96 well polystyrene microtiter plates  (Thermo Fisher)


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    Structured Review

    Thermo Fisher 96 well polystyrene microtiter plates
    Effect of soluble fibrinogen on platelet adhesion to the fibrin gel. (A) Fibrin gels were formed in <t>96-well</t> <t>microtiter</t> plates by mixing 100 μ L of 1.5 mg/mL fibrinogen with 0.15 unit/mL thrombin. After polymerization for 2 h at 37 °C, thrombin was inactivated by adding PPACK (5 mM). The gels were incubated with different concentrations of fibrinogen for 30 min at 37 °C, the solution above the gels were aspirated, and the gels were incubated for another 1 h at 37 °C. Aliquots (100 μ L) of labeled platelets (1×10 8 /mL) were added to each well. After 50 min incubation at 37 °C, the nonadherent cells were removed by two washes with PBS, and fluorescence was measured. Adhesion of platelets to intact fibrin clots (in the absence of fibrinogen)was 16.5±1.2%of added cells. The data shown are the means ± SE of four experiments with triplicate determinations at each experimental point. (B) Scanning electron microscopy of platelets adherent to the naked fibrin gel (upper panel) and to fibrin coated with 1 mg/mL soluble fibrinogen. Fibrin and fibrinogen-treated fibrin gels were prepared as described in Materials and Methods. Two representative platelets adherent to each substrate are shown.
    96 Well Polystyrene Microtiter Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96 well polystyrene microtiter plates - by Bioz Stars, 2020-04
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    Images

    1) Product Images from "Control of Integrin αIIbβ3 Outside-In Signaling and Platelet Adhesion by Sensing the Physical Properties of Fibrin(ogen) Substrates"

    Article Title: Control of Integrin αIIbβ3 Outside-In Signaling and Platelet Adhesion by Sensing the Physical Properties of Fibrin(ogen) Substrates

    Journal: Biochemistry

    doi: 10.1021/bi9016022

    Effect of soluble fibrinogen on platelet adhesion to the fibrin gel. (A) Fibrin gels were formed in 96-well microtiter plates by mixing 100 μ L of 1.5 mg/mL fibrinogen with 0.15 unit/mL thrombin. After polymerization for 2 h at 37 °C, thrombin was inactivated by adding PPACK (5 mM). The gels were incubated with different concentrations of fibrinogen for 30 min at 37 °C, the solution above the gels were aspirated, and the gels were incubated for another 1 h at 37 °C. Aliquots (100 μ L) of labeled platelets (1×10 8 /mL) were added to each well. After 50 min incubation at 37 °C, the nonadherent cells were removed by two washes with PBS, and fluorescence was measured. Adhesion of platelets to intact fibrin clots (in the absence of fibrinogen)was 16.5±1.2%of added cells. The data shown are the means ± SE of four experiments with triplicate determinations at each experimental point. (B) Scanning electron microscopy of platelets adherent to the naked fibrin gel (upper panel) and to fibrin coated with 1 mg/mL soluble fibrinogen. Fibrin and fibrinogen-treated fibrin gels were prepared as described in Materials and Methods. Two representative platelets adherent to each substrate are shown.
    Figure Legend Snippet: Effect of soluble fibrinogen on platelet adhesion to the fibrin gel. (A) Fibrin gels were formed in 96-well microtiter plates by mixing 100 μ L of 1.5 mg/mL fibrinogen with 0.15 unit/mL thrombin. After polymerization for 2 h at 37 °C, thrombin was inactivated by adding PPACK (5 mM). The gels were incubated with different concentrations of fibrinogen for 30 min at 37 °C, the solution above the gels were aspirated, and the gels were incubated for another 1 h at 37 °C. Aliquots (100 μ L) of labeled platelets (1×10 8 /mL) were added to each well. After 50 min incubation at 37 °C, the nonadherent cells were removed by two washes with PBS, and fluorescence was measured. Adhesion of platelets to intact fibrin clots (in the absence of fibrinogen)was 16.5±1.2%of added cells. The data shown are the means ± SE of four experiments with triplicate determinations at each experimental point. (B) Scanning electron microscopy of platelets adherent to the naked fibrin gel (upper panel) and to fibrin coated with 1 mg/mL soluble fibrinogen. Fibrin and fibrinogen-treated fibrin gels were prepared as described in Materials and Methods. Two representative platelets adherent to each substrate are shown.

    Techniques Used: Incubation, Labeling, Fluorescence, Electron Microscopy

    Platelet adhesion to immobilized fibrinogen. Left ordinate: The wells of 96-well microtiter plates were coated with different concentrations of fibrinogen (0.1–50 μ g/mL) for 3 h at 37 °C followed by postcoating with 1%PVP. Aliquots (100 μ L) of labeled platelets (1 × 10 8 /mL) were added to each well. After 50 min incubation at 37 °C, the nonadherent cells were removed by two washes with PBS, and fluorescence was measured. Adhesion is shown as fluorescence in arbitrary units. The data shown are the means ± SE of four experiments with triplicate determinations at each experimental point. Maximal platelet adhesion was 17.3 ± 3.5% of added cells. Right ordinate: The wells of microtiter plates were coated with different concentrations of fibrinogen as described above, and mAb 2G5 was added at 1 μ g/mL. After incubation for 1 h at 37 °C, the microtiter plates were washed, and goat anti-mouse IgG conjugated to alkaline phosphatase was added. The binding was detected by reaction with p -nitrophenyl phosphate, measuring the absorbance at 405 nm.
    Figure Legend Snippet: Platelet adhesion to immobilized fibrinogen. Left ordinate: The wells of 96-well microtiter plates were coated with different concentrations of fibrinogen (0.1–50 μ g/mL) for 3 h at 37 °C followed by postcoating with 1%PVP. Aliquots (100 μ L) of labeled platelets (1 × 10 8 /mL) were added to each well. After 50 min incubation at 37 °C, the nonadherent cells were removed by two washes with PBS, and fluorescence was measured. Adhesion is shown as fluorescence in arbitrary units. The data shown are the means ± SE of four experiments with triplicate determinations at each experimental point. Maximal platelet adhesion was 17.3 ± 3.5% of added cells. Right ordinate: The wells of microtiter plates were coated with different concentrations of fibrinogen as described above, and mAb 2G5 was added at 1 μ g/mL. After incubation for 1 h at 37 °C, the microtiter plates were washed, and goat anti-mouse IgG conjugated to alkaline phosphatase was added. The binding was detected by reaction with p -nitrophenyl phosphate, measuring the absorbance at 405 nm.

    Techniques Used: Labeling, Incubation, Fluorescence, Binding Assay

    2) Product Images from "The Oxidative Stress Agent Hypochlorite Stimulates c-di-GMP Synthesis and Biofilm Formation in Pseudomonas aeruginosa"

    Article Title: The Oxidative Stress Agent Hypochlorite Stimulates c-di-GMP Synthesis and Biofilm Formation in Pseudomonas aeruginosa

