto antibodies, from 0.06 to 0.48 μ g per well in blocking buffer, were then incubated in these wells for 90 min. After washing with TBST, the wells were incubated with HRP-linked anti-mouse IgGAM antibody (1:2,500, diluted in blocking buffer, Abcam).

    Conjugation Assay:

    Article Title: Epitope Mapping of the Immunodominant Invariable Region of Borrelia burgdorferi VlsE in Three Host Species
    Article Snippet: N-terminal conjugation to biotin was performed by the N -succinimidyl maleimide carboxylate method as per the instructions of the manufacturer (Molecular Probes, Eugene, Oreg.). .. Briefly, 96-well ELISA plates were coated with streptavidin (Pierce Chemical Company, Rockford, Ill.) in coating buffer (0.1 M carbonate buffer, pH 9.2), followed by incubation with biotinylated peptides.

    Flow Cytometry:

    Article Title: CCR7-deficiency allows Accelerated Clearance of Chlamydia from the Female Reproductive Tract
    Article Snippet: After 72 h of incubation at 37°C, supernatants were collected and added to 96-well ELISA plates (Costar, Corning, NY) that had been pre-coated with purified anti-IFN-γ (R4-6A2), anti-IL-2, anti-IL-4, anti-IL-6, anti-IL-17A, anti-IL-23 (eBiosciences) or anti-IL-10 (R & D). .. In other experiments, stimulated mononuclear cells recovered from the FRT were stimulated with HKEBs for 48 hours and stained using antibodies specific for CD4, CD44, IFN-γ, and IL-17A and examined by flow cytometry, as described above.

    Protease Inhibitor:

    Article Title: Biochemical Engineering of Cell Surface Sialic Acids Stimulates Axonal Growth
    Article Snippet: Protein was determined in 96-well ELISA plates using 200 μl of bicinchonic acid protein reagent (Pierce, Rockford, IL) and a 50 μl sample. .. Cell pellets were solubilized at 4°C for 1 hr in buffer containing 150 m m NaCl, 50 m m Tris, 1 m m CaCl2 , 1 m m MgCl2 , 1% Triton, and protease inhibitor mixture (Sigma, Deisenhofen, Germany) at pH 7.4.

    Infection:

    Article Title: Increased Susceptibility to Salmonella Infection in Signal Regulatory Protein alpha (SIRP?) Deficient Mice
    Article Snippet: Mice were infected and treated with antibiotics as described in Salmonella-specific ELISPOT assay. .. After 24-48 h incubation at 37°C, the supernatants were collected and added to 96-well ELISA plates (Costar, Corning, NY) pre-coated with purified anti-IFNγ, anti-IL-2 or anti-IL-4 (eBiosciences, San Diego, CA).

    Recombinant:

    Article Title: Identification of a mimotope for circulating anti-cytokeratin 8/18 antibody and its usage for the diagnosis of breast cancer
    Article Snippet: For enzyme-linked immunosorbent assay (ELISA), recombinant proteins (CK8, CK18 or truncated CK18 proteins) in 8 M urea were serially diluted with PBS to 0.5 M urea through four steps to allow renaturation of protein secondary structure and complex formation between CK8 and CK18 or truncated CK18. .. After complex formation between CK8 and CK18 or truncated CK18, mixtures were coated onto 96-well ELISA plates at a concentration of about 1 μ g/well (Maxisorp; NUNC, Thermo Scientific, Rochester, NY) and incubated at 4°C for 16 h. The plates were blocked with 5% skim milk in TBST at RT for 1 h. Diluted K94 auto antibodies, from 0.06 to 0.48 μ g per well in blocking buffer, were then incubated in these wells for 90 min. After washing with TBST, the wells were incubated with HRP-linked anti-mouse IgGAM antibody (1:2,500, diluted in blocking buffer, Abcam).

    Article Title: RGMb is a novel binding partner for PD-L2 and its engagement with PD-L2 promotes respiratory tolerance
    Article Snippet: .. To test the capacities of mRGMb antibodies and mPD-L2 fusion proteins to block RGMb binding to BMP-2/4, 96-well ELISA plates were coated with 1 µg/ml of recombinant mouse BMP-2 (Invitrogen) or BMP-4 (R & D Systems). mRGMb antibodies, isotype controls, mPD-L2-hIgG1/IgA, mPD-L2-mIgG2a/IgA, or control Ig fusion proteins at the indicated concentrations were preincubated with 20 µg/ml mRGMb-HIS (R & D Systems) for 45 min at 4°C, then added to the plates and incubated for 1 h at 37°C. .. Anti–penta-HIS-HRP (QIAGEN) at 1:1,000 was used for detection.

