Semiquantitative fluorescence bead assay to assess changes in endothelial adhesion molecule expression in BM microvessels before and after TBI.

(A) IVM experiments were performed to quantitate the ratio of specific/nonspecific bead binding in skull BM microvessels, as described in Figure 4. The ratio in nonirradiated BM microvessels was always higher than 1, indicating constitutive expression of all 3 molecules. This was most apparent for VCAM-1 (P < .005) and, to a lesser extent, P-selectin (P < .01), whereas specific binding of anti–E-selectin–coated beads was weak but statistically significant (P < .05). Bead accumulation was analyzed in 8 to 14 fields of view in each experiment. (B) For tissue-specific accumulation of beads in long bones, equal numbers of specific and nonspecific mAb-coated beads were injected into irradiated and nonirradiated mice. Animals were exsanguinated 15 minutes later and were perfused with heparinized ice-cold saline to remove intravascular blood. BM from both femora and tibiae was harvested as described in “Materials and methods,” and a single-cell suspension was produced to generate cytospins. The ratio of specific/nonspecific beads in 20 fields of view per cytospin was determined by fluorescence microscopy and, if necessary, corrected for differences in the bead input ratio; thus, a ratio of 1 (represented by line) indicates no specific binding above background. Bars represent mean ± SEM. ns indicates nonsignificant.