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