    In Vivo:

    Article Title: Biochemical Engineering of Cell Surface Sialic Acids Stimulates Axonal Growth
    Article Snippet: Slices (425 μm) were orientated as in vivo and fixed on microelectrode arrays (MEAs) (NMI, Reutlingen, Germany) of 60 substrate-integrated electrodes by a plasma clot. .. Protein was determined in 96-well ELISA plates using 200 μl of bicinchonic acid protein reagent (Pierce, Rockford, IL) and a 50 μl sample.

    Isolation:

    Article Title: CCR7-deficiency allows Accelerated Clearance of Chlamydia from the Female Reproductive Tract
    Article Snippet: Mice were infected with Chlamydia as described above and mononuclear cells from the FRT were isolated as previously described ( ). .. After 72 h of incubation at 37°C, supernatants were collected and added to 96-well ELISA plates (Costar, Corning, NY) that had been pre-coated with purified anti-IFN-γ (R4-6A2), anti-IL-2, anti-IL-4, anti-IL-6, anti-IL-17A, anti-IL-23 (eBiosciences) or anti-IL-10 (R & D).

    Purification:

    Article Title: Longevity of adenovirus vector immunity in mice and its implications for vaccine efficacy
    Article Snippet: .. Briefly, 96-well ELISA plates (Thermo Fisher Scientific Clear Flat-Bottom Immuno Nonsterile 96-Well Plates) were coated with 0.5 μg/ml of purified GFP protein (Upstate, Temecula, CA) or HA protein of HK/156 (MyBioSource, Inc., San Diego, CA), incubated overnight at 4°C, and blocked with 1% bovine serum albumin (BSA) in PBS. ..

    Article Title: HIV-1 subtype C superinfected individuals mount low autologous neutralizing antibody responses prior to intrasubtype superinfection
    Article Snippet: .. Briefly, 96-well ELISA plates were coated overnight with 100 μl (2 μg/ml) purified gp120 protein (GeneART) from the Zambian subtype C seroconverter ZM205F [ , ] at 4°C. ..

    Article Title: Increased Susceptibility to Salmonella Infection in Signal Regulatory Protein alpha (SIRP?) Deficient Mice
    Article Snippet: .. After 24-48 h incubation at 37°C, the supernatants were collected and added to 96-well ELISA plates (Costar, Corning, NY) pre-coated with purified anti-IFNγ, anti-IL-2 or anti-IL-4 (eBiosciences, San Diego, CA). .. Cytokine production was detected using biotinylated anti-IFNγ, anti-IL-2 and anti-IL-4 (eBiosciences, San Diego, CA), respectively, followed by ExtrAvidin peroxidase substrate (Sigma-Aldrich, St. Louis, MO).

    Article Title: CCR7-deficiency allows Accelerated Clearance of Chlamydia from the Female Reproductive Tract
    Article Snippet: .. After 72 h of incubation at 37°C, supernatants were collected and added to 96-well ELISA plates (Costar, Corning, NY) that had been pre-coated with purified anti-IFN-γ (R4-6A2), anti-IL-2, anti-IL-4, anti-IL-6, anti-IL-17A, anti-IL-23 (eBiosciences) or anti-IL-10 (R & D). .. Cytokine production was detected using biotinylated antibodies specific for each cytokine (eBiosciences), followed by the addition of ExtrAvidin-Peroxidase and TMB substrate (Sigma-Aldrich and BD Biosciences).

    Article Title: Biochemical Engineering of Cell Surface Sialic Acids Stimulates Axonal Growth
    Article Snippet: In brief, coverslips were coated overnight with 0.01% poly- l -lysine or laminin at 37°C and washed three times with H2 O. Purified small (3 × 105 ) cerebellar neurons from 6- to 7-d-old mice were seeded onto each coverslip, yielding a final volume of 400 μl. .. Protein was determined in 96-well ELISA plates using 200 μl of bicinchonic acid protein reagent (Pierce, Rockford, IL) and a 50 μl sample.

    Staining:

    Article Title: CCR7-deficiency allows Accelerated Clearance of Chlamydia from the Female Reproductive Tract
    Article Snippet: Paragraph title: Cytokine ELISA and intracellular staining ... After 72 h of incubation at 37°C, supernatants were collected and added to 96-well ELISA plates (Costar, Corning, NY) that had been pre-coated with purified anti-IFN-γ (R4-6A2), anti-IL-2, anti-IL-4, anti-IL-6, anti-IL-17A, anti-IL-23 (eBiosciences) or anti-IL-10 (R & D).

