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-IgG2a, anti-TLR2, or anti-TLR4 antibody (all from eBioscience), or 5 μM TLR9 antagonist (ODN TTAGGG; InvivoGen, San Diego, CA).
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-IgG2a, anti-TLR2, or anti-TLR4 antibody (all from eBioscience), or 5 μM TLR9 antagonist (ODN TTAGGG; InvivoGen, San Diego, CA).