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.02311

    Characterization of the DGC PA3177. (A) E. coli BL21-pET3177 and the vector control strain BL21-pET23a were grown in LB broth supplemented with 0.4 mM IPTG for 5 h at 37°C followed by nucleotide extraction and quantification of intracellular c-di-GMP by LC-MS. Levels of c-di-GMP were normalized to the total protein content of the respective sample and compared to c-di-GMP levels in empty vector control strain BL21-pET23a ( n = 6). The chromatogram shows c-di-GMP peaks of one representative measurement. (B) Attachment of P. aeruginosa during PA3177 overexpression was evaluated by crystal violet staining. PAO1-pJN3177 and empty vector control strain PAO1-pJN105 were incubated in 96-well microtiter plates for 2 h at 37°C followed by biomass quantification. For recombinant gene expression, BM2 was supplemented with 0.1% (w/v) arabinose (+Ara). Cultures without arabinose (–Ara) served as negative controls. Experiments were carried out in triplicate, each with six wells per strain and condition ( n = 18). (C) Planktonic growth during 5 h at 37°C. (D) Overnight cultures of PA3177-overexpressing strain P. aeruginosa PA01-pJN3177 and vector control PAO1-pJN105 were diluted in LB broth supplemented with 0.1% (w/v) arabinose and grown for 5 h at 37°C followed by evaluation of swimming, swarming and twitching motility. Assays were carried out with three independent bacterial cultures and at least four agar plates per experiment ( n ≥ 12). In all experiments statistical significance was evaluated by the Mann-Whitney test ( ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.01, n.s., not significant). Boxes include median (thick horizontal line), 25th and 75th percentiles. Dots indicate extreme values considered as outliers.
    Figure Legend Snippet: Characterization of the DGC PA3177. (A) E. coli BL21-pET3177 and the vector control strain BL21-pET23a were grown in LB broth supplemented with 0.4 mM IPTG for 5 h at 37°C followed by nucleotide extraction and quantification of intracellular c-di-GMP by LC-MS. Levels of c-di-GMP were normalized to the total protein content of the respective sample and compared to c-di-GMP levels in empty vector control strain BL21-pET23a ( n = 6). The chromatogram shows c-di-GMP peaks of one representative measurement. (B) Attachment of P. aeruginosa during PA3177 overexpression was evaluated by crystal violet staining. PAO1-pJN3177 and empty vector control strain PAO1-pJN105 were incubated in 96-well microtiter plates for 2 h at 37°C followed by biomass quantification. For recombinant gene expression, BM2 was supplemented with 0.1% (w/v) arabinose (+Ara). Cultures without arabinose (–Ara) served as negative controls. Experiments were carried out in triplicate, each with six wells per strain and condition ( n = 18). (C) Planktonic growth during 5 h at 37°C. (D) Overnight cultures of PA3177-overexpressing strain P. aeruginosa PA01-pJN3177 and vector control PAO1-pJN105 were diluted in LB broth supplemented with 0.1% (w/v) arabinose and grown for 5 h at 37°C followed by evaluation of swimming, swarming and twitching motility. Assays were carried out with three independent bacterial cultures and at least four agar plates per experiment ( n ≥ 12). In all experiments statistical significance was evaluated by the Mann-Whitney test ( ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.01, n.s., not significant). Boxes include median (thick horizontal line), 25th and 75th percentiles. Dots indicate extreme values considered as outliers.

    Techniques Used: Plasmid Preparation, Liquid Chromatography with Mass Spectroscopy, Over Expression, Staining, Incubation, Recombinant, Expressing, Acetylene Reduction Assay, MANN-WHITNEY

    Implication of c-di-GMP in the response of P. aeruginosa PAO1 to NaClO. (A) P. aeruginosa PAO1 was incubated with NaClO (8 μg/ml, OD 600 = 1.0, BM2) for 1h followed by nucleotide extraction and quantification of intracellular c-di-GMP by LC-MS. Levels of c-di-GMP were normalized to the total protein content of the respective sample and compared to c-di-GMP levels in untreated controls ( n = 6). The chromatogram shows c-di-GMP peaks of one representative measurement. (B) Attachment of P. aeruginosa in response to NaClO during overexpression of the c-di-GMP-degrading PDE PA2133 was evaluated by crystal violet staining. PAO1-pJN2133 and the vector control strain PAO1-pJN105 were incubated in 96-well microtiter plates either in the presence or in the absence of NaClO (2 μg/ml) for 2 h at 37°C followed by the quantification of biofilm biomass. BM2 was supplemented with 0.1% (w/v) arabinose to induce recombinant gene expression. Experiments were repeated four times, each with six wells per strain and condition ( n = 24). Absorbance at 595nm (A 595 ) was determined and obtained values for NaClO-treated samples were normalized against the A 595 of respective untreated controls (=relative biomass). Statistical significance was evaluated by the Mann–Whitney test ( ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.01, n.s., not significant). Boxes include median (thick horizontal line), 25th and 75th percentiles. Dots indicate extreme values considered as outliers.
    Figure Legend Snippet: Implication of c-di-GMP in the response of P. aeruginosa PAO1 to NaClO. (A) P. aeruginosa PAO1 was incubated with NaClO (8 μg/ml, OD 600 = 1.0, BM2) for 1h followed by nucleotide extraction and quantification of intracellular c-di-GMP by LC-MS. Levels of c-di-GMP were normalized to the total protein content of the respective sample and compared to c-di-GMP levels in untreated controls ( n = 6). The chromatogram shows c-di-GMP peaks of one representative measurement. (B) Attachment of P. aeruginosa in response to NaClO during overexpression of the c-di-GMP-degrading PDE PA2133 was evaluated by crystal violet staining. PAO1-pJN2133 and the vector control strain PAO1-pJN105 were incubated in 96-well microtiter plates either in the presence or in the absence of NaClO (2 μg/ml) for 2 h at 37°C followed by the quantification of biofilm biomass. BM2 was supplemented with 0.1% (w/v) arabinose to induce recombinant gene expression. Experiments were repeated four times, each with six wells per strain and condition ( n = 24). Absorbance at 595nm (A 595 ) was determined and obtained values for NaClO-treated samples were normalized against the A 595 of respective untreated controls (=relative biomass). Statistical significance was evaluated by the Mann–Whitney test ( ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.01, n.s., not significant). Boxes include median (thick horizontal line), 25th and 75th percentiles. Dots indicate extreme values considered as outliers.

    Techniques Used: Incubation, Liquid Chromatography with Mass Spectroscopy, Over Expression, Staining, Plasmid Preparation, Recombinant, Expressing, MANN-WHITNEY

    Attachment and DGC expression in PAO1-PA3177Ω in response to NaClO. (A) Attachment of P. aeruginosa mutant strain PAO1-PA3177Ω and wildtype PAO1 after 2 h incubation with half-MIC concentrations of NaClO (2 μg/ml) compared to untreated controls was assayed in 96-well microtiter plates by crystal violet staining. To show alterations in attachment caused by NaClO, A 595 of treated samples was normalized to A 595 of untreated controls for each strain (=relative biomass). Statistical significance was evaluated by the Mann–Whitney test ( ∗∗∗ p ≤ 0.001, n ≥ 12). Boxes include median (thick horizontal line), 25th and 75th percentiles. Dots indicate extreme values considered as outliers. (B) P. aeruginosa mutant strain PAO1-PA3177Ω was treated with 2 μg/ml NaClO and relative gene expression levels of genes encoding DGCs in comparison to untreated control cultures were analyzed by qRT-PCR. The figure shows mean averages and standard deviations calculated from three experiments, each analyzed in duplicate ( n = 6). Ct values were normalized against the expression of housekeeping genes rpoD and fabD . The red line corresponds to a relative gene expression level of 1, which means no change in gene expression. Blue bars indicate relative gene expression values ≥ 3.
    Figure Legend Snippet: Attachment and DGC expression in PAO1-PA3177Ω in response to NaClO. (A) Attachment of P. aeruginosa mutant strain PAO1-PA3177Ω and wildtype PAO1 after 2 h incubation with half-MIC concentrations of NaClO (2 μg/ml) compared to untreated controls was assayed in 96-well microtiter plates by crystal violet staining. To show alterations in attachment caused by NaClO, A 595 of treated samples was normalized to A 595 of untreated controls for each strain (=relative biomass). Statistical significance was evaluated by the Mann–Whitney test ( ∗∗∗ p ≤ 0.001, n ≥ 12). Boxes include median (thick horizontal line), 25th and 75th percentiles. Dots indicate extreme values considered as outliers. (B) P. aeruginosa mutant strain PAO1-PA3177Ω was treated with 2 μg/ml NaClO and relative gene expression levels of genes encoding DGCs in comparison to untreated control cultures were analyzed by qRT-PCR. The figure shows mean averages and standard deviations calculated from three experiments, each analyzed in duplicate ( n = 6). Ct values were normalized against the expression of housekeeping genes rpoD and fabD . The red line corresponds to a relative gene expression level of 1, which means no change in gene expression. Blue bars indicate relative gene expression values ≥ 3.

