Elevated levels of homocysteine compromise blood-brain barrier integrity in mice -- Kamath et al. 107 (2): 591 Data Supplement - Supplemental materials for: Kamath et al -- Blood

Elevated levels of homocysteine compromise blood-brain barrier integrity in mice
Blood Kamath et al. 107: 591

Supplemental materials for: Kamath et al

Materials
Bovine serum albumin (BSA), MES buffer were from Sigma (St. Louis, MO). Carboxylate-modified microspheres (1.0 µm diameter) were from Molecular Probes (Eugene, OR). Monoclonal antibody against mouse P-selectin mAb RB40.34 and control mAb rat IgG1 were purchased from BD Bioscience (San Diego, CA).

Method
In vivo detection of P-selectin on endothelial surface
Yellow green (excitation/emission, 505 nm/515 nm) and red (excitation/emission, 580 nm/605 nm) carboxylate-modified microspheres (1.0 µm diameter) were covalently coupled to anti-P-selectin monoclonal antibody RB40.34 or control rat IgG₁. 500 µg antibody in 1 mL 50 mM MES buffer (pH 6.0) was coupled to 1.35 × 10¹⁰ microspheres according to the manufacturer instructions (Molecular Probes). Mice were infused with 1 × 10⁹ microspheres of each color and mesenteric venules observed immediately by fluorescence intravital microscopy. The order of infusion of yellow green and red microspheres was swapped between experiments. The shear rate (approx. 90-125 s^-1) of the venules was not statistically different among the groups. P < 0.05 was considered as statistically significant by ANOVA followed by Kruskal-Wallis test.

Files in this Data Supplement:

Figure S1. P-selectin expression in mesenteric venules (200-300 µm) (PDF, 24.4 KB) - Fluorescent microspheres (1.0 µm) coupled to anti-P-selectin antibody were infused through retro-orbital venous plexus, and their binding to venules was analyzed. Seven venules from 3 WT mice, and 9 venules from 4 WT and 6 venules from 3 CBS+/- on hyperhomocysteinemic diet (Diet) were used for analysis. A. Higher number of microspheres was observed binding to mesenteric venules of CBS+/- mice on Diet (right) compared to venules of WT and WT/Diet mice. Representative photographs are shown. Lines delineate the blood vessel. B. Quantification of the number of anti-P-selectin beads binding per mm².