Journal: PLoS ONE

Article Title: Toll-Like Receptor 4–Mediated Nuclear Factor Kappa B Activation Is Essential for Sensing Exogenous Oxidants to Propagate and Maintain Oxidative/Nitrosative Cellular Stress

doi: 10.1371/journal.pone.0073840

Figure Lengend Snippet: Cells were stimulated with 1 µg/ml of LPS from Porphyromonas gingivalis (LPS-PG) (1 µg/ml), MPLA (57 nM), PPC (1 µM), PDO (50 µM), or PN (1 mM) for 16 h. SEAP released into the supernatant was quantified using Quanti-Blue (Fig. 4A) [n = means ± SEM from 5 independent experiments; # p ≤0.01 vs treatments with LPG-PG or PPC alone; + p ≤0.01 versus treatment with LPS-PG alone]. Fig. 4B - A representative qualitative representation of LPS-PG concentration-dependent changes in purple/blue color development (absorbance) indicative of increased SEAP release (i.e., enhanced with increasing NF-κB) with increasing LPS-PG concentration after Quanti-Blue reaction. HEK-Blue mTLR2 cells incubated with different concentrations of LPS-PG at 0.1, 1.0 and 10 µg/ml for 16 h were compared to treatments with different prooxidants assumed to be potential TLR2 activators.

Article Snippet: HEK-Blue-Null1-v, HEK-Blue mouse TLR2 (mTLR2) and HEK-Blue mTLR4 cells were purchased from InvivoGen (San Diego, CA).

Techniques: Concentration Assay, Incubation

Journal: PLoS ONE

Article Title: TLR2, TLR4 and CD14 Recognize Venom-Associated Molecular Patterns from Tityus serrulatus to Induce Macrophage-Derived Inflammatory Mediators

doi: 10.1371/journal.pone.0088174

Figure Lengend Snippet: Peritoneal macrophages from C57Bl/6 (WT) mice, TLR2 −/− , TLR4 −/− or CD14 −/− mice were stimulated with TsV (50 µg/ml) (a, b) for 30 min or 24 h or with TsV (e, f) or Ts1 (50 µg/ml) (c, d, g, h) for 24 h in a 5% CO 2 atmosphere at 37°C. The concentrations of IL-6 (a, c), TNF-α (b, d), PGE 2 (e, g) and LTB 4 (f, h) in the culture supernatants were determined by ELISA. * p <0.05 (one-way ANOVA) compared to the WT mice. The values represent the means ± SD ( n = 8), and the data are from 2 independent experiments.

Article Snippet: The cells were seeded in 96-well micro culture plates at a density of 2×10 5 cells/well in DMEM supplemented with Normocin™ (50 mg/mL) and cultured at 37°C in a humidified 5% CO 2 atmosphere for 18 h. After this period, the cells were incubated with 10 ng/ml of LPS from Rhodobacter sphaeroides , a TLR4 antagonist (LPS-RS - InvivoGen), with or without LPS from E. coli (0.5 µg/ml), and with TsV (50 µg/ml) or Ts1 (50 µg/ml) for 24 h. In another experiment, the cells were pre-incubated with 100 ng/ml of a purified monoclonal IgG antibody against mouse TLR2 (anti-mTLR2-IgG – InvivoGen) for 30 min and then stimulated with LPS from E. coli (0.5 µg/ml) and TsV or Ts1 (50 µg/ml) for 24 h. After 24 h of stimulation, the medium was harvested, and 50 µl samples were mixed with QUANTI-Blue™ (InvivoGen), which is a SEAP detection medium (150 µl), in 96-well plates at 37°C for 2 h. The optical density was then measured at 650 nm using an ELISA reader.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: TLR2, TLR4 and CD14 Recognize Venom-Associated Molecular Patterns from Tityus serrulatus to Induce Macrophage-Derived Inflammatory Mediators

doi: 10.1371/journal.pone.0088174

Figure Lengend Snippet: Adherent peritoneal macrophages from WT (C57Bl/6), TLR2 −/− , TLR4 −/− or MyD88 −/− mice were stimulated with TsV (50 µg/mL) for 10 or 120 min in a 5% CO 2 atmosphere at 37°C. The p-NF-κB (a, d), p-IκBα (b, e) and p-c-Jun (c, f) protein levels were determined using the PathScan Inflammation Multi-Target Sandwich ELISA kit. The results are presented as a percentage of the phosphoprotein level in non-stimulated control cell lysate (dashed line). * p <0.05 (one-way ANOVA) compared to WT. The values represent the means ± SD ( n = 4), and the data are from 2 independent experiments.