    Techniques Used: Expressing, Mutagenesis, Incubation, Staining, MANN-WHITNEY, Quantitative RT-PCR

    Attachment assays with PAO1 mutants. (A) Attachment of P. aeruginosa PAO1 wildtype (WT) or PAO1 mutant strains during overexpression of PA3177 was evaluated by crystal violet staining. Bacteria were incubated in 96-well microtiter plates for 2 h at 37°C prior to biomass quantification. For recombinant gene expression, L was supplemented with 0.1% (w/v) arabinose (+). Cultures without arabinose (-) served as negative controls. (B) Attachment of P. aeruginosa PAO1 WT or PAO1 deletion mutants during NaClO treatment (2 μg/ml in BM2, 2 h) was evaluated by crystal violet staining. All experiments were carried out in triplicate, each with six wells per strain and condition ( n = 18). Statistical significance was evaluated by the Mann–Whitney test ( ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.01, n.s., not significant). Boxes include median (thick horizontal line), 25th and 75th percentiles. Dots indicate extreme values considered as outliers.
    Figure Legend Snippet: Attachment assays with PAO1 mutants. (A) Attachment of P. aeruginosa PAO1 wildtype (WT) or PAO1 mutant strains during overexpression of PA3177 was evaluated by crystal violet staining. Bacteria were incubated in 96-well microtiter plates for 2 h at 37°C prior to biomass quantification. For recombinant gene expression, L was supplemented with 0.1% (w/v) arabinose (+). Cultures without arabinose (-) served as negative controls. (B) Attachment of P. aeruginosa PAO1 WT or PAO1 deletion mutants during NaClO treatment (2 μg/ml in BM2, 2 h) was evaluated by crystal violet staining. All experiments were carried out in triplicate, each with six wells per strain and condition ( n = 18). Statistical significance was evaluated by the Mann–Whitney test ( ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.01, n.s., not significant). Boxes include median (thick horizontal line), 25th and 75th percentiles. Dots indicate extreme values considered as outliers.

    Techniques Used: Mutagenesis, Over Expression, Staining, Incubation, Recombinant, Expressing, MANN-WHITNEY

    Attachment of P. aeruginosa PAO1 in the presence of different oxidants. Attachment of P. aeruginosa PAO1 during 2 h incubation in the presence of sublethal NaClO concentrations (A–C) or half-MIC concentrations of oxidants NaClO (2 μg/ml), Ca(ClO) 2 (2 μg/ml), NH 2 Cl (equivalent to 2 μg/ml NaClO), H 2 O 2 (50 μg/ml) and paraquat (1 μg/ml) (D) . (A,B,D) Attachment was assayed in 96-well microtiter plates by crystal violet staining and subsequent measurement of A 595 . Obtained values for treated samples were normalized against A 595 of untreated controls (=relative biomass). Experiments were performed at least in triplicate, each with multiple wells per condition ( n ≥ 18). (C) P. aeruginosa PAO1 was incubated with sub-MIC concentrations of NaClO (2 μg/ml) in BM2 for 2 h in 50 ml reaction tubes containing glass microscope slides. Adhered bacteria were stained with the DNA-intercalating dye SYTO9 prior to visualization by fluorescence microscopy at 100× magnification (scale bar: 100 μm). Average surface coverages from 36 pictures per condition were calculated using ImageJ . Experiments were performed in triplicate. For all experiments, statistical significance was evaluated by the Mann-Whitney test ( ∗∗∗ p ≤ 0.001, n.s., not significant). Boxes include median (thick horizontal line), 25th and 75th percentiles. Dots indicate extreme values considered as outliers.
    Figure Legend Snippet: Attachment of P. aeruginosa PAO1 in the presence of different oxidants. Attachment of P. aeruginosa PAO1 during 2 h incubation in the presence of sublethal NaClO concentrations (A–C) or half-MIC concentrations of oxidants NaClO (2 μg/ml), Ca(ClO) 2 (2 μg/ml), NH 2 Cl (equivalent to 2 μg/ml NaClO), H 2 O 2 (50 μg/ml) and paraquat (1 μg/ml) (D) . (A,B,D) Attachment was assayed in 96-well microtiter plates by crystal violet staining and subsequent measurement of A 595 . Obtained values for treated samples were normalized against A 595 of untreated controls (=relative biomass). Experiments were performed at least in triplicate, each with multiple wells per condition ( n ≥ 18). (C) P. aeruginosa PAO1 was incubated with sub-MIC concentrations of NaClO (2 μg/ml) in BM2 for 2 h in 50 ml reaction tubes containing glass microscope slides. Adhered bacteria were stained with the DNA-intercalating dye SYTO9 prior to visualization by fluorescence microscopy at 100× magnification (scale bar: 100 μm). Average surface coverages from 36 pictures per condition were calculated using ImageJ . Experiments were performed in triplicate. For all experiments, statistical significance was evaluated by the Mann-Whitney test ( ∗∗∗ p ≤ 0.001, n.s., not significant). Boxes include median (thick horizontal line), 25th and 75th percentiles. Dots indicate extreme values considered as outliers.

    Techniques Used: Incubation, Staining, Microscopy, Fluorescence, MANN-WHITNEY

    3) Product Images from "Sensor Kinase PA4398 Modulates Swarming Motility and Biofilm Formation in Pseudomonas aeruginosa PA14"

    Article Title: Sensor Kinase PA4398 Modulates Swarming Motility and Biofilm Formation in Pseudomonas aeruginosa PA14

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.02832-14

    Biofilm formation and rapid attachment. Bacteria were grown for 24 h (A) or for 1 h (B) at 37°C in 96-well microtiter plates containing BM2 biofilm medium. Biofilm formation and adhesion ability were analyzed by staining of the adherent biomass with crystal violet, followed by quantification ( A 595 ) for the stained wells. Shown are the results of at least three independent biological experiments, each with six technical repeats. The statistical significance of differences between the PA4398 − mutant and WT PA14, as well as between the PA4398c and pUCP20 strains, was determined by the Mann-Whitney test (***, P ≤ 0.001; n. s., not significant). The highest and lowest outliers are indicated by “×.”
    Figure Legend Snippet: Biofilm formation and rapid attachment. Bacteria were grown for 24 h (A) or for 1 h (B) at 37°C in 96-well microtiter plates containing BM2 biofilm medium. Biofilm formation and adhesion ability were analyzed by staining of the adherent biomass with crystal violet, followed by quantification ( A 595 ) for the stained wells. Shown are the results of at least three independent biological experiments, each with six technical repeats. The statistical significance of differences between the PA4398 − mutant and WT PA14, as well as between the PA4398c and pUCP20 strains, was determined by the Mann-Whitney test (***, P ≤ 0.001; n. s., not significant). The highest and lowest outliers are indicated by “×.”

    Techniques Used: Staining, Mutagenesis, MANN-WHITNEY

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    Article Snippet: The protein was purified, and the identity was determined by Western blot analysis with EBNA1-specific antibody (MAB8173; Chemicon International). .. 96-well polystyrene plates (Thermo Fisher Scientific) were coated with 1 μg/well of rEBNA1 protein in PBS, or infected and noninfected cell lysates, or PBS alone overnight at 4°C.

    Transfection:

    Article Title: EBNA1-specific T cells from patients with multiple sclerosis cross react with myelin antigens and co-produce IFN-? and IL-2
    Article Snippet: The C-terminal domain of EBNA1 (aa 458–641) inserted in the expression vector pET15b (Novagen) was transfected into Escherichia coli BL21 (DE3) pLysS cells and expression was induced with 1 mM IPTG (Invitrogen). .. 96-well polystyrene plates (Thermo Fisher Scientific) were coated with 1 μg/well of rEBNA1 protein in PBS, or infected and noninfected cell lysates, or PBS alone overnight at 4°C.

