96 well elisa plate  (Thermo Fisher)


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    Structured Review

    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: 99/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96 well elisa plate - by Bioz Stars, 2020-04
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    Images

    1) Product Images from "Total and Proteinase K-Resistant α-Synuclein Levels in Erythrocytes, Determined by their Ability to Bind Phospholipids, Associate with Parkinson’s Disease"

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

    Journal: Scientific Reports

    doi: 10.1038/srep11120

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

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

    2) Product Images from "Role for Bcl-xL as an inhibitor of cytosolic cytochrome C accumulation in DNA damage-induced apoptosis"

    Article Title: Role for Bcl-xL as an inhibitor of cytosolic cytochrome C accumulation in DNA damage-induced apoptosis

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    ( A ) Column-purified H 6 -Bcl-x L (lanes: 1, 5 μg; 2, 2.5 μg) and Bcl-x ST (lane 3, 5 μg) proteins were resolved by SDS/PAGE and transferred to a nitrocellulose filter. Purified MAPK protein was used as a negative control (lane 4). The filter was incubated with purified cytochrome C (bovine heart cytochrome C) for 1 h at room temperature. The filters then were analyzed by immunoblotting with anti-cytochrome C. ( B ) Purified cytochrome C was separated by SDS/PAGE and transferred to three nitrocellulose filters. The filters were incubated with purified, eluted H 6 , H 6 -Bcl-x LT . or Bcl-x ST for 1 h at room temperature. The filters then were analyzed by immunoblotting with anti-Bcl-x. ( C ) Cytochrome C was adsorbed onto the plastic surface of a 96-well ELISA plate. Various concentrations of GST (▴), GST–Bcl-x LT (▵), or GST–Bcl-x ST (▪) were added to the wells. Binding of Bcl-x to cytochrome C was measured by ELISA immunoassay. ( D ) Cytochrome C adsorbed to plates was incubated with 1 μg/ml GST, 1 μg/ml GST–Bcl-x LT , 5 μg/ml GST–Bcl-x ST , or a mixture of 1 μg/ml GST–Bcl-x LT and 5 μg/ml GST–Bcl-x ST . Binding of Bcl-x was assessed by ELISA. Hatched bars represent controls with no cytochrome C. Solid bars represent assays performed in the presence of cytochrome C.
    Figure Legend Snippet: ( A ) Column-purified H 6 -Bcl-x L (lanes: 1, 5 μg; 2, 2.5 μg) and Bcl-x ST (lane 3, 5 μg) proteins were resolved by SDS/PAGE and transferred to a nitrocellulose filter. Purified MAPK protein was used as a negative control (lane 4). The filter was incubated with purified cytochrome C (bovine heart cytochrome C) for 1 h at room temperature. The filters then were analyzed by immunoblotting with anti-cytochrome C. ( B ) Purified cytochrome C was separated by SDS/PAGE and transferred to three nitrocellulose filters. The filters were incubated with purified, eluted H 6 , H 6 -Bcl-x LT . or Bcl-x ST for 1 h at room temperature. The filters then were analyzed by immunoblotting with anti-Bcl-x. ( C ) Cytochrome C was adsorbed onto the plastic surface of a 96-well ELISA plate. Various concentrations of GST (▴), GST–Bcl-x LT (▵), or GST–Bcl-x ST (▪) were added to the wells. Binding of Bcl-x to cytochrome C was measured by ELISA immunoassay. ( D ) Cytochrome C adsorbed to plates was incubated with 1 μg/ml GST, 1 μg/ml GST–Bcl-x LT , 5 μg/ml GST–Bcl-x ST , or a mixture of 1 μg/ml GST–Bcl-x LT and 5 μg/ml GST–Bcl-x ST . Binding of Bcl-x was assessed by ELISA. Hatched bars represent controls with no cytochrome C. Solid bars represent assays performed in the presence of cytochrome C.

    Techniques Used: Purification, SDS Page, Negative Control, Incubation, Enzyme-linked Immunosorbent Assay, Binding Assay

    Related Articles

    Centrifugation:

    Article Title: Identification of Genes Required for Secretion of the Francisella Oxidative Burst-Inhibiting Acid Phosphatase AcpA
    Article Snippet: The conditions included three different centrifugation schemes: (1) 16,100 × g once for 30 min in 1.5 ml microfuge tubes, (2) in a 96-well format, 2095 × g once for 40 min, and (3) in a 96-well format, 2095 × g for 40 min with the transfer of supernatants to another plate and centrifugation at 2095 × g for an additional 40 min. .. A volume of 50 μl of supernatant was dried in a 96-well immune-plate (Thermo Scientific, Rochester, NY, USA) overnight in the presence of protease inhibitor and then used for AcpA ELISA analysis as described below.

    Positive Control:

    Article Title: Inhibition of Pyrimidine Biosynthesis Pathway Suppresses Viral Growth through Innate Immunity
    Article Snippet: For each plate, columns 1 and 12 were dedicated to controls: culture wells were alternatively spiked with 1 µl of DMSO alone (negative control) or supplemented with recombinant IFN-β so that final concentration equals 1,000 IU/ml (positive control). .. For one 96-well culture plate, 17 µg of plasmid were diluted in 500 µl of DMEM (Gibco-Invitrogen).

