Recombinant vcam 1

We coated 96-well microplates with fibronectin (10 μg/ml in PBS; Millipore, Billerica, MA), collagen I (10 μg/ml in PBS; Sigma-Aldrich), laminin (10 μg/ml in PBS; BD Biosciences, Franklin Lakes, NJ), or 10% FBS as control. For the VCAM-1 adhesion assay, 10 μg/ml recombinant VCAM-1 (R&D) was used to coat wells. Cells were labeled for 20 min with 2 μM calcein AM fluorescent dye (Invitrogen) in Hank's buffered salt solution (HBSS). After two washes with HBSS, the cells were plated at 100,000 cells/well and incubated at 37°C for 2 h. The microplate was washed to remove nonadherent cells, and the remaining adherent cells were measured using a fluorescence plate reader with excitation wavelength of 488 nm and emission detected at 512 nm. Fluorescence data were then normalized to the mean fluorescence obtained for control cells. To measure α4β1-specific adhesion, cells were either treated with dimethyl sulfoxide or blocked with the monovalent peptide LDV (1 μM), which was a generous gift from Larry Sklar and Tione Buranda (University of New Mexico).

For adhesion assays, WT donor LSK cells regenerated in WT and Wnt5a^+/− primary recipients were sorted out after 16 wk, and 500 cells were spotted on slides coated with recombinant VCAM1 (R&D Systems) in PBS. The cells were stimulated 15 min after depositing with murine rCXCL12 (150 ng/ml: R&D Systems) and imaged on a DM RBE fluorescent microscope (Leica). Adhesion was calculated by measurement of light incidence with ImageJ.