    Cell Culture:

    Article Title: Collagen-Like Proteins (ClpA, ClpB, ClpC, and ClpD) Are Required for Biofilm Formation and Adhesion to Plant Roots by Bacillus amyloliquefaciens FZB42
    Article Snippet: Biofilm formation assay For the biofilm formation assays, cells were cultured from single colonies and then resuspended in 3 mL of LB at 37°C with shaking. .. To quantify biofilm growth, we applied the crystal violet staining method in 96-well polystyrene plates (Thermo) [ – ].

    other:

    Article Title: Manganese acquisition is essential for virulence of Enterococcus faecalis
    Article Snippet: Biofilm assays on fibrinogen-coated silicone catheters and 96-well polystyrene plates Silicone catheters (1 cm, Nalgene 50 silicone tubing, Brand Products) or 96-well polystyrene plates (Grenier CellSTAR) were coated overnight at 4°C with 100 μg ml-1 human fibrinogen free of plasminogen and von Willebrand Factor (Enzyme Research Laboratory).

    Injection:

    Article Title: Sodium Solute Symporter and Cadherin Proteins Act as Bacillus thuringiensis Cry3Ba Toxin Functional Receptors in Tribolium castaneum *
    Article Snippet: The assays were performed in 96-well polystyrene plates (Sterilin, Thermofisher) with one flour disc and one larva per well. .. For toxicity assays in gene silencing experiments, 4 days after dsRNA or control buffer injection, larvae were weighed and exposed to 12.5 μg/μl Cry3Ba spore-toxin mixtures in flour discs.

    Binding Assay:

    Article Title: Identification of the Actin and Plasminogen Binding Regions of Group B Streptococcal Phosphoglycerate Kinase *
    Article Snippet: Actin and plasminogen binding by full-length and truncated rGBS-PGK molecules were assayed using ELISA as described previously ( ). .. Actin (1 μg/well; Sigma-Aldrich) and plasminogen (0.1 μg/well; Sigma-Aldrich) were resuspended in 0.1 m sodium carbonate solution (pH 9.5) and immobilized to wells of a 96-well polystyrene plate (Maxi-sorp; NUNC, Thermo Fisher Scientific, Nepean, ON, CA) via 8 h of incubation at room temperature.

    Fluorescence:

    Article Title: Efficacy of Ozonated Water, Chlorine, Chlorine Dioxide, Quaternary Ammonium Compounds and Peroxyacetic Acid Against Listeria monocytogenes Biofilm on Polystyrene Surfaces
    Article Snippet: Live/Dead Staining of Biofilm The biofilms formed on wells of 96-well polystyrene plate were subjected to the BacLight Live/Dead® staining kit (Molecular Probes) per the manufacture’s manual. .. Briefly, cells in biofilms were incubated with 1:1 mixture of SYTO 9 and propidium iodide dyes in the dark for 15 min, rinsed with phosphate buffered saline, pH 7.4 (PBS), and then visualized using EVOS FL fluorescence microscope (Life Technologies).

    Article Title: Short-term Exposure to Triclosan Decreases Thyroxine In Vivo via Upregulation of Hepatic Catabolism in Young Long-Evans Rats
    Article Snippet: Each well in polystyrene 96-well plates (Nunc Thermo Fisher Scientific, Rochester, NY) contained a total of 235 μl, including 50 μl of substrate (1.5nM ethoxyresorufin or pentoxyresorufin), 50 μl of diluted microsomes, and 110 μl of 0.05mM Tris buffer (pH 8.0). .. The fluorescence signal was measured every 33 s for 5 min at 37°C after reaction initiation.

    Article Title: Efficacy of Ozonated Water, Chlorine, Chlorine Dioxide, Quaternary Ammonium Compounds and Peroxyacetic Acid Against Listeria monocytogenes Biofilm on Polystyrene Surfaces
    Article Snippet: The biofilms formed on wells of 96-well polystyrene plate were subjected to the BacLight Live/Dead® staining kit (Molecular Probes) per the manufacture’s manual. .. Briefly, cells in biofilms were incubated with 1:1 mixture of SYTO 9 and propidium iodide dyes in the dark for 15 min, rinsed with phosphate buffered saline, pH 7.4 (PBS), and then visualized using EVOS FL fluorescence microscope (Life Technologies).

    Microscopy:

    Article Title: Efficacy of Ozonated Water, Chlorine, Chlorine Dioxide, Quaternary Ammonium Compounds and Peroxyacetic Acid Against Listeria monocytogenes Biofilm on Polystyrene Surfaces
    Article Snippet: Live/Dead Staining of Biofilm The biofilms formed on wells of 96-well polystyrene plate were subjected to the BacLight Live/Dead® staining kit (Molecular Probes) per the manufacture’s manual. .. Briefly, cells in biofilms were incubated with 1:1 mixture of SYTO 9 and propidium iodide dyes in the dark for 15 min, rinsed with phosphate buffered saline, pH 7.4 (PBS), and then visualized using EVOS FL fluorescence microscope (Life Technologies).

    Article Title: Efficacy of Ozonated Water, Chlorine, Chlorine Dioxide, Quaternary Ammonium Compounds and Peroxyacetic Acid Against Listeria monocytogenes Biofilm on Polystyrene Surfaces
    Article Snippet: The biofilms formed on wells of 96-well polystyrene plate were subjected to the BacLight Live/Dead® staining kit (Molecular Probes) per the manufacture’s manual. .. Briefly, cells in biofilms were incubated with 1:1 mixture of SYTO 9 and propidium iodide dyes in the dark for 15 min, rinsed with phosphate buffered saline, pH 7.4 (PBS), and then visualized using EVOS FL fluorescence microscope (Life Technologies).

    Purification:

    Article Title: Danger signals derived from stressed and necrotic epithelial cells activate human eosinophils
    Article Snippet: Eosinophils diluted in KRG (50 000 cells/well) were put in 96-well polystyrene plates with v-shaped bottoms (Nunc A/S). .. As a control, purified lipopolysaccharide (LPS, smooth Escherichia coli 055, kind gift from Dr Liliana Håversen, Department of Clinical Bacteriology, Göteborg University) at final concentrations of 1 µg/ml, 10 ng/ml, 1 ng/ml, 0·1 ng/ml was added to the eosinophils either in the presence or absence of 2·5% heat-inactivated human serum.

    Article Title: EBNA1-specific T cells from patients with multiple sclerosis cross react with myelin antigens and co-produce IFN-? and IL-2
    Article Snippet: Paragraph title: ELISA and expression and purification of recombinant EBNA1. ... 96-well polystyrene plates (Thermo Fisher Scientific) were coated with 1 μg/well of rEBNA1 protein in PBS, or infected and noninfected cell lysates, or PBS alone overnight at 4°C.

    Labeling:

    Article Title: Immunogens Modeling a Fusion-Intermediate Conformation of gp41 Elicit Antibodies to the Membrane Proximal External Region of the HIV Envelope Glycoprotein
    Article Snippet: Briefly, 96-well polystyrene plates (Immulon II HB, Thermo Scientific) were coated with 50 μl/well of a 1.0 μM solution of the various recombinant proteins in PBS and incubated overnight at RT. .. Plates were washed and 50 μl /well of peroxidase labeled goat anti-mouse-IgG (H+L) (KPL) was applied and incubated 30 minutes at room temperature.

    Staining:

    Article Title: Efficacy of Ozonated Water, Chlorine, Chlorine Dioxide, Quaternary Ammonium Compounds and Peroxyacetic Acid Against Listeria monocytogenes Biofilm on Polystyrene Surfaces
    Article Snippet: .. Live/Dead Staining of Biofilm The biofilms formed on wells of 96-well polystyrene plate were subjected to the BacLight Live/Dead® staining kit (Molecular Probes) per the manufacture’s manual. .. Briefly, cells in biofilms were incubated with 1:1 mixture of SYTO 9 and propidium iodide dyes in the dark for 15 min, rinsed with phosphate buffered saline, pH 7.4 (PBS), and then visualized using EVOS FL fluorescence microscope (Life Technologies).