Article Snippet: The cells were seeded in 96-well micro culture plates at a density of 2×10 5 cells/well in DMEM supplemented with Normocin™ (50 mg/mL) and cultured at 37°C in a humidified 5% CO 2 atmosphere for 18 h. After this period, the cells were incubated with 10 ng/ml of LPS from Rhodobacter sphaeroides , a TLR4 antagonist (LPS-RS - InvivoGen), with or without LPS from E. coli (0.5 µg/ml), and with TsV (50 µg/ml) or Ts1 (50 µg/ml) for 24 h. In another experiment, the cells were pre-incubated with 100 ng/ml of a purified monoclonal IgG antibody against mouse TLR2 (anti-mTLR2-IgG – InvivoGen) for 30 min and then stimulated with LPS from E. coli (0.5 µg/ml) and TsV or Ts1 (50 µg/ml) for 24 h. After 24 h of stimulation, the medium was harvested, and 50 µl samples were mixed with QUANTI-Blue™ (InvivoGen), which is a SEAP detection medium (150 µl), in 96-well plates at 37°C for 2 h. The optical density was then measured at 650 nm using an ELISA reader.

Techniques: Sandwich ELISA

Journal: PLoS ONE

Article Title: TLR2, TLR4 and CD14 Recognize Venom-Associated Molecular Patterns from Tityus serrulatus to Induce Macrophage-Derived Inflammatory Mediators

doi: 10.1371/journal.pone.0088174

Figure Lengend Snippet: These cells were derived from RAW 264.7 macrophages and contain a secreted embryonic alkaline phosphatase (SEAP) reporter construct that is integrated into the cellular DNA and that can be induced by NF-κB. The cells were incubated with either (A) anti-mTLR2-IgG (100 ng/ml) or (B) LPS-RS (10 ng/ml) for 30 min, with or without LPS (0.5 µg/ml), and TsV or Ts1 (50 µg/ml) for 24 h. The QUANTI-Blue™ substrate was used to measure the SEAP at 650 nm with an ELISA reader. The measurements were performed in triplicate, and a representative experiment is shown. * p <0.05 (one-way ANOVA) compared to medium alone (dashed line). The values represent the means ± SD ( n = 8), and the data are from 2 independent experiments.

Article Snippet: The cells were seeded in 96-well micro culture plates at a density of 2×10 5 cells/well in DMEM supplemented with Normocin™ (50 mg/mL) and cultured at 37°C in a humidified 5% CO 2 atmosphere for 18 h. After this period, the cells were incubated with 10 ng/ml of LPS from Rhodobacter sphaeroides , a TLR4 antagonist (LPS-RS - InvivoGen), with or without LPS from E. coli (0.5 µg/ml), and with TsV (50 µg/ml) or Ts1 (50 µg/ml) for 24 h. In another experiment, the cells were pre-incubated with 100 ng/ml of a purified monoclonal IgG antibody against mouse TLR2 (anti-mTLR2-IgG – InvivoGen) for 30 min and then stimulated with LPS from E. coli (0.5 µg/ml) and TsV or Ts1 (50 µg/ml) for 24 h. After 24 h of stimulation, the medium was harvested, and 50 µl samples were mixed with QUANTI-Blue™ (InvivoGen), which is a SEAP detection medium (150 µl), in 96-well plates at 37°C for 2 h. The optical density was then measured at 650 nm using an ELISA reader.