    Synthesized:

    Article Title: Protein Kinase C Regulates the Cell Surface Activity of Endothelin-Converting Enzyme-1
    Article Snippet: Cell-based Quenched Fluorescent Substrate Assay EA.hy926 cells grown in 96-well microtiter plates were washed with Opti-MEM (Invitrogen Australia, Mt. .. Following pre-incubation at 37°C for 30 min, a bradykinin-based quenched fluorescent substrate (QFS), (7-methoxycoumarin-4-yl)acetyl-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(2, 4-dinitrophenyl) (custom synthesized by Auspep; Johnson and Ahn, ), was added to a final concentration of 40 μM.

    Blocking Assay:

    Article Title: Critical role of amino acid position 343 of surfactant protein-D in the selective binding of glycolipids from Mycobacterium tuberculosis
    Article Snippet: Bacterial single cell suspensions (5 × 105 ) in 50 μL TBS (50 mM Tris-hydrochloride + 150 mM sodium chloride, pH 7.5) were added and dried onto triplicate wells of a 96-well microtiter plate (Immulon 1; Thermo Electron Corporation, Milford, MA) followed by overnight UV irradiation treatment to kill the bacteria. .. All plates were blocked with the blocking buffer (3% fatty acid free BSA, 20 mM Tris, 140 mM sodium chloride, 5 mM calcium chloride, pH 7.4) for 1 h at room temperature (RT) under humid conditions.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Identification of Genes Required for Secretion of the Francisella Oxidative Burst-Inhibiting Acid Phosphatase AcpA
    Article Snippet: .. A volume of 50 μl of supernatant was dried in a 96-well immune-plate (Thermo Scientific, Rochester, NY, USA) overnight in the presence of protease inhibitor and then used for AcpA ELISA analysis as described below. .. The bacterial membrane protein FopA was included as a negative control.

    Article Title: Critical role of amino acid position 343 of surfactant protein-D in the selective binding of glycolipids from Mycobacterium tuberculosis
    Article Snippet: Paragraph title: ELISA ... Bacterial single cell suspensions (5 × 105 ) in 50 μL TBS (50 mM Tris-hydrochloride + 150 mM sodium chloride, pH 7.5) were added and dried onto triplicate wells of a 96-well microtiter plate (Immulon 1; Thermo Electron Corporation, Milford, MA) followed by overnight UV irradiation treatment to kill the bacteria.

    Article Title: Dendrimer-RNA nanoparticles generate protective immunity against lethal Ebola, H1N1 influenza, and Toxoplasma gondii challenges with a single dose
    Article Snippet: Splenocytes were isolated from mice and plated at a density of 10 million cells per milliliter in 96-well culture plates in the presence of growth medium [all components were from Life Technologies unless otherwise indicated: RPMI 1620 with GlutaMAX supplemented with 8% FBS, 1 mM nonessential amino acids, 1 mM sodium pyruvate, 10 mM Hepes, 50 μM 2-mercaptoethanol (Sigma), and penicillin/streptomycin] only or 2 μg/mL OVA-derived peptide in growth medium. .. After 5 d in culture, the concentration of 1:20 diluted supernatant IFN-γ was quantified using IFN-γ ELISA kits (BD Biosciences).

    Article Title: Generation and characterization of monoclonal antibodies against the N-terminus of alpha-2-antiplasmin
    Article Snippet: Paragraph title: Characterization of selected mAbs directed against Met-α2AP by one-site non-competitive ELISA ... 96-well microtiter plates (Nunc A/S, Roskilde, Denmark) were coated with 2 μg/ml (200 μl per well in PBS containing 137 mM NaCl, 2.7 mM KCL, 6.5 mM Na2 HPO4 and 1 mM KH2 PO4 (pH 7.4)) of recombinant Met(R6)-α2AP, Met(W6)-α2AP or Asn-α2AP for 24 hours at 4°C.

    Incubation:

    Article Title: Neuroprotective effects of Rhizoma Dioscoreae polysaccharides against neuronal apoptosis induced by in vitro hypoxia
    Article Snippet: Subsequently, 0.1 ml cells were seeded at a density of 5×105 cells/ml into 96-well culture plates coated with polylysine, and were subsequently stored in a 5% CO2 incubator (Thermo Fisher Scientific, Inc.) at 37°C with saturated humidity. .. After 4 h, Gibco Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Inc.), supplemented with 10% equine serum, was removed from the plates, and the cell cultures were incubated with 2.0% B-27 neurobasal medium for 4 days.

    Article Title: Inhibition of Pyrimidine Biosynthesis Pathway Suppresses Viral Growth through Innate Immunity
    Article Snippet: For one 96-well culture plate, 17 µg of plasmid were diluted in 500 µl of DMEM (Gibco-Invitrogen). .. PEI and plasmids were mixed together and incubated for 30 min at room temperature.

