tlr2 blocking antibodies  (R&D Systems)

Journal: Glycobiology

Article Title: Essential role of Toll-like receptor 2 in macrophage activation by glycogen.

doi: 10.1093/glycob/cwr122

Figure Lengend Snippet: Fig. 1. The macrophage-stimulating activity of ESG. (A and B) Participation of the macrophage in ESG activity. CD11b+ and CD11b−cells (1 × 106 per 1 mL) derived from BALB/c PEC were incubated with saline, ESG (50–500 μg/mL) or Pam3CSK4 (TLR2 ligand; 10 ng/mL) in the presence of IFN-γ (10 ng/mL) for 48 h, then NO (A) and IL-6 (B) concentrations in the supernatants were determined. (C and D) Role of IFN-γ in the macrophage activation by ESG. CD11b+

Article Snippet: A 96-well plate (Costar Co., Cambridge, MA, USA) was coated with a monoclonal immunoglobulin M (IgM) antibody against glycogen (Baba 1993) at 4°C for 16 h. The plate was washed with PBS containing 0.05% Tween-20 and blocked with PBS containing 1% bovine serum albumin at 37°C for 1 h. It was then washed and treated with 100–1000 μg/mL of ESG at 37° C for 2 h. After washing, the plate was incubated with 0.15-20 μg/mL of purified recombinant mouse TLR2 (R&D Systems) at 37°C for 2 h. It was then treated with 0.5 μg/mL of a biotinylated anti-mouse TLR2 antibody (R&D Systems) at 37°C for 1 h and thereafter incubated with peroxidaseconjugated streptavidin (Becton Dickinson) at 37°C for 1 h. Finally, the plates were developed with a TMB substrate system (KPL, Inc., MD).

Techniques: Activity Assay, Derivative Assay, Incubation, Saline, Activation Assay

Journal: Glycobiology

Article Title: Essential role of Toll-like receptor 2 in macrophage activation by glycogen.

doi: 10.1093/glycob/cwr122

Figure Lengend Snippet: Fig. 3. The activity of ESG on TLR2-expressing HEK293 cells. HEK293 cells transfected with TLR2 expression plasmid and reporter plasmids for 24 h were stimulated by either Pam3CSK4 (0–5 ng/mL), ESG (0–400 μg/mL), LPS (0–1000 ng/mL) or CpG-ODN (TLR9 ligand; 0–1000 ng/mL) at each indicated concentration for 24 h. The relative activity was calculated as the ratio of pNF-κB-Luc (firefly) activity to RL-TK (Renilla) activity. Values are the mean ± SD. Data are representative of three independent experiments (each experiment was performed in quadruplicate). **Significantly different (P < 0.01) from non-specimen group using one-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: A 96-well plate (Costar Co., Cambridge, MA, USA) was coated with a monoclonal immunoglobulin M (IgM) antibody against glycogen (Baba 1993) at 4°C for 16 h. The plate was washed with PBS containing 0.05% Tween-20 and blocked with PBS containing 1% bovine serum albumin at 37°C for 1 h. It was then washed and treated with 100–1000 μg/mL of ESG at 37° C for 2 h. After washing, the plate was incubated with 0.15-20 μg/mL of purified recombinant mouse TLR2 (R&D Systems) at 37°C for 2 h. It was then treated with 0.5 μg/mL of a biotinylated anti-mouse TLR2 antibody (R&D Systems) at 37°C for 1 h and thereafter incubated with peroxidaseconjugated streptavidin (Becton Dickinson) at 37°C for 1 h. Finally, the plates were developed with a TMB substrate system (KPL, Inc., MD).

Techniques: Activity Assay, Expressing, Transfection, Plasmid Preparation, Concentration Assay

Journal: Glycobiology

Article Title: Essential role of Toll-like receptor 2 in macrophage activation by glycogen.

doi: 10.1093/glycob/cwr122

Figure Lengend Snippet: Fig. 4. The cell-stimulating activity of ESG on TLR2-deficient mice PEC. PECs derived from TLR2-deficient and WT (BALB/c) mice (2 × 105 per 200 μL) were cultured with either saline, ESG (200 μg/mL), Pam3CSK4 (10 ng/mL), imiquimod (TLR7 ligand; 5 μg/mL) or LPS (100 ng/mL) in the presence of IFN-γ (10 ng/ mL). After 48 h, the culture fluid was withdrawn and the amounts of NO, IL-6 and MIP-2 in the supernatants were determined. White and black bars indicate the results for WT and TLR2KO PECs, respectively. Pam3 and IMQ in the graph indicate Pam3CSK4 and imiquimod, respectively. Values are the mean ± SD. Data are representative of three separate experiments (each experiment was performed in triplicate). ***Significantly different (P < 0.001) between WT and TLR2KO by an unpaired t-test.