Techniques: Derivative Assay, Construct, Incubation, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: TLR2, TLR4 and CD14 Recognize Venom-Associated Molecular Patterns from Tityus serrulatus to Induce Macrophage-Derived Inflammatory Mediators

doi: 10.1371/journal.pone.0088174

Figure Lengend Snippet: Adherent peritoneal macrophages from WT (C57Bl/6), TLR2 −/− , TLR4 −/− or MYD88 −/− mice were stimulated with Ts1 (50 µg/ml) for 10 or 120 min in a 5% CO 2 atmosphere at 37°C. The p-NF-κB (a, d), p-IκBα (b, e) and p-c-Jun (c, f) protein levels were determined using the PathScan Inflammation Multi-Target Sandwich ELISA kit, as described in the Materials and Methods section. The results are presented as a percentage of the phosphoprotein level in non-stimulated control cell lysate (dashed line). * p <0.05 (one-way ANOVA) compared to WT. The values represent the means ± SD ( n = 4), and the data are from 2 independent experiments.

Article Snippet: The cells were seeded in 96-well micro culture plates at a density of 2×10 5 cells/well in DMEM supplemented with Normocin™ (50 mg/mL) and cultured at 37°C in a humidified 5% CO 2 atmosphere for 18 h. After this period, the cells were incubated with 10 ng/ml of LPS from Rhodobacter sphaeroides , a TLR4 antagonist (LPS-RS - InvivoGen), with or without LPS from E. coli (0.5 µg/ml), and with TsV (50 µg/ml) or Ts1 (50 µg/ml) for 24 h. In another experiment, the cells were pre-incubated with 100 ng/ml of a purified monoclonal IgG antibody against mouse TLR2 (anti-mTLR2-IgG – InvivoGen) for 30 min and then stimulated with LPS from E. coli (0.5 µg/ml) and TsV or Ts1 (50 µg/ml) for 24 h. After 24 h of stimulation, the medium was harvested, and 50 µl samples were mixed with QUANTI-Blue™ (InvivoGen), which is a SEAP detection medium (150 µl), in 96-well plates at 37°C for 2 h. The optical density was then measured at 650 nm using an ELISA reader.

Techniques: Sandwich ELISA

Journal: PLoS ONE

Article Title: TLR2, TLR4 and CD14 Recognize Venom-Associated Molecular Patterns from Tityus serrulatus to Induce Macrophage-Derived Inflammatory Mediators

doi: 10.1371/journal.pone.0088174

Figure Lengend Snippet: Pro-inflammatory cytokine production occurs via the following two routes: (1) MyD88-dependent signaling, where TsV and Ts1 are recognized by TLR4/CD14/TLR2, resulting in NF-kB nuclear translocation; and (2) MyD88-independent signaling, where TsV is recognized by TLR4/CD14 and activates ERK1/2 and p38 phosphorylation and c-Fos/Jun expression.

Article Snippet: The cells were seeded in 96-well micro culture plates at a density of 2×10 5 cells/well in DMEM supplemented with Normocin™ (50 mg/mL) and cultured at 37°C in a humidified 5% CO 2 atmosphere for 18 h. After this period, the cells were incubated with 10 ng/ml of LPS from Rhodobacter sphaeroides , a TLR4 antagonist (LPS-RS - InvivoGen), with or without LPS from E. coli (0.5 µg/ml), and with TsV (50 µg/ml) or Ts1 (50 µg/ml) for 24 h. In another experiment, the cells were pre-incubated with 100 ng/ml of a purified monoclonal IgG antibody against mouse TLR2 (anti-mTLR2-IgG – InvivoGen) for 30 min and then stimulated with LPS from E. coli (0.5 µg/ml) and TsV or Ts1 (50 µg/ml) for 24 h. After 24 h of stimulation, the medium was harvested, and 50 µl samples were mixed with QUANTI-Blue™ (InvivoGen), which is a SEAP detection medium (150 µl), in 96-well plates at 37°C for 2 h. The optical density was then measured at 650 nm using an ELISA reader.

Techniques: Translocation Assay, Expressing