    Article Title: Growth inhibitory and chemo-sensitization effects of naringenin, a natural flavanone purified from Thymus vulgaris, on human breast and colorectal cancer
    Article Snippet: Colorectal and breast cancer cells (5 × 105 cells/ml) were plated in 24-well plates and incubated in a non-CO2 incubator for 18 h. The cells were then treated with Nar (3 mM) for 24 h. mRNA was extracted using the nucleospin RNAII ready-to-use system (MACHEREY-NAGEL), and 200 ng/μl of mRNA was used in the RT reaction. .. For each sample, 2.5 μl of cDNA and 12.5 μl of Taqman Universal Master Mix (2×) were used, and the final volume was adjusted to 25 μl with nuclease-free water on an optical 96-well reaction plate (Applied Biosystems).

    Article Title: Tumor cells and their crosstalk with endothelial cells in 3D spheroids
    Article Snippet: .. Spheroids formation using multi-well agarose-coated plates Agarose hydrogel 1.5% (100 µl) was added to each well of a 96-well culture plate (Thermo Fisher Scientific, Denmark), and incubated at 37 °C for 2 hours. .. A375, BxPC3, PANC1 and MDA-MB-231cells were seeded in 100 µl growth medium at a concentration of 5,000 cells per well.

    Article Title: The Evolutionary Conserved γ-Core Motif Influences the Anti-Candida Activity of the Penicillium chrysogenum Antifungal Protein PAF
    Article Snippet: Antifungal Activity Determination The MIC of all protein and peptide variants was determined on C. albicans with broth microdilution assays in 96-well microtiter plates (Nunclon Delta, Thermo Fisher Scientific). .. In each well, 103 cells were incubated with increasing concentrations of antifungal proteins or peptides in a final volume of 200 μL 0.05× PDB.

    Article Title: Generation and characterization of monoclonal antibodies against the N-terminus of alpha-2-antiplasmin
    Article Snippet: 96-well microtiter plates (Nunc A/S, Roskilde, Denmark) were coated with 2 μg/ml (200 μl per well in PBS containing 137 mM NaCl, 2.7 mM KCL, 6.5 mM Na2 HPO4 and 1 mM KH2 PO4 (pH 7.4)) of recombinant Met(R6)-α2AP, Met(W6)-α2AP or Asn-α2AP for 24 hours at 4°C. .. Non-specific sites were blocked with 200 μl 1% bovine serum albumin (Sigma, Steinheim, Germany) in PBS and incubated for two hours at RT.

    Activity Assay:

    Article Title: The Evolutionary Conserved γ-Core Motif Influences the Anti-Candida Activity of the Penicillium chrysogenum Antifungal Protein PAF
    Article Snippet: .. The antifungal activity against C. albicans biofilms was determined in 96-well microtiter plates (Nunclon Delta, Thermo Fisher Scientific). .. Cells (105 ) in 100 μL 0.05× PDB were distributed per well and plates were incubated at 37°C for up to 24 h to allow biofilm formation.

    Article Title: The Evolutionary Conserved γ-Core Motif Influences the Anti-Candida Activity of the Penicillium chrysogenum Antifungal Protein PAF
    Article Snippet: .. Antifungal Activity Determination The MIC of all protein and peptide variants was determined on C. albicans with broth microdilution assays in 96-well microtiter plates (Nunclon Delta, Thermo Fisher Scientific). .. From a fresh C. albicans overnight culture on YPD agar, cells were harvested with an inoculation loop and suspended in 0.05× PDB (Sigma-Aldrich) ( ).

    Expressing:

    Article Title: Identification of Genes Required for Secretion of the Francisella Oxidative Burst-Inhibiting Acid Phosphatase AcpA
    Article Snippet: Both WT bacteria and the WT strain carrying the acpA expression plasmid (pacpA -Flag) were tested. .. A volume of 50 μl of supernatant was dried in a 96-well immune-plate (Thermo Scientific, Rochester, NY, USA) overnight in the presence of protease inhibitor and then used for AcpA ELISA analysis as described below.

    Article Title: Differential Expression of Prostaglandin I2 Synthase Associated with Arachidonic Acid Pathway in the Oral Squamous Cell Carcinoma
    Article Snippet: .. Gene expression quantification was performed using TaqMan Array Human CYP450 and other Oxygenases 96-well plate (Applied Biosystems) by real time qPCR. ..

    Article Title: Growth inhibitory and chemo-sensitization effects of naringenin, a natural flavanone purified from Thymus vulgaris, on human breast and colorectal cancer
    Article Snippet: For the comparative Ct method, we performed a two-step RT-PCR to obtain cDNA and real-time quantification using the target gene expression assays and Taqman Universal Master Mix (Applied Biosystems). .. For each sample, 2.5 μl of cDNA and 12.5 μl of Taqman Universal Master Mix (2×) were used, and the final volume was adjusted to 25 μl with nuclease-free water on an optical 96-well reaction plate (Applied Biosystems).