    Peptide ELISA:

    Article Title: Epitope Mapping of the Immunodominant Invariable Region of Borrelia burgdorferi VlsE in Three Host Species
    Article Snippet: VlsE consists of two invariable domains at the amino and carboxyl termini and one variable domain at the center ( ) and 297 ( ( The peptide-based ELISA was performed as previously described ( ). .. Briefly, 96-well ELISA plates were coated with streptavidin (Pierce Chemical Company, Rockford, Ill.) in coating buffer (0.1 M carbonate buffer, pH 9.2), followed by incubation with biotinylated peptides.

    Mouse Assay:

    Article Title: In vivo rescue of recombinant Zika virus from an infectious cDNA clone and its implications in vaccine development
    Article Snippet: Enzyme-linked immunosorbent assay (ELISA) To assess the levels of virus-specific antibodies present in the sera of vaccinated mice, ELISAs were performed as previously described , , . .. Briefly, 96-well ELISA plates (ThermoFisher) were coated with extracts from ZIKV-infected Vero cells, and incubated overnight at 4 °C.

    Article Title: Increased Susceptibility to Salmonella Infection in Signal Regulatory Protein alpha (SIRP?) Deficient Mice
    Article Snippet: Mice were infected and treated with antibiotics as described in Salmonella-specific ELISPOT assay. .. After 24-48 h incubation at 37°C, the supernatants were collected and added to 96-well ELISA plates (Costar, Corning, NY) pre-coated with purified anti-IFNγ, anti-IL-2 or anti-IL-4 (eBiosciences, San Diego, CA).

    Article Title: Biochemical Engineering of Cell Surface Sialic Acids Stimulates Axonal Growth
    Article Snippet: Slices of entorhinal cortex and dentate gyrus were prepared from 6-d-old BALBC mice of either sex. .. Protein was determined in 96-well ELISA plates using 200 μl of bicinchonic acid protein reagent (Pierce, Rockford, IL) and a 50 μl sample.

    SDS Page:

    Article Title: Identification of a mimotope for circulating anti-cytokeratin 8/18 antibody and its usage for the diagnosis of breast cancer
    Article Snippet: Intact CK8, CK18 and truncated CK18s were separated by SDS-PAGE and transferred to PVDF membranes. .. After complex formation between CK8 and CK18 or truncated CK18, mixtures were coated onto 96-well ELISA plates at a concentration of about 1 μ g/well (Maxisorp; NUNC, Thermo Scientific, Rochester, NY) and incubated at 4°C for 16 h. The plates were blocked with 5% skim milk in TBST at RT for 1 h. Diluted K94 auto antibodies, from 0.06 to 0.48 μ g per well in blocking buffer, were then incubated in these wells for 90 min. After washing with TBST, the wells were incubated with HRP-linked anti-mouse IgGAM antibody (1:2,500, diluted in blocking buffer, Abcam).

    Binding Assay:

    Article Title: HIV-1 subtype C superinfected individuals mount low autologous neutralizing antibody responses prior to intrasubtype superinfection
    Article Snippet: Paragraph title: gp120 binding ELISA ... Briefly, 96-well ELISA plates were coated overnight with 100 μl (2 μg/ml) purified gp120 protein (GeneART) from the Zambian subtype C seroconverter ZM205F [ , ] at 4°C.

    Article Title: RGMb is a novel binding partner for PD-L2 and its engagement with PD-L2 promotes respiratory tolerance
    Article Snippet: .. To test the capacities of mRGMb antibodies and mPD-L2 fusion proteins to block RGMb binding to BMP-2/4, 96-well ELISA plates were coated with 1 µg/ml of recombinant mouse BMP-2 (Invitrogen) or BMP-4 (R & D Systems). mRGMb antibodies, isotype controls, mPD-L2-hIgG1/IgA, mPD-L2-mIgG2a/IgA, or control Ig fusion proteins at the indicated concentrations were preincubated with 20 µg/ml mRGMb-HIS (R & D Systems) for 45 min at 4°C, then added to the plates and incubated for 1 h at 37°C. .. Anti–penta-HIS-HRP (QIAGEN) at 1:1,000 was used for detection.

