Anti tlr2

hEC were incubated with or without 50 μg/mL histone for 5 h. U937 cells (1×10⁶ cells/mL) were added onto the hEC layer for 30 min. The non-adherent cells were collected. The adherent round U937 cells were enumerated under a light microscope (Olympus).
For neutralizing histone, histone was pre-mixed with 62.5 μg/mL polysialic acid (Sigma-Aldrich) for 1 h, 100 U/mL heparin (Sigma-Aldrich) for 10 min, and 100 nM activated protein C (APC; Haematologic Technologies Inc., Essex Junction, VA) for 30 min. The mixtures were then added to hEC.
Anti-E-selectin antibody (50 μg/mL), anti-ICAM-1 antibody (10 μg/mL), or anti-VCAM-1 antibody (30 μg/mL) (all from R&D Systems, Minneapolis, MN) was incubated with histone–treated hEC for 10 min. Then U937 cells were added.
Before stimulated with histone, hEC were pre-treated for 1 h with 50 μg/mL isotype-IgG_2a, anti-TLR2, or anti-TLR4 antibody (all from eBioscience), or 5 μM TLR9 antagonist (ODN TTAGGG; InvivoGen, San Diego, CA).

Human PBMCs were isolated by density centrifugation using Histopaque-1077 (Sigma) as described (Endres et al., 1988 (link)). For stimulation experiments, the interactions were performed in round-bottom 96-well microplates with 100 μL of cells adjusted to 5 × 10⁵ PBMCs in RPMI 1640 Dutch modification (added with 2 mM glutamine, 0.1 mM pyruvate and 0.05 mg/mL gentamycin; all reagents from Sigma) and 100 μL with 1 × 10⁵ fungal cells freshly harvested or treated. The interactions were incubated for 24 h at 37°C with 5% (v/v) CO₂. In some experiments, PBMCs were preincubated for 1 h at 37°C with either laminarin (200 μg/mL), anti-TLR2 (10 μg/mL, eBioscience, Cat. No. 16-9922) or antibodies to TLR4 (10 μg/mL, Santa Cruz Biotechnology, Cat. No. sc-293072) prior to stimulation with Candida cells. Isotype matched, irrelevant antibodies, IgG₂aκ (10 μg/mL, eBioscience, Cat. No. 14-4724-85) and IgG₁(10 μg/mL, Santa Cruz Biotechnology, Cat. No. sc-52003) were used as controls for experiments assessing TLR2 and TLR4, respectively. All reagents used for the pre-incubation experiments were negative to contamination with LPS (tested with the Limulus amebocyte lysate from Sigma), and all reactions were performed in presence of 5 μ g/mL polymyxin B (Sigma) (Schwartz et al., 1972 (link)). Plates were centrifuged for 10 min at 3000 × g at 4°C, the supernatant saved and kept at −20°C until used.

Immunocytochemistry was undertaken as reported previously 24 (link) using Vectastain ABC Universal kit (Vector Laboratories, Peterborough, UK). Cells on coverslips were thawed, hydrated and endogenous peroxidase activity was quenched in 0·3% H₂O₂ in methanol for 30 min. Coverslips were incubated with the following primary antibodies (for 1 h at room temperature or overnight at 4°C): anti-BerEP4 (Dako, Glostrup, Denmark), anti-α-smooth muscle actin (abcam, Cambridge, UK), anti-vimentin (abcam), anti-desmin (abcam), anti-TLR-2 (eBioscience, San Diego, CA, USA), anti-TLR-4 (eBioscience) or anti-TLR-5 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Following incubation with biotinylated secondary antibody and avidin–biotin complex, peroxidase activity was developed with diaminobenzidene (DAB) solution as per the manufacturer's instructions.

Antibody blocking assays were performed to investigate the recognition receptor of HP0459 in RAW264.7 cells using the anti-TLR2 (eBioscience) and anti-TLR4 (BioLegend) antibodies. Briefly, RAW264.7 cells were pretreated using 8 μg of anti-TLR2 and anti-TLR4 antibody for 30 min respectively and then incubated with 10 μg ml^-1 HP0459 for 10 h. The expression levels of various cytokines were determined by ELISA. According to the conditions of cytokine activation, the recognition receptor of the HP0459 was analyzed. In addition, TLR2-/- macrophages were isolated from TLR2-/- mice to verify the results of the blocking assays.

Disaggregated crypt epithelial cells (10⁶) or colonic myofibroblasts were washed in PBS and incubated in CelLytic M reagent (Sigma) supplemented with phosphatase inhibitor cocktail 2 (Sigma) and protease inhibitor cocktail (Sigma), according to the manufacturer's instructions. The lysate was centrifuged at 10 000 g and the protein-containing supernatant was stored at −80°C until required.
Aliquots of total protein, mixed in a 1:1 ratio with Laemmli buffer (Bio-Rad, Hercules, CA, USA), were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) before transfer to a polyvinylidene difluoride (PVDF) membrane (GE Healthcare, Little Chalfont, UK).
The PVDF membrane was incubated (at 4°C) overnight with or without the following antibodies: anti-β-actin (Sigma), anti-TLR-2 (eBioscience) and anti-TLR-4 (abcam). Immunostaining was performed using a Vectastain ABC Universal kit (Vector Laboratories), according to the manufacturer's instructions.