    Modification:

    Article Title: Neuroprotective effects of Rhizoma Dioscoreae polysaccharides against neuronal apoptosis induced by in vitro hypoxia
    Article Snippet: The sections were then placed into a 0.25% pancreatic enzyme EDTA solution at 37°C for 20 min, followed by termination of digestion by addition of 5 ml Dulbecco's Modified Eagle medium (DMEM; Gibco, Thermo Fisher Scientific, Inc.) supplemented with 10% horse serum and 10% fetal bovine serum for 5 min. .. Subsequently, 0.1 ml cells were seeded at a density of 5×105 cells/ml into 96-well culture plates coated with polylysine, and were subsequently stored in a 5% CO2 incubator (Thermo Fisher Scientific, Inc.) at 37°C with saturated humidity.

    Real-time Polymerase Chain Reaction:

    Article Title: Differential Expression of Prostaglandin I2 Synthase Associated with Arachidonic Acid Pathway in the Oral Squamous Cell Carcinoma
    Article Snippet: .. Gene expression quantification was performed using TaqMan Array Human CYP450 and other Oxygenases 96-well plate (Applied Biosystems) by real time qPCR. ..

    Article Title: Growth inhibitory and chemo-sensitization effects of naringenin, a natural flavanone purified from Thymus vulgaris, on human breast and colorectal cancer
    Article Snippet: Paragraph title: Relative quantitative Real-Time PCR assay ... For each sample, 2.5 μl of cDNA and 12.5 μl of Taqman Universal Master Mix (2×) were used, and the final volume was adjusted to 25 μl with nuclease-free water on an optical 96-well reaction plate (Applied Biosystems).

    Transfection:

    Article Title: Inhibition of Pyrimidine Biosynthesis Pathway Suppresses Viral Growth through Innate Immunity
    Article Snippet: Cell transfection was performed with a 1 mg/ml poly-ethylenimine (PEI “Max”; Polyscience) solution adjusted at pH = 7. .. For one 96-well culture plate, 17 µg of plasmid were diluted in 500 µl of DMEM (Gibco-Invitrogen).

    Activation Assay:

    Article Title: Triptolide, a constituent of immunosuppressive Chinese herbal medicine, is a potent suppressor of dendritic-cell maturation and trafficking
    Article Snippet: Paragraph title: Allogeneic and anti-CD3/CD28–induced CD4+ T-cell activation ... Purified CD4+ T cells (105 /well) were cultured in 96-well flat-bottom plates with CD3/CD28 T-cell expansion Dynabeads (5 × 103 /well; Dynal ASA, Oslo, Norway).

    Article Title: Dendrimer-RNA nanoparticles generate protective immunity against lethal Ebola, H1N1 influenza, and Toxoplasma gondii challenges with a single dose
    Article Snippet: Paragraph title: T-Cell Activation Assay. ... Splenocytes were isolated from mice and plated at a density of 10 million cells per milliliter in 96-well culture plates in the presence of growth medium [all components were from Life Technologies unless otherwise indicated: RPMI 1620 with GlutaMAX supplemented with 8% FBS, 1 mM nonessential amino acids, 1 mM sodium pyruvate, 10 mM Hepes, 50 μM 2-mercaptoethanol (Sigma), and penicillin/streptomycin] only or 2 μg/mL OVA-derived peptide in growth medium.

    Protease Inhibitor:

    Article Title: Identification of Genes Required for Secretion of the Francisella Oxidative Burst-Inhibiting Acid Phosphatase AcpA
    Article Snippet: .. A volume of 50 μl of supernatant was dried in a 96-well immune-plate (Thermo Scientific, Rochester, NY, USA) overnight in the presence of protease inhibitor and then used for AcpA ELISA analysis as described below. .. The bacterial membrane protein FopA was included as a negative control.

    Transferring:

    Article Title: Neuroprotective effects of Rhizoma Dioscoreae polysaccharides against neuronal apoptosis induced by in vitro hypoxia
    Article Snippet: The cells were isolated from the sections by a mechanical method using a Pasteur pipette, and passed through a 200 mesh stainless steel sieve. .. Subsequently, 0.1 ml cells were seeded at a density of 5×105 cells/ml into 96-well culture plates coated with polylysine, and were subsequently stored in a 5% CO2 incubator (Thermo Fisher Scientific, Inc.) at 37°C with saturated humidity.

    Cell Culture:

    Article Title: Triptolide, a constituent of immunosuppressive Chinese herbal medicine, is a potent suppressor of dendritic-cell maturation and trafficking
    Article Snippet: .. Purified CD4+ T cells (105 /well) were cultured in 96-well flat-bottom plates with CD3/CD28 T-cell expansion Dynabeads (5 × 103 /well; Dynal ASA, Oslo, Norway). ..

    Generated:

    Article Title: Protein Kinase C Regulates the Cell Surface Activity of Endothelin-Converting Enzyme-1
    Article Snippet: Cell-based Quenched Fluorescent Substrate Assay EA.hy926 cells grown in 96-well microtiter plates were washed with Opti-MEM (Invitrogen Australia, Mt. .. Fluorescence generated by cleavage of the QFS was continuously monitored (λ ex = 320 nm, λ em = 405 nm) in a FLUOStar Optima plate reader (BMG LabTech, Offenburg, Germany) maintained at 37°C.