    Spectrophotometry:

    Article Title: CCR7-deficiency allows Accelerated Clearance of Chlamydia from the Female Reproductive Tract
    Article Snippet: After 72 h of incubation at 37°C, supernatants were collected and added to 96-well ELISA plates (Costar, Corning, NY) that had been pre-coated with purified anti-IFN-γ (R4-6A2), anti-IL-2, anti-IL-4, anti-IL-6, anti-IL-17A, anti-IL-23 (eBiosciences) or anti-IL-10 (R & D). .. Developed ELISA plates were analyzed using a spectrophotometer (SpectraMax M5; Molecular Devices), and cytokine concentrations calculated according to standard curves.

    Concentration Assay:

    Article Title: Identification of a mimotope for circulating anti-cytokeratin 8/18 antibody and its usage for the diagnosis of breast cancer
    Article Snippet: .. After complex formation between CK8 and CK18 or truncated CK18, mixtures were coated onto 96-well ELISA plates at a concentration of about 1 μ g/well (Maxisorp; NUNC, Thermo Scientific, Rochester, NY) and incubated at 4°C for 16 h. The plates were blocked with 5% skim milk in TBST at RT for 1 h. Diluted K94 auto antibodies, from 0.06 to 0.48 μ g per well in blocking buffer, were then incubated in these wells for 90 min. After washing with TBST, the wells were incubated with HRP-linked anti-mouse IgGAM antibody (1:2,500, diluted in blocking buffer, Abcam). .. Visualization was performed with 3, 3′, 5, 5′-tetramethylbenzidine (TMB, Pierce) at 100 μ l per well.

    Enzyme-linked Immunospot:

    Article Title: Increased Susceptibility to Salmonella Infection in Signal Regulatory Protein alpha (SIRP?) Deficient Mice
    Article Snippet: Mice were infected and treated with antibiotics as described in Salmonella-specific ELISPOT assay. .. After 24-48 h incubation at 37°C, the supernatants were collected and added to 96-well ELISA plates (Costar, Corning, NY) pre-coated with purified anti-IFNγ, anti-IL-2 or anti-IL-4 (eBiosciences, San Diego, CA).

    Lysis:

    Article Title: Increased Susceptibility to Salmonella Infection in Signal Regulatory Protein alpha (SIRP?) Deficient Mice
    Article Snippet: After RBC lysis, one million splenocytes were incubated in the presence of 10μM Salmonella specific peptide (SseI, SseJ, FliC or PagC, ) or serial diluted HKST in 96-well round-bottom plates. .. After 24-48 h incubation at 37°C, the supernatants were collected and added to 96-well ELISA plates (Costar, Corning, NY) pre-coated with purified anti-IFNγ, anti-IL-2 or anti-IL-4 (eBiosciences, San Diego, CA).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90
    Thermo Fisher 96 well elisa plate
    The phospholipid <t>ELISA</t> assay specifically detects α-Syn in S-PRBC samples. ( a ) Samples of human S-PRBC (0–4 μg protein) were applied to a <t>96-well</t> microtiter dish containing the indicated phospholipids (Phosphatidylinositol (PI); phosphatidylserine (PS); phosphatidylethanolamine (PE)) at a final amount of 100 μg/well, or without phospholipids (-PL) and processed for the detection of α-Syn using anti human α-Syn antibody, α-Syn #10 19 ). Graph illustrates the means ± SD, n = 3 replicates. ( b ) Samples of mouse PRBC or S-PRBC from wt or α-Syn-/- (1 μg protein), analyzed by Western blotting using anti- α-Syn antibody, α-Syn #3. Arrows indicate non-specific immunoreactive bands. ( c ) S-PRBC from wt and α-Syn-/- mice were applied in increasing amounts (0–4 μg protein) into wells of a microtiter dish coated with PI-PS-PE phospholipids (as in ( a )), using anti α-Syn antibody, α-Syn#3 or without a primary detecting ab (−1 ° ab). Graph illustrates the means ± SD of n = 3 repeats.
    96 Well Elisa Plate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/96 well elisa plate/product/Thermo Fisher
    Average 90 stars, based on 43 article reviews
    Price from $9.99 to $1999.99
    96 well elisa plate - by Bioz Stars, 2020-02
    90/100 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, 2020-02
    99/100 stars
      Buy from Supplier