    Article Title: Efficacy of Ozonated Water, Chlorine, Chlorine Dioxide, Quaternary Ammonium Compounds and Peroxyacetic Acid Against Listeria monocytogenes Biofilm on Polystyrene Surfaces
    Article Snippet: .. The biofilms formed on wells of 96-well polystyrene plate were subjected to the BacLight Live/Dead® staining kit (Molecular Probes) per the manufacture’s manual. .. Briefly, cells in biofilms were incubated with 1:1 mixture of SYTO 9 and propidium iodide dyes in the dark for 15 min, rinsed with phosphate buffered saline, pH 7.4 (PBS), and then visualized using EVOS FL fluorescence microscope (Life Technologies).

    Article Title: Collagen-Like Proteins (ClpA, ClpB, ClpC, and ClpD) Are Required for Biofilm Formation and Adhesion to Plant Roots by Bacillus amyloliquefaciens FZB42
    Article Snippet: .. To quantify biofilm growth, we applied the crystal violet staining method in 96-well polystyrene plates (Thermo) [ – ]. ..

    Plasmid Preparation:

    Article Title: EBNA1-specific T cells from patients with multiple sclerosis cross react with myelin antigens and co-produce IFN-? and IL-2
    Article Snippet: The C-terminal domain of EBNA1 (aa 458–641) inserted in the expression vector pET15b (Novagen) was transfected into Escherichia coli BL21 (DE3) pLysS cells and expression was induced with 1 mM IPTG (Invitrogen). .. 96-well polystyrene plates (Thermo Fisher Scientific) were coated with 1 μg/well of rEBNA1 protein in PBS, or infected and noninfected cell lysates, or PBS alone overnight at 4°C.

    Negative Control:

    Article Title: Identification of the Actin and Plasminogen Binding Regions of Group B Streptococcal Phosphoglycerate Kinase *
    Article Snippet: Actin (1 μg/well; Sigma-Aldrich) and plasminogen (0.1 μg/well; Sigma-Aldrich) were resuspended in 0.1 m sodium carbonate solution (pH 9.5) and immobilized to wells of a 96-well polystyrene plate (Maxi-sorp; NUNC, Thermo Fisher Scientific, Nepean, ON, CA) via 8 h of incubation at room temperature. .. Wells incubated with sodium carbonate solution containing no protein were used as a negative control.

    Recombinant:

    Article Title: Immunogens Modeling a Fusion-Intermediate Conformation of gp41 Elicit Antibodies to the Membrane Proximal External Region of the HIV Envelope Glycoprotein
    Article Snippet: .. Briefly, 96-well polystyrene plates (Immulon II HB, Thermo Scientific) were coated with 50 μl/well of a 1.0 μM solution of the various recombinant proteins in PBS and incubated overnight at RT. ..

    Article Title: EBNA1-specific T cells from patients with multiple sclerosis cross react with myelin antigens and co-produce IFN-? and IL-2
    Article Snippet: Paragraph title: ELISA and expression and purification of recombinant EBNA1. ... 96-well polystyrene plates (Thermo Fisher Scientific) were coated with 1 μg/well of rEBNA1 protein in PBS, or infected and noninfected cell lysates, or PBS alone overnight at 4°C.

    Tube Formation Assay:

    Article Title: Collagen-Like Proteins (ClpA, ClpB, ClpC, and ClpD) Are Required for Biofilm Formation and Adhesion to Plant Roots by Bacillus amyloliquefaciens FZB42
    Article Snippet: Paragraph title: Biofilm formation assay ... To quantify biofilm growth, we applied the crystal violet staining method in 96-well polystyrene plates (Thermo) [ – ].

    Concentration Assay:

    Article Title: The effects of nicotine in the neonatal quinpirole rodent model of psychosis: Neural plasticity mechanisms and nicotinic receptor changes
    Article Snippet: For the BDNF assay, anti-BDNF monoclonal antibody (mAb) was added to a carbonate coating buffer (pH 9.7, per specifications included with the Promega protocol for BDNF), and 100 μl of the coating buffer was added to each well of a 96-well polystyrene ELISA plate (MaxiSorb, Nalge Nunc International, Rochester, NY) and incubated overnight at 4 °C. .. The standard was diluted in Block & Sample 1× buffer to achieve a concentration range of 0–500 pg/ml.

    Lysis:

    Article Title: The effects of nicotine in the neonatal quinpirole rodent model of psychosis: Neural plasticity mechanisms and nicotinic receptor changes
    Article Snippet: In brief, 250 μl of RIPA cell lysis buffer (150 mM NaCL, 50 mM Tris-HCl, 1.0% NP-40, 0.5% Sodium deoxycholate and 0.1% SDS) plus protease and phosphatase inhibitors (P5726, P8340, P0044, Sigma-Aldrich, St. Louis, MO) was added to each tissue sample and homogenized using a Fisher Scientific sonic dismembrator 500 (Fisher Scientific, Inc, Atlanta, GA). .. For the BDNF assay, anti-BDNF monoclonal antibody (mAb) was added to a carbonate coating buffer (pH 9.7, per specifications included with the Promega protocol for BDNF), and 100 μl of the coating buffer was added to each well of a 96-well polystyrene ELISA plate (MaxiSorb, Nalge Nunc International, Rochester, NY) and incubated overnight at 4 °C.

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  • 99
    Thermo Fisher 96 well polystyrene microtiter plates
    Characterization of the DGC PA3177. (A) E. coli BL21-pET3177 and the vector control strain BL21-pET23a were grown in LB broth supplemented with 0.4 mM IPTG for 5 h at 37°C followed by nucleotide extraction and quantification of intracellular c-di-GMP by LC-MS. Levels of c-di-GMP were normalized to the total protein content of the respective sample and compared to c-di-GMP levels in empty vector control strain BL21-pET23a ( n = 6). The chromatogram shows c-di-GMP peaks of one representative measurement. (B) Attachment of P. aeruginosa during PA3177 overexpression was evaluated by crystal violet staining. PAO1-pJN3177 and empty vector control strain PAO1-pJN105 were incubated in <t>96-well</t> <t>microtiter</t> plates for 2 h at 37°C followed by biomass quantification. For recombinant gene expression, BM2 was supplemented with 0.1% (w/v) arabinose (+Ara). Cultures without arabinose (–Ara) served as negative controls. Experiments were carried out in triplicate, each with six wells per strain and condition ( n = 18). (C) Planktonic growth during 5 h at 37°C. (D) Overnight cultures of PA3177-overexpressing strain P. aeruginosa PA01-pJN3177 and vector control PAO1-pJN105 were diluted in LB broth supplemented with 0.1% (w/v) arabinose and grown for 5 h at 37°C followed by evaluation of swimming, swarming and twitching motility. Assays were carried out with three independent bacterial cultures and at least four agar plates per experiment ( n ≥ 12). In all experiments statistical significance was evaluated by the Mann-Whitney test ( ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.01, n.s., not significant). Boxes include median (thick horizontal line), 25th and 75th percentiles. Dots indicate extreme values considered as outliers.
    96 Well Polystyrene Microtiter Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/96 well polystyrene microtiter plates/product/Thermo Fisher
    Average 99 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    96 well polystyrene microtiter plates - by Bioz Stars, 2020-04
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    87
    Thermo Fisher black welled polystyrene microtitre plate
    Expression of the viscA gene per biomass unit in biofilms (blue diamonds) and planktonic cells (red squares) in the initial state of biofilms grown in <t>microtitre</t> wells. Data represent mean ± sd for a representative experiment with five replicates. *Significant difference between biofilm and planktonic cells at each time point ( P
    Black Welled Polystyrene Microtitre Plate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/black welled polystyrene microtitre plate/product/Thermo Fisher
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    Characterization of the DGC PA3177. (A) E. coli BL21-pET3177 and the vector control strain BL21-pET23a were grown in LB broth supplemented with 0.4 mM IPTG for 5 h at 37°C followed by nucleotide extraction and quantification of intracellular c-di-GMP by LC-MS. Levels of c-di-GMP were normalized to the total protein content of the respective sample and compared to c-di-GMP levels in empty vector control strain BL21-pET23a ( n = 6). The chromatogram shows c-di-GMP peaks of one representative measurement. (B) Attachment of P. aeruginosa during PA3177 overexpression was evaluated by crystal violet staining. PAO1-pJN3177 and empty vector control strain PAO1-pJN105 were incubated in 96-well microtiter plates for 2 h at 37°C followed by biomass quantification. For recombinant gene expression, BM2 was supplemented with 0.1% (w/v) arabinose (+Ara). Cultures without arabinose (–Ara) served as negative controls. Experiments were carried out in triplicate, each with six wells per strain and condition ( n = 18). (C) Planktonic growth during 5 h at 37°C. (D) Overnight cultures of PA3177-overexpressing strain P. aeruginosa PA01-pJN3177 and vector control PAO1-pJN105 were diluted in LB broth supplemented with 0.1% (w/v) arabinose and grown for 5 h at 37°C followed by evaluation of swimming, swarming and twitching motility. Assays were carried out with three independent bacterial cultures and at least four agar plates per experiment ( n ≥ 12). In all experiments statistical significance was evaluated by the Mann-Whitney test ( ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.01, n.s., not significant). Boxes include median (thick horizontal line), 25th and 75th percentiles. Dots indicate extreme values considered as outliers.

