Journal:

Article Title: Lipopolysaccharide-Trap-Fc, a Multifunctional Agent To Battle Gram-Negative Bacteria ▿

doi: 10.1128/IAI.00004-09

Figure Lengend Snippet: LPS-Trap-Fc1 to -Fc4 bind LPS and block LPS-mediated IL-6 production in Mono Mac 6 cells. (A) Supernatants of HEK293T cells transfected with LPS-Trap-Fc1 were incubated in the presence (lane 1) or absence (lane 3) of biotin-LPS. Additionally, supernatants were preincubated with an anti-TLR4/MD-2 MAb (lane 2) or an excess of LPS (lane 4). Complexes were immunoprecipitated with streptavidin-Sepharose, subjected to SDS-PAGE, and analyzed by Western blotting with an anti-FLAG MAb. (B) RAW 264.7 cells were incubated with LPS-Trap-Fc1 or a medium control. Cells were stimulated with 100 ng/ml LPS overnight, and IL-6 levels in supernatants were determined by ELISA. *, P < 0.002 compared to control (Mann-Whitney U test, n = 6).

Article Snippet: For specificity control, some supernatants were preincubated with either 50 μg/ml LPS ( Salmonella enterica serovar Minnesota; Sigma), which has a high binding activity for TLR4/MD-2, or 10 μg/ml anti-mouse TLR4/MD-2 MTS510 MAb (Hycult Biotechnology, Uden, The Netherlands) for 1 h at room temperature.

Techniques: Blocking Assay, Transfection, Incubation, Immunoprecipitation, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Frontiers in Medicine

Article Title: Toll-like receptor 4 and Syk kinase shape dendritic cell-induced immune activation to major house dust mite allergens

doi: 10.3389/fmed.2023.1105538

Figure Lengend Snippet: The role of TLR4 in HDM-induced moDC activation. (A) A HEK-293/TLR4 reporter cell line and the parental HEK-293 control cell line were incubated for 1 h at 37°C with either TLR4-blocking antibody or the respective isotype control antibody. Subsequently, cultures were exposed for 24 h to 10 ng/mL LPS or 10 μg/mL HDM extract, after which the culture supernatants were collected and screened for by ELISA for IL-8, that is expressed upon TLR4 engagement. The measured data are shown relative to levels obtained from LPS stimulation. The data are presented as the mean ± SD ( n = 3–4 donors). (B) MoDCs were incubated for 1 h at 37°C with either TLR4-blocking antibody or the respective isotype controls and subsequently exposed for 24 h to 10 ng/mL LPS or 50 μg/mL HDM extract. Culture supernatants were collected and screened for the cytokines IL-6, IL-10, and IL-12p70 by ELISA. The measured data are shown relative to levels obtained from untreated (control group) cells stimulated with HDM extract. The data are presented as the mean ± SD ( n = 5 donors). ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (RM-ANOVA analysis with Tukey’s multiple comparison test).

Article Snippet: After seeding the cultures into 96-well flat-bottom culture plates (Corning) and letting them adhere for 24 h, the cells were treated for 1 h with 30 μg/mL of either anti-human TLR4/MD-2 (clone 7E3, Hycult Biotech) or mouse IgG1 control antibody (Invivogen) prior to stimulation with 10 μg/mL HDM extract or LPS as a positive control.

Techniques: Activation Assay, Control, Incubation, Blocking Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: FEMS Immunology & Medical Microbiology

Article Title: Toll-like receptor 4 signaling plays a role in triggering periodontal infection

doi: 10.1111/j.1574-695x.2008.00386.x

Figure Lengend Snippet: Fig. 1. Expression of TLR4 mRNA in HPDLCs. Four passage HPDLCs were obtained as described in the ‘Materials and methods’. Total RNA was extracted, and the mRNA expression of TLR4 was analyzed by reverse transcriptase PCR (a). Levels of b-actin mRNA served as internal controls (b). Lanes: M, molecular size marker; 1, sample A; 2, sample B; 3, sample C; 4, negative controls. The results were representative of three different experiments.