    79
    Thermo Fisher sdab 1a6
    Characterization of labile heme in plasma following acute hemolysis. (A) Number of RBC in C57BL/6 mice receiving Phenylhydrazine (PHX) and control (CTRL) mice receiving PBS. (B) Heme concentration in the plasma of C57BL/6 mice receiving phenylhydrazine. (C) Correlation between circulating RBC numbers (data from A) and heme concentration in plasma (data from B). (D) Concentration of bioavailable heme in plasma of C57BL/6 mice receiving phenylhydrazine, quantified by a heme reporter assay [ 31 ]. (E) Correlation between circulating RBC numbers (data from A) and concentration of bioavailable heme in plasma (data from D). (F) Soluble hemin quantified by a sandwich ELISA in which the sdAbs <t>1A6</t> and 2H7 are used to capture and reveal heme, respectively. (G) Detection of soluble heme versus heme bound to HPX using the same <t>sdAb-based</t> ELISA as in (F). Note that heme bound to HPX is not detected by ELISA. (H) A pull-down assay using streptavidin-beads to capture heme-biotin. The sdAb 2H10 bound to heme-biotin was added to HPX at 1/6 SdAb/HPX molar ratio. Streptavidin-beads pulled down the sdAb 2H10 as well as HPX bound to heme-biotin, demonstrating that HPX can bind heme-bound to sdAb 2H10. This is consistent with the higher affinity of HPX toward heme as compared to the sdAb 2H10. Coomassie-based stain of 15% SDS/PAGE gel loaded with streptavidin-beads used to pull-down heme-biotin from different reaction mixtures. Grey arrowheads indicate the molecular weight of the protein ladder (NZYColour Protein Marker II, Nzytech ® ) in kDa loaded in the first lane of the gel. Gel is representative of two independent experiments with similar trend. (I) Plasma HBC 1/2 in C57BL/6 mice receiving phenylhydrazine. Circles in A, B, C, D, E, and I correspond to individual mice. Red dash line represents mean ± STD. * P
    Sdab 1a6, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sdab 1a6/product/Thermo Fisher
    Average 79 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sdab 1a6 - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    Image Search Results


    The phospholipid ELISA assay specifically detects α-Syn in S-PRBC samples. ( a ) Samples of human S-PRBC (0–4 μg protein) were applied to a 96-well microtiter dish containing the indicated phospholipids (Phosphatidylinositol (PI); phosphatidylserine (PS); phosphatidylethanolamine (PE)) at a final amount of 100 μg/well, or without phospholipids (-PL) and processed for the detection of α-Syn using anti human α-Syn antibody, α-Syn #10 19 ). Graph illustrates the means ± SD, n = 3 replicates. ( b ) Samples of mouse PRBC or S-PRBC from wt or α-Syn-/- (1 μg protein), analyzed by Western blotting using anti- α-Syn antibody, α-Syn #3. Arrows indicate non-specific immunoreactive bands. ( c ) S-PRBC from wt and α-Syn-/- mice were applied in increasing amounts (0–4 μg protein) into wells of a microtiter dish coated with PI-PS-PE phospholipids (as in ( a )), using anti α-Syn antibody, α-Syn#3 or without a primary detecting ab (−1 ° ab). Graph illustrates the means ± SD of n = 3 repeats.

    Journal: Scientific Reports

    Article Title: Total and Proteinase K-Resistant α-Synuclein Levels in Erythrocytes, Determined by their Ability to Bind Phospholipids, Associate with Parkinson’s Disease

    doi: 10.1038/srep11120

    Figure Lengend Snippet: The phospholipid ELISA assay specifically detects α-Syn in S-PRBC samples. ( a ) Samples of human S-PRBC (0–4 μg protein) were applied to a 96-well microtiter dish containing the indicated phospholipids (Phosphatidylinositol (PI); phosphatidylserine (PS); phosphatidylethanolamine (PE)) at a final amount of 100 μg/well, or without phospholipids (-PL) and processed for the detection of α-Syn using anti human α-Syn antibody, α-Syn #10 19 ). Graph illustrates the means ± SD, n = 3 replicates. ( b ) Samples of mouse PRBC or S-PRBC from wt or α-Syn-/- (1 μg protein), analyzed by Western blotting using anti- α-Syn antibody, α-Syn #3. Arrows indicate non-specific immunoreactive bands. ( c ) S-PRBC from wt and α-Syn-/- mice were applied in increasing amounts (0–4 μg protein) into wells of a microtiter dish coated with PI-PS-PE phospholipids (as in ( a )), using anti α-Syn antibody, α-Syn#3 or without a primary detecting ab (−1 ° ab). Graph illustrates the means ± SD of n = 3 repeats.

    Article Snippet: Phospholipid ELISA assay A PolySorp, 96-well ELISA plate (Thermo Scientific) was coated with a mixture of phospholipids dissolved in methanol in a final amount of 100 μg/well and incubated overnight at 4 °C for complete evaporation of methanol.

    Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Mouse Assay

    Total and proteinase K-resistant α-Syn are detected in samples of SPRBC by a standard sandwich-ELISA but with a lower efficacy. Samples of human S-PRBC (0–2.5 μg protein) were applied to a 96-well microtiter dishes and α- Syn levels were determined by phospholipid (PL)-ELISA (as in Fig. 2a ) or a sandwich- ELISA, using anti α- Syn ab, Syn - 1 (Transduction laboratories) as a capturing antibody. α-Syn detection was obtained with α-Syn #3 antibody (PL-ELISA, a and c) or C-20 antibody (Santa Cruz) (sandwich ELISA, b and d). Graph illustrates the means ± SD (n = 3 replicates) of total α-Syn (filled line) and proteinase K-resistant α-Syn (dashed line) levels of a representative sample in each group.

    Journal: Scientific Reports

    Article Title: Total and Proteinase K-Resistant α-Synuclein Levels in Erythrocytes, Determined by their Ability to Bind Phospholipids, Associate with Parkinson’s Disease

    doi: 10.1038/srep11120

    Figure Lengend Snippet: Total and proteinase K-resistant α-Syn are detected in samples of SPRBC by a standard sandwich-ELISA but with a lower efficacy. Samples of human S-PRBC (0–2.5 μg protein) were applied to a 96-well microtiter dishes and α- Syn levels were determined by phospholipid (PL)-ELISA (as in Fig. 2a ) or a sandwich- ELISA, using anti α- Syn ab, Syn - 1 (Transduction laboratories) as a capturing antibody. α-Syn detection was obtained with α-Syn #3 antibody (PL-ELISA, a and c) or C-20 antibody (Santa Cruz) (sandwich ELISA, b and d). Graph illustrates the means ± SD (n = 3 replicates) of total α-Syn (filled line) and proteinase K-resistant α-Syn (dashed line) levels of a representative sample in each group.

    Article Snippet: Phospholipid ELISA assay A PolySorp, 96-well ELISA plate (Thermo Scientific) was coated with a mixture of phospholipids dissolved in methanol in a final amount of 100 μg/well and incubated overnight at 4 °C for complete evaporation of methanol.

    Techniques: Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Transduction

    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

    Characterization of labile heme in plasma following acute hemolysis. (A) Number of RBC in C57BL/6 mice receiving Phenylhydrazine (PHX) and control (CTRL) mice receiving PBS. (B) Heme concentration in the plasma of C57BL/6 mice receiving phenylhydrazine. (C) Correlation between circulating RBC numbers (data from A) and heme concentration in plasma (data from B). (D) Concentration of bioavailable heme in plasma of C57BL/6 mice receiving phenylhydrazine, quantified by a heme reporter assay [ 31 ]. (E) Correlation between circulating RBC numbers (data from A) and concentration of bioavailable heme in plasma (data from D). (F) Soluble hemin quantified by a sandwich ELISA in which the sdAbs 1A6 and 2H7 are used to capture and reveal heme, respectively. (G) Detection of soluble heme versus heme bound to HPX using the same sdAb-based ELISA as in (F). Note that heme bound to HPX is not detected by ELISA. (H) A pull-down assay using streptavidin-beads to capture heme-biotin. The sdAb 2H10 bound to heme-biotin was added to HPX at 1/6 SdAb/HPX molar ratio. Streptavidin-beads pulled down the sdAb 2H10 as well as HPX bound to heme-biotin, demonstrating that HPX can bind heme-bound to sdAb 2H10. This is consistent with the higher affinity of HPX toward heme as compared to the sdAb 2H10. Coomassie-based stain of 15% SDS/PAGE gel loaded with streptavidin-beads used to pull-down heme-biotin from different reaction mixtures. Grey arrowheads indicate the molecular weight of the protein ladder (NZYColour Protein Marker II, Nzytech ® ) in kDa loaded in the first lane of the gel. Gel is representative of two independent experiments with similar trend. (I) Plasma HBC 1/2 in C57BL/6 mice receiving phenylhydrazine. Circles in A, B, C, D, E, and I correspond to individual mice. Red dash line represents mean ± STD. * P