    Journal: Frontiers in Microbiology

    Article Title: The Oxidative Stress Agent Hypochlorite Stimulates c-di-GMP Synthesis and Biofilm Formation in Pseudomonas aeruginosa

    doi: 10.3389/fmicb.2017.02311

    Figure Lengend Snippet: Characterization of the DGC PA3177. (A) E. coli BL21-pET3177 and the vector control strain BL21-pET23a were grown in LB broth supplemented with 0.4 mM IPTG for 5 h at 37°C followed by nucleotide extraction and quantification of intracellular c-di-GMP by LC-MS. Levels of c-di-GMP were normalized to the total protein content of the respective sample and compared to c-di-GMP levels in empty vector control strain BL21-pET23a ( n = 6). The chromatogram shows c-di-GMP peaks of one representative measurement. (B) Attachment of P. aeruginosa during PA3177 overexpression was evaluated by crystal violet staining. PAO1-pJN3177 and empty vector control strain PAO1-pJN105 were incubated in 96-well microtiter plates for 2 h at 37°C followed by biomass quantification. For recombinant gene expression, BM2 was supplemented with 0.1% (w/v) arabinose (+Ara). Cultures without arabinose (–Ara) served as negative controls. Experiments were carried out in triplicate, each with six wells per strain and condition ( n = 18). (C) Planktonic growth during 5 h at 37°C. (D) Overnight cultures of PA3177-overexpressing strain P. aeruginosa PA01-pJN3177 and vector control PAO1-pJN105 were diluted in LB broth supplemented with 0.1% (w/v) arabinose and grown for 5 h at 37°C followed by evaluation of swimming, swarming and twitching motility. Assays were carried out with three independent bacterial cultures and at least four agar plates per experiment ( n ≥ 12). In all experiments statistical significance was evaluated by the Mann-Whitney test ( ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.01, n.s., not significant). Boxes include median (thick horizontal line), 25th and 75th percentiles. Dots indicate extreme values considered as outliers.

    Article Snippet: Attachment Assay in Microtiter Plates Attachment assays in 96-well polystyrene microtiter plates (Nunc, Thermo Fisher Scientific, St. Leon-Rot, Germany) were performed as previously described ( ) with the following minor modifications.

    Techniques: Plasmid Preparation, Liquid Chromatography with Mass Spectroscopy, Over Expression, Staining, Incubation, Recombinant, Expressing, Acetylene Reduction Assay, MANN-WHITNEY

    Implication of c-di-GMP in the response of P. aeruginosa PAO1 to NaClO. (A) P. aeruginosa PAO1 was incubated with NaClO (8 μg/ml, OD 600 = 1.0, BM2) for 1h followed by nucleotide extraction and quantification of intracellular c-di-GMP by LC-MS. Levels of c-di-GMP were normalized to the total protein content of the respective sample and compared to c-di-GMP levels in untreated controls ( n = 6). The chromatogram shows c-di-GMP peaks of one representative measurement. (B) Attachment of P. aeruginosa in response to NaClO during overexpression of the c-di-GMP-degrading PDE PA2133 was evaluated by crystal violet staining. PAO1-pJN2133 and the vector control strain PAO1-pJN105 were incubated in 96-well microtiter plates either in the presence or in the absence of NaClO (2 μg/ml) for 2 h at 37°C followed by the quantification of biofilm biomass. BM2 was supplemented with 0.1% (w/v) arabinose to induce recombinant gene expression. Experiments were repeated four times, each with six wells per strain and condition ( n = 24). Absorbance at 595nm (A 595 ) was determined and obtained values for NaClO-treated samples were normalized against the A 595 of respective untreated controls (=relative biomass). Statistical significance was evaluated by the Mann–Whitney test ( ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.01, n.s., not significant). Boxes include median (thick horizontal line), 25th and 75th percentiles. Dots indicate extreme values considered as outliers.

    Journal: Frontiers in Microbiology

    Article Title: The Oxidative Stress Agent Hypochlorite Stimulates c-di-GMP Synthesis and Biofilm Formation in Pseudomonas aeruginosa

    doi: 10.3389/fmicb.2017.02311

    Figure Lengend Snippet: Implication of c-di-GMP in the response of P. aeruginosa PAO1 to NaClO. (A) P. aeruginosa PAO1 was incubated with NaClO (8 μg/ml, OD 600 = 1.0, BM2) for 1h followed by nucleotide extraction and quantification of intracellular c-di-GMP by LC-MS. Levels of c-di-GMP were normalized to the total protein content of the respective sample and compared to c-di-GMP levels in untreated controls ( n = 6). The chromatogram shows c-di-GMP peaks of one representative measurement. (B) Attachment of P. aeruginosa in response to NaClO during overexpression of the c-di-GMP-degrading PDE PA2133 was evaluated by crystal violet staining. PAO1-pJN2133 and the vector control strain PAO1-pJN105 were incubated in 96-well microtiter plates either in the presence or in the absence of NaClO (2 μg/ml) for 2 h at 37°C followed by the quantification of biofilm biomass. BM2 was supplemented with 0.1% (w/v) arabinose to induce recombinant gene expression. Experiments were repeated four times, each with six wells per strain and condition ( n = 24). Absorbance at 595nm (A 595 ) was determined and obtained values for NaClO-treated samples were normalized against the A 595 of respective untreated controls (=relative biomass). Statistical significance was evaluated by the Mann–Whitney test ( ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.01, n.s., not significant). Boxes include median (thick horizontal line), 25th and 75th percentiles. Dots indicate extreme values considered as outliers.

    Article Snippet: Attachment Assay in Microtiter Plates Attachment assays in 96-well polystyrene microtiter plates (Nunc, Thermo Fisher Scientific, St. Leon-Rot, Germany) were performed as previously described ( ) with the following minor modifications.