    Imaging:

    Article Title: Plaque2.0—A High-Throughput Analysis Framework to Score Virus-Cell Transmission and Clonal Cell Expansion
    Article Snippet: .. All live imaging experiments were performed in 96-well black plates (Matrix; Thermo Fisher Scientific, Lausanne, Switzerland) and the entire wells were acquired in four sites by a motorized stage, allowing precise, high-speed selection of overlapping tiles. .. Confocal microscopy Confocal fluorescence microscopy was conducted with a Leica SP5 confocal laser scanning microscope (Leica Microsystems, Germany) equipped with a 63x objective (oil immersion; NA 1.4), a UV laser (355 nm), an argon laser (488 nm), and a diode laser (561 nm).

    Protein Concentration:

    Article Title: The Evolutionary Conserved γ-Core Motif Influences the Anti-Candida Activity of the Penicillium chrysogenum Antifungal Protein PAF
    Article Snippet: The MIC was defined as the protein concentration that inhibited growth by ≥90% compared to the untreated control, which was referred as 100% growth. .. The antifungal activity against C. albicans biofilms was determined in 96-well microtiter plates (Nunclon Delta, Thermo Fisher Scientific).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Growth inhibitory and chemo-sensitization effects of naringenin, a natural flavanone purified from Thymus vulgaris, on human breast and colorectal cancer
    Article Snippet: For the comparative Ct method, we performed a two-step RT-PCR to obtain cDNA and real-time quantification using the target gene expression assays and Taqman Universal Master Mix (Applied Biosystems). .. For each sample, 2.5 μl of cDNA and 12.5 μl of Taqman Universal Master Mix (2×) were used, and the final volume was adjusted to 25 μl with nuclease-free water on an optical 96-well reaction plate (Applied Biosystems).

    Recombinant:

    Article Title: Inhibition of Pyrimidine Biosynthesis Pathway Suppresses Viral Growth through Innate Immunity
    Article Snippet: For each plate, columns 1 and 12 were dedicated to controls: culture wells were alternatively spiked with 1 µl of DMSO alone (negative control) or supplemented with recombinant IFN-β so that final concentration equals 1,000 IU/ml (positive control). .. For one 96-well culture plate, 17 µg of plasmid were diluted in 500 µl of DMEM (Gibco-Invitrogen).

    Article Title: Generation and characterization of monoclonal antibodies against the N-terminus of alpha-2-antiplasmin
    Article Snippet: .. 96-well microtiter plates (Nunc A/S, Roskilde, Denmark) were coated with 2 μg/ml (200 μl per well in PBS containing 137 mM NaCl, 2.7 mM KCL, 6.5 mM Na2 HPO4 and 1 mM KH2 PO4 (pH 7.4)) of recombinant Met(R6)-α2AP, Met(W6)-α2AP or Asn-α2AP for 24 hours at 4°C. .. Non-specific sites were blocked with 200 μl 1% bovine serum albumin (Sigma, Steinheim, Germany) in PBS and incubated for two hours at RT.

    MTT Assay:

    Article Title: Neuroprotective effects of Rhizoma Dioscoreae polysaccharides against neuronal apoptosis induced by in vitro hypoxia
    Article Snippet: Subsequently, 0.1 ml cells were seeded at a density of 5×105 cells/ml into 96-well culture plates coated with polylysine, and were subsequently stored in a 5% CO2 incubator (Thermo Fisher Scientific, Inc.) at 37°C with saturated humidity. .. Subsequently, the cells were incubated with RDPS (0.025, 0.05, 0.10, 0.25, 0.50, 1.0, 2.0, 4.0 or 8.0 g/l) for 48 h, after which the culture medium (0.1 ml) was removed and added to 96-well plates containing 0.5% MTT for 4 h. Following removal of the culture media, 0.15 ml dimethyl sulfoxide (DMSO; Sigma-Aldrich) was added to the wells, and the plates were agitated for 10 min. Absorbance [optical density (OD)] values were measured at 490 nm using a microplate reader (EL×800™; BioTek Instruments, Inc., Winooski, VT, USA).

    Fluorescence:

    Article Title: Protein Kinase C Regulates the Cell Surface Activity of Endothelin-Converting Enzyme-1
    Article Snippet: Cell-based Quenched Fluorescent Substrate Assay EA.hy926 cells grown in 96-well microtiter plates were washed with Opti-MEM (Invitrogen Australia, Mt. .. Fluorescence generated by cleavage of the QFS was continuously monitored (λ ex = 320 nm, λ em = 405 nm) in a FLUOStar Optima plate reader (BMG LabTech, Offenburg, Germany) maintained at 37°C.

    Multiple Displacement Amplification:

    Article Title: Tumor cells and their crosstalk with endothelial cells in 3D spheroids
    Article Snippet: Spheroids formation using multi-well agarose-coated plates Agarose hydrogel 1.5% (100 µl) was added to each well of a 96-well culture plate (Thermo Fisher Scientific, Denmark), and incubated at 37 °C for 2 hours. .. A375, BxPC3, PANC1 and MDA-MB-231cells were seeded in 100 µl growth medium at a concentration of 5,000 cells per well.