    Journal: The FEBS journal

    Article Title: Characterization of plasma labile heme in hemolytic conditions

    doi: 10.1111/febs.14192

    Figure Lengend Snippet: Characterization of labile heme in plasma following acute hemolysis. (A) Number of RBC in C57BL/6 mice receiving Phenylhydrazine (PHX) and control (CTRL) mice receiving PBS. (B) Heme concentration in the plasma of C57BL/6 mice receiving phenylhydrazine. (C) Correlation between circulating RBC numbers (data from A) and heme concentration in plasma (data from B). (D) Concentration of bioavailable heme in plasma of C57BL/6 mice receiving phenylhydrazine, quantified by a heme reporter assay [ 31 ]. (E) Correlation between circulating RBC numbers (data from A) and concentration of bioavailable heme in plasma (data from D). (F) Soluble hemin quantified by a sandwich ELISA in which the sdAbs 1A6 and 2H7 are used to capture and reveal heme, respectively. (G) Detection of soluble heme versus heme bound to HPX using the same sdAb-based ELISA as in (F). Note that heme bound to HPX is not detected by ELISA. (H) A pull-down assay using streptavidin-beads to capture heme-biotin. The sdAb 2H10 bound to heme-biotin was added to HPX at 1/6 SdAb/HPX molar ratio. Streptavidin-beads pulled down the sdAb 2H10 as well as HPX bound to heme-biotin, demonstrating that HPX can bind heme-bound to sdAb 2H10. This is consistent with the higher affinity of HPX toward heme as compared to the sdAb 2H10. Coomassie-based stain of 15% SDS/PAGE gel loaded with streptavidin-beads used to pull-down heme-biotin from different reaction mixtures. Grey arrowheads indicate the molecular weight of the protein ladder (NZYColour Protein Marker II, Nzytech ® ) in kDa loaded in the first lane of the gel. Gel is representative of two independent experiments with similar trend. (I) Plasma HBC 1/2 in C57BL/6 mice receiving phenylhydrazine. Circles in A, B, C, D, E, and I correspond to individual mice. Red dash line represents mean ± STD. * P

    Article Snippet: To measure labile heme, 96 well plates were coated with SdAb 1A6 (0.3–5 μg·mL−1 ) in 50 mm carbonate/bicarbonate buffer, pH 9.6 (16 h, 4 °C), washed (5×, PBS 0.1% Tween 20) and blocked (2 h, RT) with protein-free blocking buffer (Pierce from Thermo Fischer Scientific).

    Techniques: Mouse Assay, Concentration Assay, Reporter Assay, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Pull Down Assay, Staining, SDS Page, Molecular Weight, Marker

    Selection of heme-binding sdAbs using phage display technology. (A) MALDI-TOF/TOF analysis of biotinylated-heme. Peak of mass-to-charge ( m/z ) 969.2 Da with characteristic isotopic cluster pattern, corresponding to biotinylation of a single hemin carboxylic acid residue. (B) Schematic representation (Accelrys draw 4.1 (BIOVIA, San Diego, CA, USA) and 3D representations; PYMOL software, PyMOL Molecular Graphics System, Version 1.3, Schroödinger, LLC, New York, NY, USA) of heme and biotinylated-heme. (C) Elution cycles outputs of bacteria infected with phages displaying sdAbs recognizing heme. (D) Ratio of heme binding to protein expression of 1721 sdAb (circles) screened by ELISA, as described in Experimental procedures. SdAbs 2H10, 2H7, and 1A6, with highest heme binding to protein expression ratio, are highlighted. (E) SDS/PAGE of purified sdAbs stained by coomassie-based stain or detected by western blot using an anti-HA mAb. (F) ELISA for recognition of solid-phase heme by purified sdAb. Ctrl: Control sdAb that does not recognize heme. (G) SdAb CDR1 aminoacid sequences, as determined by DNA sequencing. Binding affinity of SdAbs toward heme, determined by BIAcore surface Plasmon resonance. ND, not detectable.