    Techniques: Incubation, Liquid Chromatography with Mass Spectroscopy, Over Expression, Staining, Plasmid Preparation, Recombinant, Expressing, MANN-WHITNEY

    Attachment and DGC expression in PAO1-PA3177Ω in response to NaClO. (A) Attachment of P. aeruginosa mutant strain PAO1-PA3177Ω and wildtype PAO1 after 2 h incubation with half-MIC concentrations of NaClO (2 μg/ml) compared to untreated controls was assayed in 96-well microtiter plates by crystal violet staining. To show alterations in attachment caused by NaClO, A 595 of treated samples was normalized to A 595 of untreated controls for each strain (=relative biomass). Statistical significance was evaluated by the Mann–Whitney test ( ∗∗∗ p ≤ 0.001, n ≥ 12). Boxes include median (thick horizontal line), 25th and 75th percentiles. Dots indicate extreme values considered as outliers. (B) P. aeruginosa mutant strain PAO1-PA3177Ω was treated with 2 μg/ml NaClO and relative gene expression levels of genes encoding DGCs in comparison to untreated control cultures were analyzed by qRT-PCR. The figure shows mean averages and standard deviations calculated from three experiments, each analyzed in duplicate ( n = 6). Ct values were normalized against the expression of housekeeping genes rpoD and fabD . The red line corresponds to a relative gene expression level of 1, which means no change in gene expression. Blue bars indicate relative gene expression values ≥ 3.

    Journal: Frontiers in Microbiology

    Article Title: The Oxidative Stress Agent Hypochlorite Stimulates c-di-GMP Synthesis and Biofilm Formation in Pseudomonas aeruginosa

    doi: 10.3389/fmicb.2017.02311

    Figure Lengend Snippet: Attachment and DGC expression in PAO1-PA3177Ω in response to NaClO. (A) Attachment of P. aeruginosa mutant strain PAO1-PA3177Ω and wildtype PAO1 after 2 h incubation with half-MIC concentrations of NaClO (2 μg/ml) compared to untreated controls was assayed in 96-well microtiter plates by crystal violet staining. To show alterations in attachment caused by NaClO, A 595 of treated samples was normalized to A 595 of untreated controls for each strain (=relative biomass). Statistical significance was evaluated by the Mann–Whitney test ( ∗∗∗ p ≤ 0.001, n ≥ 12). Boxes include median (thick horizontal line), 25th and 75th percentiles. Dots indicate extreme values considered as outliers. (B) P. aeruginosa mutant strain PAO1-PA3177Ω was treated with 2 μg/ml NaClO and relative gene expression levels of genes encoding DGCs in comparison to untreated control cultures were analyzed by qRT-PCR. The figure shows mean averages and standard deviations calculated from three experiments, each analyzed in duplicate ( n = 6). Ct values were normalized against the expression of housekeeping genes rpoD and fabD . The red line corresponds to a relative gene expression level of 1, which means no change in gene expression. Blue bars indicate relative gene expression values ≥ 3.

    Article Snippet: Attachment Assay in Microtiter Plates Attachment assays in 96-well polystyrene microtiter plates (Nunc, Thermo Fisher Scientific, St. Leon-Rot, Germany) were performed as previously described ( ) with the following minor modifications.

    Techniques: Expressing, Mutagenesis, Incubation, Staining, MANN-WHITNEY, Quantitative RT-PCR

    Attachment assays with PAO1 mutants. (A) Attachment of P. aeruginosa PAO1 wildtype (WT) or PAO1 mutant strains during overexpression of PA3177 was evaluated by crystal violet staining. Bacteria were incubated in 96-well microtiter plates for 2 h at 37°C prior to biomass quantification. For recombinant gene expression, L was supplemented with 0.1% (w/v) arabinose (+). Cultures without arabinose (-) served as negative controls. (B) Attachment of P. aeruginosa PAO1 WT or PAO1 deletion mutants during NaClO treatment (2 μg/ml in BM2, 2 h) was evaluated by crystal violet staining. All experiments were carried out in triplicate, each with six wells per strain and condition ( n = 18). Statistical significance was evaluated by the Mann–Whitney test ( ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.01, n.s., not significant). Boxes include median (thick horizontal line), 25th and 75th percentiles. Dots indicate extreme values considered as outliers.

    Journal: Frontiers in Microbiology

    Article Title: The Oxidative Stress Agent Hypochlorite Stimulates c-di-GMP Synthesis and Biofilm Formation in Pseudomonas aeruginosa

    doi: 10.3389/fmicb.2017.02311

    Figure Lengend Snippet: Attachment assays with PAO1 mutants. (A) Attachment of P. aeruginosa PAO1 wildtype (WT) or PAO1 mutant strains during overexpression of PA3177 was evaluated by crystal violet staining. Bacteria were incubated in 96-well microtiter plates for 2 h at 37°C prior to biomass quantification. For recombinant gene expression, L was supplemented with 0.1% (w/v) arabinose (+). Cultures without arabinose (-) served as negative controls. (B) Attachment of P. aeruginosa PAO1 WT or PAO1 deletion mutants during NaClO treatment (2 μg/ml in BM2, 2 h) was evaluated by crystal violet staining. All experiments were carried out in triplicate, each with six wells per strain and condition ( n = 18). Statistical significance was evaluated by the Mann–Whitney test ( ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.01, n.s., not significant). Boxes include median (thick horizontal line), 25th and 75th percentiles. Dots indicate extreme values considered as outliers.

    Article Snippet: Attachment Assay in Microtiter Plates Attachment assays in 96-well polystyrene microtiter plates (Nunc, Thermo Fisher Scientific, St. Leon-Rot, Germany) were performed as previously described ( ) with the following minor modifications.

    Techniques: Mutagenesis, Over Expression, Staining, Incubation, Recombinant, Expressing, MANN-WHITNEY

    Attachment of P. aeruginosa PAO1 in the presence of different oxidants. Attachment of P. aeruginosa PAO1 during 2 h incubation in the presence of sublethal NaClO concentrations (A–C) or half-MIC concentrations of oxidants NaClO (2 μg/ml), Ca(ClO) 2 (2 μg/ml), NH 2 Cl (equivalent to 2 μg/ml NaClO), H 2 O 2 (50 μg/ml) and paraquat (1 μg/ml) (D) . (A,B,D) Attachment was assayed in 96-well microtiter plates by crystal violet staining and subsequent measurement of A 595 . Obtained values for treated samples were normalized against A 595 of untreated controls (=relative biomass). Experiments were performed at least in triplicate, each with multiple wells per condition ( n ≥ 18). (C) P. aeruginosa PAO1 was incubated with sub-MIC concentrations of NaClO (2 μg/ml) in BM2 for 2 h in 50 ml reaction tubes containing glass microscope slides. Adhered bacteria were stained with the DNA-intercalating dye SYTO9 prior to visualization by fluorescence microscopy at 100× magnification (scale bar: 100 μm). Average surface coverages from 36 pictures per condition were calculated using ImageJ . Experiments were performed in triplicate. For all experiments, statistical significance was evaluated by the Mann-Whitney test ( ∗∗∗ p ≤ 0.001, n.s., not significant). Boxes include median (thick horizontal line), 25th and 75th percentiles. Dots indicate extreme values considered as outliers.

    Journal: Frontiers in Microbiology

    Article Title: The Oxidative Stress Agent Hypochlorite Stimulates c-di-GMP Synthesis and Biofilm Formation in Pseudomonas aeruginosa

    doi: 10.3389/fmicb.2017.02311

    Figure Lengend Snippet: Attachment of P. aeruginosa PAO1 in the presence of different oxidants. Attachment of P. aeruginosa PAO1 during 2 h incubation in the presence of sublethal NaClO concentrations (A–C) or half-MIC concentrations of oxidants NaClO (2 μg/ml), Ca(ClO) 2 (2 μg/ml), NH 2 Cl (equivalent to 2 μg/ml NaClO), H 2 O 2 (50 μg/ml) and paraquat (1 μg/ml) (D) . (A,B,D) Attachment was assayed in 96-well microtiter plates by crystal violet staining and subsequent measurement of A 595 . Obtained values for treated samples were normalized against A 595 of untreated controls (=relative biomass). Experiments were performed at least in triplicate, each with multiple wells per condition ( n ≥ 18). (C) P. aeruginosa PAO1 was incubated with sub-MIC concentrations of NaClO (2 μg/ml) in BM2 for 2 h in 50 ml reaction tubes containing glass microscope slides. Adhered bacteria were stained with the DNA-intercalating dye SYTO9 prior to visualization by fluorescence microscopy at 100× magnification (scale bar: 100 μm). Average surface coverages from 36 pictures per condition were calculated using ImageJ . Experiments were performed in triplicate. For all experiments, statistical significance was evaluated by the Mann-Whitney test ( ∗∗∗ p ≤ 0.001, n.s., not significant). Boxes include median (thick horizontal line), 25th and 75th percentiles. Dots indicate extreme values considered as outliers.