    Isolation:

    Article Title: Neuroprotective effects of Rhizoma Dioscoreae polysaccharides against neuronal apoptosis induced by in vitro hypoxia
    Article Snippet: The cells were isolated from the sections by a mechanical method using a Pasteur pipette, and passed through a 200 mesh stainless steel sieve. .. Subsequently, 0.1 ml cells were seeded at a density of 5×105 cells/ml into 96-well culture plates coated with polylysine, and were subsequently stored in a 5% CO2 incubator (Thermo Fisher Scientific, Inc.) at 37°C with saturated humidity.

    Article Title: Dendrimer-RNA nanoparticles generate protective immunity against lethal Ebola, H1N1 influenza, and Toxoplasma gondii challenges with a single dose
    Article Snippet: .. Splenocytes were isolated from mice and plated at a density of 10 million cells per milliliter in 96-well culture plates in the presence of growth medium [all components were from Life Technologies unless otherwise indicated: RPMI 1620 with GlutaMAX supplemented with 8% FBS, 1 mM nonessential amino acids, 1 mM sodium pyruvate, 10 mM Hepes, 50 μM 2-mercaptoethanol (Sigma), and penicillin/streptomycin] only or 2 μg/mL OVA-derived peptide in growth medium. .. Peptides used were the immunodominant H-2Kb –restricted MHC class I OVA-derived peptide SIINFEKL (InvivoGen) or the H-2 I-Ab MHC class II-restricted peptide ISQAVHAAHAEINEAGR (InvivoGen).

    Detection Assay:

    Article Title: Sulforaphane induces apoptosis in rhabdomyosarcoma and restores TRAIL-sensitivity in the aggressive alveolar subtype leading to tumor elimination in mice
    Article Snippet: Cell viability was determined using the Luminescence ATP Detection Assay System (ATPlite) (PerkinElmer). .. Cells were plated in triplicate using 96-well cluster plates: 20 × 103 CCA cells were plated with 100 μL of OPTI-MEM (GIBCO BRL) containing 4% FCS, 2 mM l -glutamine, and 1% penicillin/streptomycin.

    Microscopy:

    Article Title: Plaque2.0—A High-Throughput Analysis Framework to Score Virus-Cell Transmission and Clonal Cell Expansion
    Article Snippet: Paragraph title: Time-lapse multi-site, multi-channel microscopy ... All live imaging experiments were performed in 96-well black plates (Matrix; Thermo Fisher Scientific, Lausanne, Switzerland) and the entire wells were acquired in four sites by a motorized stage, allowing precise, high-speed selection of overlapping tiles.

    Purification:

    Article Title: Triptolide, a constituent of immunosuppressive Chinese herbal medicine, is a potent suppressor of dendritic-cell maturation and trafficking
    Article Snippet: .. Purified CD4+ T cells (105 /well) were cultured in 96-well flat-bottom plates with CD3/CD28 T-cell expansion Dynabeads (5 × 103 /well; Dynal ASA, Oslo, Norway). ..

    Article Title: Critical role of amino acid position 343 of surfactant protein-D in the selective binding of glycolipids from Mycobacterium tuberculosis
    Article Snippet: Bacterial single cell suspensions (5 × 105 ) in 50 μL TBS (50 mM Tris-hydrochloride + 150 mM sodium chloride, pH 7.5) were added and dried onto triplicate wells of a 96-well microtiter plate (Immulon 1; Thermo Electron Corporation, Milford, MA) followed by overnight UV irradiation treatment to kill the bacteria. .. Alternatively, 50 μL containing 0.1 μg/μL of purified mycobacterial cell wall components were dried in ethanol onto duplicate or triplicate wells of a 96-well microtiter plate (EIA/RIA, CoStar, Corning, NY).

    Article Title: Generation and characterization of monoclonal antibodies against the N-terminus of alpha-2-antiplasmin
    Article Snippet: Purified mAbs were tested for their specific reactivities with α2AP by one-site non-competitive ELISA. .. 96-well microtiter plates (Nunc A/S, Roskilde, Denmark) were coated with 2 μg/ml (200 μl per well in PBS containing 137 mM NaCl, 2.7 mM KCL, 6.5 mM Na2 HPO4 and 1 mM KH2 PO4 (pH 7.4)) of recombinant Met(R6)-α2AP, Met(W6)-α2AP or Asn-α2AP for 24 hours at 4°C.

    Selection:

    Article Title: Plaque2.0—A High-Throughput Analysis Framework to Score Virus-Cell Transmission and Clonal Cell Expansion
    Article Snippet: .. All live imaging experiments were performed in 96-well black plates (Matrix; Thermo Fisher Scientific, Lausanne, Switzerland) and the entire wells were acquired in four sites by a motorized stage, allowing precise, high-speed selection of overlapping tiles. .. Confocal microscopy Confocal fluorescence microscopy was conducted with a Leica SP5 confocal laser scanning microscope (Leica Microsystems, Germany) equipped with a 63x objective (oil immersion; NA 1.4), a UV laser (355 nm), an argon laser (488 nm), and a diode laser (561 nm).