    Journal: The FEBS journal

    Article Title: Characterization of plasma labile heme in hemolytic conditions

    doi: 10.1111/febs.14192

    Figure Lengend Snippet: Selection of heme-binding sdAbs using phage display technology. (A) MALDI-TOF/TOF analysis of biotinylated-heme. Peak of mass-to-charge ( m/z ) 969.2 Da with characteristic isotopic cluster pattern, corresponding to biotinylation of a single hemin carboxylic acid residue. (B) Schematic representation (Accelrys draw 4.1 (BIOVIA, San Diego, CA, USA) and 3D representations; PYMOL software, PyMOL Molecular Graphics System, Version 1.3, Schroödinger, LLC, New York, NY, USA) of heme and biotinylated-heme. (C) Elution cycles outputs of bacteria infected with phages displaying sdAbs recognizing heme. (D) Ratio of heme binding to protein expression of 1721 sdAb (circles) screened by ELISA, as described in Experimental procedures. SdAbs 2H10, 2H7, and 1A6, with highest heme binding to protein expression ratio, are highlighted. (E) SDS/PAGE of purified sdAbs stained by coomassie-based stain or detected by western blot using an anti-HA mAb. (F) ELISA for recognition of solid-phase heme by purified sdAb. Ctrl: Control sdAb that does not recognize heme. (G) SdAb CDR1 aminoacid sequences, as determined by DNA sequencing. Binding affinity of SdAbs toward heme, determined by BIAcore surface Plasmon resonance. ND, not detectable.

    Article Snippet: To measure labile heme, 96 well plates were coated with SdAb 1A6 (0.3–5 μg·mL−1 ) in 50 mm carbonate/bicarbonate buffer, pH 9.6 (16 h, 4 °C), washed (5×, PBS 0.1% Tween 20) and blocked (2 h, RT) with protein-free blocking buffer (Pierce from Thermo Fischer Scientific).

    Techniques: Selection, Binding Assay, Software, Infection, Expressing, Enzyme-linked Immunosorbent Assay, SDS Page, Purification, Staining, Western Blot, DNA Sequencing, SPR Assay

    Analysis of heme binding by SdAbs. (A) SdAbs bound to biotinylated-heme in solution were pooled-down using streptavidin (SA) beads and detected by western blot using anti-HA mAb. Input was measured by Coomassie-based stain. (B) UV-Visible spectra of hemin. Soret region at approximately 364 and 383 nm and a CT band at 622 nm are shown, representative of three independent experiments. (C) UV-visible spectra of sdAb 2H10, 1A6 and 2H7 bound to heme at different concentrations. Soret (412 nm), Q 1 (530 nm), Q 0 (565 nm), and CT (624 nm) bands are highlighted. (D) Far UV CD spectra of sdAb 2H10 in the apo (black) and heme-bound (red) forms. Shift from 212 to 218 is due to heme-driven conformational rearrangement of the sdAb secondary structure. The inset shows the Soret region, with the appearance of the 412 nm band, due to heme binding to the sdAb. (E) ATR FTIR absorption spectra (top) and second derivative (bottom) of sdAb 2H10 in the apo (black) and heme-bound (red) forms in the amide I region (1700–1610 cm −1 ), showing structural modification upon heme coordination. (F) High frequency Resonance Raman spectra of hemin and sdAb 2H10 bound to hemin, obtained with 413 nm excitation.

    Journal: The FEBS journal

    Article Title: Characterization of plasma labile heme in hemolytic conditions

    doi: 10.1111/febs.14192

    Figure Lengend Snippet: Analysis of heme binding by SdAbs. (A) SdAbs bound to biotinylated-heme in solution were pooled-down using streptavidin (SA) beads and detected by western blot using anti-HA mAb. Input was measured by Coomassie-based stain. (B) UV-Visible spectra of hemin. Soret region at approximately 364 and 383 nm and a CT band at 622 nm are shown, representative of three independent experiments. (C) UV-visible spectra of sdAb 2H10, 1A6 and 2H7 bound to heme at different concentrations. Soret (412 nm), Q 1 (530 nm), Q 0 (565 nm), and CT (624 nm) bands are highlighted. (D) Far UV CD spectra of sdAb 2H10 in the apo (black) and heme-bound (red) forms. Shift from 212 to 218 is due to heme-driven conformational rearrangement of the sdAb secondary structure. The inset shows the Soret region, with the appearance of the 412 nm band, due to heme binding to the sdAb. (E) ATR FTIR absorption spectra (top) and second derivative (bottom) of sdAb 2H10 in the apo (black) and heme-bound (red) forms in the amide I region (1700–1610 cm −1 ), showing structural modification upon heme coordination. (F) High frequency Resonance Raman spectra of hemin and sdAb 2H10 bound to hemin, obtained with 413 nm excitation.

    Article Snippet: To measure labile heme, 96 well plates were coated with SdAb 1A6 (0.3–5 μg·mL−1 ) in 50 mm carbonate/bicarbonate buffer, pH 9.6 (16 h, 4 °C), washed (5×, PBS 0.1% Tween 20) and blocked (2 h, RT) with protein-free blocking buffer (Pierce from Thermo Fischer Scientific).

    Techniques: Binding Assay, Western Blot, Staining, Modification