    Article Snippet: Attachment Assay in Microtiter Plates Attachment assays in 96-well polystyrene microtiter plates (Nunc, Thermo Fisher Scientific, St. Leon-Rot, Germany) were performed as previously described ( ) with the following minor modifications.

    Techniques: Incubation, Staining, Microscopy, Fluorescence, MANN-WHITNEY

    Adherence of S. pneumoniae to human epithelial cells ( A ) and in vitro growth ( B ). Detroit nasopharyngeal epithelial cell lines were exposed to non-Ec- Sp containing or lacking ICE 1 Sp ST344 and RD 1 Sp ST344, respectively ( A ). Adherence was determined at 30 min and calculated as the proportion of recovered bacteria to the inoculum. Experiments were repeated three times (three times on three different days: Indicated are mean and SD (standard deviation)). Swiss and Thai strains were classic and sporadic non-Ec- Sp , respectively. For the nine adherence experiments, ordinary one-way ANOVA resulted in P = 0.0052. See text for details. MLST, multilocus sequence type. Isolates of the BC3-NT lineage have been recently defined ( Chewapreecha et al. 2014 ). Measurement of planktonic growth was done in 96-well microtiter polystyrene plates and OD450nm was measured on an ELISA plate reader every 30 min for 22 h ( B ). Each experiment included three technical replicates and each experiment was performed three times.

    Journal: Genome Biology and Evolution

    Article Title: Global Phylogenomic Analysis of Nonencapsulated Streptococcus pneumoniae Reveals a Deep-Branching Classic Lineage That Is Distinct from Multiple Sporadic Lineages

    doi: 10.1093/gbe/evu263

    Figure Lengend Snippet: Adherence of S. pneumoniae to human epithelial cells ( A ) and in vitro growth ( B ). Detroit nasopharyngeal epithelial cell lines were exposed to non-Ec- Sp containing or lacking ICE 1 Sp ST344 and RD 1 Sp ST344, respectively ( A ). Adherence was determined at 30 min and calculated as the proportion of recovered bacteria to the inoculum. Experiments were repeated three times (three times on three different days: Indicated are mean and SD (standard deviation)). Swiss and Thai strains were classic and sporadic non-Ec- Sp , respectively. For the nine adherence experiments, ordinary one-way ANOVA resulted in P = 0.0052. See text for details. MLST, multilocus sequence type. Isolates of the BC3-NT lineage have been recently defined ( Chewapreecha et al. 2014 ). Measurement of planktonic growth was done in 96-well microtiter polystyrene plates and OD450nm was measured on an ELISA plate reader every 30 min for 22 h ( B ). Each experiment included three technical replicates and each experiment was performed three times.

    Article Snippet: Adherence to Human Epithelial Cell Line Detroit 562 and Analysis of Planktonic Growth As for measuring planktonic growth, 96-well microtiter polystyrene plates (Thermo Fisher Scientific, Denmark) were used for the isolates Switzerland 4, 11, Thailand 1 and 2, respectively.

    Techniques: In Vitro, Standard Deviation, Sequencing, Enzyme-linked Immunosorbent Assay

    Biofilm formation and rapid attachment. Bacteria were grown for 24 h (A) or for 1 h (B) at 37°C in 96-well microtiter plates containing BM2 biofilm medium. Biofilm formation and adhesion ability were analyzed by staining of the adherent biomass with crystal violet, followed by quantification ( A 595 ) for the stained wells. Shown are the results of at least three independent biological experiments, each with six technical repeats. The statistical significance of differences between the PA4398 − mutant and WT PA14, as well as between the PA4398c and pUCP20 strains, was determined by the Mann-Whitney test (***, P ≤ 0.001; n. s., not significant). The highest and lowest outliers are indicated by “×.”

    Journal: Applied and Environmental Microbiology

    Article Title: Sensor Kinase PA4398 Modulates Swarming Motility and Biofilm Formation in Pseudomonas aeruginosa PA14

    doi: 10.1128/AEM.02832-14

    Figure Lengend Snippet: Biofilm formation and rapid attachment. Bacteria were grown for 24 h (A) or for 1 h (B) at 37°C in 96-well microtiter plates containing BM2 biofilm medium. Biofilm formation and adhesion ability were analyzed by staining of the adherent biomass with crystal violet, followed by quantification ( A 595 ) for the stained wells. Shown are the results of at least three independent biological experiments, each with six technical repeats. The statistical significance of differences between the PA4398 − mutant and WT PA14, as well as between the PA4398c and pUCP20 strains, was determined by the Mann-Whitney test (***, P ≤ 0.001; n. s., not significant). The highest and lowest outliers are indicated by “×.”

    Article Snippet: Biofilm formation was analyzed in 96-well polystyrene microtiter plates (Nunc, Thermo Fisher Scientific, St. Leon-Rot, Germany) by using an abiotic solid-surface assay ( ).

    Techniques: Staining, Mutagenesis, MANN-WHITNEY

    Expression of the viscA gene per biomass unit in biofilms (blue diamonds) and planktonic cells (red squares) in the initial state of biofilms grown in microtitre wells. Data represent mean ± sd for a representative experiment with five replicates. *Significant difference between biofilm and planktonic cells at each time point ( P

    Journal: Microbiology

    Article Title: Lipopeptide biosurfactant viscosin enhances dispersal of Pseudomonas fluorescens SBW25 biofilms

    doi: 10.1099/mic.0.000191

    Figure Lengend Snippet: Expression of the viscA gene per biomass unit in biofilms (blue diamonds) and planktonic cells (red squares) in the initial state of biofilms grown in microtitre wells. Data represent mean ± sd for a representative experiment with five replicates. *Significant difference between biofilm and planktonic cells at each time point ( P

    Article Snippet: The culture was aliquoted (125 μl) into wells of a black-welled polystyrene microtitre plate (MicroWell 96-well optical-bottom, non-treated; Thermo Scientific Nunc) and biofilm was allowed to develop at room temperature as described previously.

    Techniques: Expressing

    Biofilm formation of P. fluorescens SBW25 (dark bars) and P. fluorescens SBW25Δ viscA (light bars) in AB minimal medium with citrate. The biofilm was quantified from 7.5 to 17.5 h using the crystal violet assay. The A 590 value represents crystal violet-stained biofilm attached to the walls of the microtitre wells and is an indirect measure of the biofilm formed. Data represent mean ± sd for a representative experiment with eight replicates. *Significant difference between strains at each time point ( P

    Journal: Microbiology

    Article Title: Lipopeptide biosurfactant viscosin enhances dispersal of Pseudomonas fluorescens SBW25 biofilms

    doi: 10.1099/mic.0.000191

    Figure Lengend Snippet: Biofilm formation of P. fluorescens SBW25 (dark bars) and P. fluorescens SBW25Δ viscA (light bars) in AB minimal medium with citrate. The biofilm was quantified from 7.5 to 17.5 h using the crystal violet assay. The A 590 value represents crystal violet-stained biofilm attached to the walls of the microtitre wells and is an indirect measure of the biofilm formed. Data represent mean ± sd for a representative experiment with eight replicates. *Significant difference between strains at each time point ( P

    Article Snippet: The culture was aliquoted (125 μl) into wells of a black-welled polystyrene microtitre plate (MicroWell 96-well optical-bottom, non-treated; Thermo Scientific Nunc) and biofilm was allowed to develop at room temperature as described previously.

    Techniques: Crystal Violet Assay, Staining