    Mouse Assay:

    Article Title: Dendrimer-RNA nanoparticles generate protective immunity against lethal Ebola, H1N1 influenza, and Toxoplasma gondii challenges with a single dose
    Article Snippet: .. Splenocytes were isolated from mice and plated at a density of 10 million cells per milliliter in 96-well culture plates in the presence of growth medium [all components were from Life Technologies unless otherwise indicated: RPMI 1620 with GlutaMAX supplemented with 8% FBS, 1 mM nonessential amino acids, 1 mM sodium pyruvate, 10 mM Hepes, 50 μM 2-mercaptoethanol (Sigma), and penicillin/streptomycin] only or 2 μg/mL OVA-derived peptide in growth medium. .. Peptides used were the immunodominant H-2Kb –restricted MHC class I OVA-derived peptide SIINFEKL (InvivoGen) or the H-2 I-Ab MHC class II-restricted peptide ISQAVHAAHAEINEAGR (InvivoGen).

    Viability Assay:

    Article Title: Sulforaphane induces apoptosis in rhabdomyosarcoma and restores TRAIL-sensitivity in the aggressive alveolar subtype leading to tumor elimination in mice
    Article Snippet: Paragraph title: Viability assay ... Cells were plated in triplicate using 96-well cluster plates: 20 × 103 CCA cells were plated with 100 μL of OPTI-MEM (GIBCO BRL) containing 4% FCS, 2 mM l -glutamine, and 1% penicillin/streptomycin.

    Plasmid Preparation:

    Article Title: Identification of Genes Required for Secretion of the Francisella Oxidative Burst-Inhibiting Acid Phosphatase AcpA
    Article Snippet: Both WT bacteria and the WT strain carrying the acpA expression plasmid (pacpA -Flag) were tested. .. A volume of 50 μl of supernatant was dried in a 96-well immune-plate (Thermo Scientific, Rochester, NY, USA) overnight in the presence of protease inhibitor and then used for AcpA ELISA analysis as described below.

    Article Title: Inhibition of Pyrimidine Biosynthesis Pathway Suppresses Viral Growth through Innate Immunity
    Article Snippet: .. For one 96-well culture plate, 17 µg of plasmid were diluted in 500 µl of DMEM (Gibco-Invitrogen). .. In parallel, 53 µg of poly(ethyleneimine) from Sigma-Aldrich (PEI) were diluted in 500 µl of DMEM (Gibco-BRL).

    Software:

    Article Title: Growth inhibitory and chemo-sensitization effects of naringenin, a natural flavanone purified from Thymus vulgaris, on human breast and colorectal cancer
    Article Snippet: For each sample, 2.5 μl of cDNA and 12.5 μl of Taqman Universal Master Mix (2×) were used, and the final volume was adjusted to 25 μl with nuclease-free water on an optical 96-well reaction plate (Applied Biosystems). .. Real-time PCR was performed on an ABI 7000 SDS system using ABI Prism's SDS collection software version 1.1 (Applied Biosystems).

    Irradiation:

    Article Title: Critical role of amino acid position 343 of surfactant protein-D in the selective binding of glycolipids from Mycobacterium tuberculosis
    Article Snippet: .. Bacterial single cell suspensions (5 × 105 ) in 50 μL TBS (50 mM Tris-hydrochloride + 150 mM sodium chloride, pH 7.5) were added and dried onto triplicate wells of a 96-well microtiter plate (Immulon 1; Thermo Electron Corporation, Milford, MA) followed by overnight UV irradiation treatment to kill the bacteria. .. To confirm that NCRD binding was independent of the UV exposure, ELISA experiments were also performed without UV treatment with no significant differences seen in the results (data not shown).

    Negative Control:

    Article Title: Identification of Genes Required for Secretion of the Francisella Oxidative Burst-Inhibiting Acid Phosphatase AcpA
    Article Snippet: A volume of 50 μl of supernatant was dried in a 96-well immune-plate (Thermo Scientific, Rochester, NY, USA) overnight in the presence of protease inhibitor and then used for AcpA ELISA analysis as described below. .. The bacterial membrane protein FopA was included as a negative control.

    Article Title: Inhibition of Pyrimidine Biosynthesis Pathway Suppresses Viral Growth through Innate Immunity
    Article Snippet: For each plate, columns 1 and 12 were dedicated to controls: culture wells were alternatively spiked with 1 µl of DMSO alone (negative control) or supplemented with recombinant IFN-β so that final concentration equals 1,000 IU/ml (positive control). .. For one 96-well culture plate, 17 µg of plasmid were diluted in 500 µl of DMEM (Gibco-Invitrogen).

    Binding Assay:

    Article Title: Critical role of amino acid position 343 of surfactant protein-D in the selective binding of glycolipids from Mycobacterium tuberculosis
    Article Snippet: Binding of SP-D NCRD mutants to mycobacteria and/or their cell envelope components was performed by ELISA (Schlesinger et al. ). .. Bacterial single cell suspensions (5 × 105 ) in 50 μL TBS (50 mM Tris-hydrochloride + 150 mM sodium chloride, pH 7.5) were added and dried onto triplicate wells of a 96-well microtiter plate (Immulon 1; Thermo Electron Corporation, Milford, MA) followed by overnight UV irradiation treatment to kill the bacteria.