Article Snippet: The endogenous peroxidase signal was quenched by incubation in 1.5% H2O2 for 15 min. After 30 min of blocking with normal goat serum, the cells were incubated with mouse anti-TLR4 mAb (Serotec) at a dilution of 1 : 200 overnight at 4 1C.

Techniques: Expressing, Reverse Transcription, Marker

Journal: FEMS Immunology & Medical Microbiology

Article Title: Toll-like receptor 4 signaling plays a role in triggering periodontal infection

doi: 10.1111/j.1574-695x.2008.00386.x

Figure Lengend Snippet: Fig. 2. Expression of TLR4 in HPDLCs detected by immunofluorescence staining. Four passage HPDLCs from three individual healthy subjects were obtained as described in the ‘Materials and methods’. After fixation, the cells were treated with monoclonal antibody to TLR4 (anti- TLR4 mAb) (green), and the nuclei were counterstained with PI (red) (a). The other cells were treated with PBS, which was substituted for anti- TLR4 mAb, and served as controls (b). Scale bars = 20 mm.

Article Snippet: The endogenous peroxidase signal was quenched by incubation in 1.5% H2O2 for 15 min. After 30 min of blocking with normal goat serum, the cells were incubated with mouse anti-TLR4 mAb (Serotec) at a dilution of 1 : 200 overnight at 4 1C.

Techniques: Expressing, Staining

Journal: FEMS Immunology & Medical Microbiology

Article Title: Toll-like receptor 4 signaling plays a role in triggering periodontal infection

doi: 10.1111/j.1574-695x.2008.00386.x

Figure Lengend Snippet: Fig. 3. Gene expression changes in HPDLCs stimulated with lipopolysaccharide analyzed by real-time PCR. HPDLCs were cultured in DMEM with or without 1 mg mL1 lipopolysaccharide for 6 h. cDNA was prepared. Real-time PCR was used to quantify TLR4 (a), IL-6 (b), Fos (c) and LY64 (d) mRNA expression levels. For each transcript, conventional PCR-amplified products were used as standards (from 1 100 to 1 106) to generate a standard curve for absolute real-time quantification. The absolute mRNA levels of all the genes were then normalized to b-actin levels of individual samples. Three different experiments were performed, and the representative results were shown. HPDLCs from three individual persons were pooled for each experiment. Lipopolysaccharide induced the expression of TLR4 and IL-6, while it repressed the expression of Fos and LY64.

Article Snippet: The endogenous peroxidase signal was quenched by incubation in 1.5% H2O2 for 15 min. After 30 min of blocking with normal goat serum, the cells were incubated with mouse anti-TLR4 mAb (Serotec) at a dilution of 1 : 200 overnight at 4 1C.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Cell Culture, Expressing

Journal: FEMS Immunology & Medical Microbiology

Article Title: Toll-like receptor 4 signaling plays a role in triggering periodontal infection

doi: 10.1111/j.1574-695x.2008.00386.x

Figure Lengend Snippet: Fig. 4. Secretion of IL-6 in HPDLCs stimulated with lipopolysaccharide in the absence or presence of anti-TLR4 mAb. (a) HPDLCs were plated at a density of 5 104 cells mL1 in 96-well plates. After 2 days, the cells were exposed to Escherichia coli lipopolysaccharide (100 ng mL1, 1 mg mL1, 10 mg mL1). Following incubation for various periods of time (6, 12, 24, 48 h) at 37 1C, the supernatants were harvested and assayed via ELISA for the concentration of IL-6. (b) HPDLCs (5 104 cells mL1 in 96-well plates) were incubated with 1 mg mL1 lipopolysaccharide for 24 h in the absence or presence of various titers of anti-TLR4 mAb (500–25-fold dilutions). The levels of IL-6 in the culture supernatants were measured by ELISA. P o 0.05, significantly different from levels in the absence of anti-TLR4 mAb. Three different experiments were performed, and each data point represents the mean SD. HPDLCs from three individual people were pooled for each experiment.

Article Snippet: The endogenous peroxidase signal was quenched by incubation in 1.5% H2O2 for 15 min. After 30 min of blocking with normal goat serum, the cells were incubated with mouse anti-TLR4 mAb (Serotec) at a dilution of 1 : 200 overnight at 4 1C.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Concentration Assay