Anti vcam 1

HUVECs were plated in 6 or 12-well plates (1.5.10⁵ cells/well) and cultured overnight to reach confluency. Confluent cell monolayers were then preincubated for 1h with NPs (10 μM) or controls (PDTC 200 μM; CsA 10 μM) or 18h with ZA (10 μM) before addition of TNF (200 U/mL for 6h) or IFNγ (100 U/mL for 48h). Cells were immunostained using anti-VCAM-1, anti-E/P-selectin, anti-ICAM-1-FITC (R&D Systems), anti-pan HLA class I (anti-HLA-A, -B and -C; clone W6/32), anti-pan HLA class II (anti-HLA-DR, -DP, -DQ; clone L243), anti-MICA (clone AMO1) (BamOmab, Tubingen, Germany), anti-HLA-E-APC (clone 3D12) (Miltenyi Biotec, Paris, France) mouse IgG and anti-mouse IgG+IgM (H+L)-FITC (Jackson Immunoresearch Laboratories, West Grove, PA, USA) antibodies at 10 μg/mL or diluted at 1/100. An isotype-matched IgG was used as a non-specific control and binding of antibodies to the cells was evaluated by flow cytometry using 10 000 cells/sample and a BD FACSCanto^™ II flow cytometer (Becton Dickinson, San Jose, CA, USA). Cells were gated on forward scattered light (FSC) versus side scattered light (SSC) parameters to select live cells. Data were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR, USA) and depicted in histograms plotting geometric mean of fluorescence intensity (GFI) on a four-decade logarithmic scale (x-axis) versus cell number (y-axis).

Western-Blotting was performed as described before^{21 (link)}. Samples (20 µg protein extract) were separated on 10% SDS-polyacryamide gels followed by semi-dry blotting onto PVDF membranes (Roche, Basel, Switzerland). Membranes were blocked for 1 hour at room temperature in tris buffered saline (TBS) (Bio-Rad, Hercules, USA) and incubated overnight at 4 °C with one of the following primary polyclonal antibodies: anti-VCAM-1, anti-ICAM-1 (both from R&D Systems, Wiesbaden, Germany) or anti-CIITA (Santa Cruz Biotechnology, Dallas, USA). Hereafter, the membranes were thoroughly washed with TBS 0,1% Tween-20 and incubated with the appropriate horseradish peroxidase conjugated secondary antibody (Jackson ImmunoResearch, Baltimore, USA). Proteins were visualized using enhanced chemo luminescence technology (Pierce, Rockford, USA). To confirm equal protein loading, membranes were stripped and re-probed with monoclonal anti-β-actin (Santa Cruz Biotechnology, Dallas, USA) antibody.

Aorta was harvested from rabbits, placed immediately in formaldehyde 10 %, embedded in paraffin 24 h later, cut at 5 μm, stained with hematoxylin and eosin (H&E), and then scanned to assess pathological changes. For immunohistochemical staining, sections were incubated with anti-VCAM-1 (R&D Systems, MN, USA) and anti-F4/80 (Abcam, MA, USA) at 37 °C for 1 h, color developed with 3,3′-diaminobenzidine tetrahydrochloride and counterstained with hematoxylin. Samples in the absence of the primary antibodies were used as negative controls. Slides were observed under a light microscope, and images were subjected to statistical evaluation of positively stained cells in 10 random fields of view at a magnification of × 400. The average numbers of positively stained cells were counted per high power field (HPF).

To determine whether baicalein could modify the oxLDL-induced adhesion molecule expression, HUVECs were grown to confluence, pretreated with baicalein for 2 hrs and stimulated with oxLDL (150 μg/ml) for 24 hrs. At the end of stimulation, HUVECs were harvested and incubated with FITC-conjugated anti-ICAM-1, anti-VCAM-1 and anti-E-selectin (R&D, Minneapolis, MN) for 45 min at room temperature. After the HUVECs had been washed three times, their immunofluorescence intensity was analyzed by flow cytometry using a Becton Dickinson FACScan flow cytometer (Mountain View, CA, USA).

PMN-mediated transfer of clinical isolates in 6-well plates was conducted as described before with the following modification: After PMNs were collected from the donor cultures, they were preincubated for 30 min at 37 °C with function-blocking antibodies against the cellular adhesion molecules L-selectin, very late antigen-4 (VLA-4) or lymphocyte function-associated antigen 1 (LFA-1). Similarly, recipient cultures were preincubated for 30 min at 37 °C with antibodies against the adhesion molecules E-selectin, vascular cell adhesion protein 1 (VCAM-1), or intercellular adhesion molecule 1 (ICAM-1). Therefore, PMNs were either left untreated or were treated with 10 µg/mL anti-integrin αL (hu1124; Novus Biologicals, Littleton, CO, USA) or 20 µg/mL anti-integrin β2 (R&D Systems, Minneapolis, MN, USA) (both subunits of LFA-1), 5 µg/mL anti-integrin α4 (subunit of VLA-4, R&D Systems) or 25 µg/mL L-selectin (R&D Systems). HEC-LTTs were either left untreated or were treated with 25 µg/mL anti-E-selectin, 25 µg/mL anti-VCAM-1 or 10 µg/mL anti-ICAM-1 (all R&D Systems). Concentrations were chosen as described in the literature or by the manufacturer to induce inhibitory effects. PMNs and recipient cultures were then incubated as described, and cultures were incubated overnight before fixation and immunofluorescence staining.