Article Snippet: A 96-well plate (Costar Co., Cambridge, MA, USA) was coated with a monoclonal immunoglobulin M (IgM) antibody against glycogen (Baba 1993) at 4°C for 16 h. The plate was washed with PBS containing 0.05% Tween-20 and blocked with PBS containing 1% bovine serum albumin at 37°C for 1 h. It was then washed and treated with 100–1000 μg/mL of ESG at 37° C for 2 h. After washing, the plate was incubated with 0.15-20 μg/mL of purified recombinant mouse TLR2 (R&D Systems) at 37°C for 2 h. It was then treated with 0.5 μg/mL of a biotinylated anti-mouse TLR2 antibody (R&D Systems) at 37°C for 1 h and thereafter incubated with peroxidaseconjugated streptavidin (Becton Dickinson) at 37°C for 1 h. Finally, the plates were developed with a TMB substrate system (KPL, Inc., MD).

Techniques: Activity Assay, Derivative Assay, Cell Culture, Saline

Journal: Glycobiology

Article Title: Essential role of Toll-like receptor 2 in macrophage activation by glycogen.

doi: 10.1093/glycob/cwr122

Figure Lengend Snippet: Fig. 6. The cell-stimulating activity of ESG toward TLR2/4DKO and MyD88KO PECs. PECs (2 × 105 per 200 μL) derived from TLR2/4DKO, MyD88KO and WT mice were incubated with either saline, ESG (200 μg/mL), Pam3CSK4 (10 ng/mL), imiquimod (5 μg/mL) or LPS (100 ng/mL) in the presence of IFN-γ (10 ng/mL). After 48 h, the culture fluid was withdrawn and NO, IL-6 and MIP-2 in the supernatant was measured. The results of TLR2/4DKO and WT (C57BL/6) PECs are shown in (A)–(C), and the results of MyD88KO and WT (BALB/c) PECs are shown in (D)–(F). Pam3 and IMQ in the graph indicate Pam3CSK4 and imiquimod, respectively. Values are the mean ± SD. Data are representative of two independent experiments (each experiment was performed in triplicate). ***Significantly different (P < 0.001) between WT and KO mice by an unpaired t-test.

Article Snippet: A 96-well plate (Costar Co., Cambridge, MA, USA) was coated with a monoclonal immunoglobulin M (IgM) antibody against glycogen (Baba 1993) at 4°C for 16 h. The plate was washed with PBS containing 0.05% Tween-20 and blocked with PBS containing 1% bovine serum albumin at 37°C for 1 h. It was then washed and treated with 100–1000 μg/mL of ESG at 37° C for 2 h. After washing, the plate was incubated with 0.15-20 μg/mL of purified recombinant mouse TLR2 (R&D Systems) at 37°C for 2 h. It was then treated with 0.5 μg/mL of a biotinylated anti-mouse TLR2 antibody (R&D Systems) at 37°C for 1 h and thereafter incubated with peroxidaseconjugated streptavidin (Becton Dickinson) at 37°C for 1 h. Finally, the plates were developed with a TMB substrate system (KPL, Inc., MD).

Techniques: Activity Assay, Derivative Assay, Incubation, Saline

Journal: Glycobiology

Article Title: Essential role of Toll-like receptor 2 in macrophage activation by glycogen.

doi: 10.1093/glycob/cwr122

Figure Lengend Snippet: Fig. 9. The binding activities of TLR2 to glycogen. (A) Bindings of ESG (100–1000 μg/mL) to TLR2 (2.5 μg/mL) were tested by ELISA. Pam3CSK4 (100 μg/mL), LPS (500 μg/mL) or imiquimod (500 μg/mL) was used as a control compound. Pam3 and IMQ in the graph indicate Pam3CSK4 and imiquimod, respectively. (B) Dose response binding curve at the indicated concentration of TLR2 to ESG (500 μg/mL). (C) Bindings of ESG to dectin-1 (β-glucan receptor) and comparison with other glucans such as oyster glycogen (natural-source glycogen; α-1,4/1,6 glucan), soluble starch (α-1,4/ 1,6 glucan), dextran (α-1,6 glucan), SPG (soluble β-1,3/1,6 glucan) and OX-CA (particle β-1,3/1,6 glucan). All glucans, Pam3CSK4, TLR2 and dectin-1 were tested with concentrations of 1000, 100, 2.5 and 1 μg/mL, respectively. OysGly and Pam3 in the graph indicate oyster glycogen and Pam3CSK4, respectively. Data are represented as the mean ± SD from three independent experiments (each experiment was performed in quadruplicate). **Significantly different (P < 0.01) from the negative control by one-way ANOVA followed by Dunnett’s multiple comparison test.

Article Snippet: A 96-well plate (Costar Co., Cambridge, MA, USA) was coated with a monoclonal immunoglobulin M (IgM) antibody against glycogen (Baba 1993) at 4°C for 16 h. The plate was washed with PBS containing 0.05% Tween-20 and blocked with PBS containing 1% bovine serum albumin at 37°C for 1 h. It was then washed and treated with 100–1000 μg/mL of ESG at 37° C for 2 h. After washing, the plate was incubated with 0.15-20 μg/mL of purified recombinant mouse TLR2 (R&D Systems) at 37°C for 2 h. It was then treated with 0.5 μg/mL of a biotinylated anti-mouse TLR2 antibody (R&D Systems) at 37°C for 1 h and thereafter incubated with peroxidaseconjugated streptavidin (Becton Dickinson) at 37°C for 1 h. Finally, the plates were developed with a TMB substrate system (KPL, Inc., MD).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Control, Concentration Assay, Comparison, Starch, Negative Control