    Concentration Assay:

    Article Title: Neuroprotective effects of Rhizoma Dioscoreae polysaccharides against neuronal apoptosis induced by in vitro hypoxia
    Article Snippet: Next, the cells were counted and adjusted to a concentration of 5×106 /ml using DMEM. .. Subsequently, 0.1 ml cells were seeded at a density of 5×105 cells/ml into 96-well culture plates coated with polylysine, and were subsequently stored in a 5% CO2 incubator (Thermo Fisher Scientific, Inc.) at 37°C with saturated humidity.

    Article Title: Protein Kinase C Regulates the Cell Surface Activity of Endothelin-Converting Enzyme-1
    Article Snippet: Cell-based Quenched Fluorescent Substrate Assay EA.hy926 cells grown in 96-well microtiter plates were washed with Opti-MEM (Invitrogen Australia, Mt. .. Following pre-incubation at 37°C for 30 min, a bradykinin-based quenched fluorescent substrate (QFS), (7-methoxycoumarin-4-yl)acetyl-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(2, 4-dinitrophenyl) (custom synthesized by Auspep; Johnson and Ahn, ), was added to a final concentration of 40 μM.

    Article Title: Inhibition of Pyrimidine Biosynthesis Pathway Suppresses Viral Growth through Innate Immunity
    Article Snippet: For each plate, columns 1 and 12 were dedicated to controls: culture wells were alternatively spiked with 1 µl of DMSO alone (negative control) or supplemented with recombinant IFN-β so that final concentration equals 1,000 IU/ml (positive control). .. For one 96-well culture plate, 17 µg of plasmid were diluted in 500 µl of DMEM (Gibco-Invitrogen).

    Article Title: Dendrimer-RNA nanoparticles generate protective immunity against lethal Ebola, H1N1 influenza, and Toxoplasma gondii challenges with a single dose
    Article Snippet: Splenocytes were isolated from mice and plated at a density of 10 million cells per milliliter in 96-well culture plates in the presence of growth medium [all components were from Life Technologies unless otherwise indicated: RPMI 1620 with GlutaMAX supplemented with 8% FBS, 1 mM nonessential amino acids, 1 mM sodium pyruvate, 10 mM Hepes, 50 μM 2-mercaptoethanol (Sigma), and penicillin/streptomycin] only or 2 μg/mL OVA-derived peptide in growth medium. .. After 5 d in culture, the concentration of 1:20 diluted supernatant IFN-γ was quantified using IFN-γ ELISA kits (BD Biosciences).

    Article Title: Tumor cells and their crosstalk with endothelial cells in 3D spheroids
    Article Snippet: Spheroids formation using multi-well agarose-coated plates Agarose hydrogel 1.5% (100 µl) was added to each well of a 96-well culture plate (Thermo Fisher Scientific, Denmark), and incubated at 37 °C for 2 hours. .. A375, BxPC3, PANC1 and MDA-MB-231cells were seeded in 100 µl growth medium at a concentration of 5,000 cells per well.

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  • 99
    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: 99/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher elisa plates
    Description and characterization of the chimeric human <t>FasL-derived</t> constructs Panel A: Schematic representation of soluble FasL (sFasL), Flag-tagged sFasL (sfFasL), polymeric Flag-tagged soluble FasL (pfFasL), polymeric TCR γ4 and δ5 Flag-tagged soluble FasL generating the TCR-pfFasL upon cotransfection, and beta2-microglobulin-fused HLA-A*02: 01 Flag-tagged soluble FasL (HLA-pfFasL). The f and p symbols represent the flag epitope and the LIF receptor-derived domain triggering the polymerisation of the FasL oligomers, respectively. Panel B: direct immunoblot of the supernatants from COS cells transfected with the empty vector (control) or the FasL constructs sFasL, sfFasL and pfFasL. Panel C: immunoprecipitation of the TCR-pfFasL chimera from transfected HEK cells, using an irrelevant IgG1 antibody, the anti-Flag (clone M2), the anti-FasL (clone 10F2), the anti-TCRγδ (clone IMU-510) or the anti-TCRδ5 (clone 12C7) antibodies. Panel D: immunoprecipitation of the HLA-pfFasL chimera from the supernatant of COS cells, with anti-Flag, anti-FasL or anti-β2microglobulin antibodies. As controls, the same experiment was performed with irrelevant IgG1 and IgG2 antibodies. Panel E: cytotoxic effect of the FasL chimeras. The indicated chimeras, as supernatants from transfected cells and quantitated using the <t>ELISA</t> for FasL, were incubated at the indicated concentrations with Jurkat cells. After 18 h, the MTT cell viability assay was performed. The anti-Flag M2 antibody at 0.5 µg/ml was added to sfFasL to render it cytotoxic.
    Elisa Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 430 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    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: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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