Immunomagnetic enrichment kit

Bone marrow was flushed from femur and tibia, and suspended in phosphate buffered saline. RBCs were removed by ammonium chloride-lysis solution. Lineage-negative (Lin⁻) population of cells was isolated by negative selection by using immunomagnetic enrichment kit (StemCell Technologies) as per the manufacturer’s instructions. Briefly, the mononuclear cells were suspended in the recommended medium and incubated with specific antibodies for labeling unwanted nonhematopoietic stem/progenitor cells which include CD5, CD11b, CD19, CD45R, 7–4, Ly-6G/C (Gr-1), and TER119-expressing cells. Antibody-treated cells were then labeled by Tetrameric Antibody Complexes that recognize biotin and dextran-coated magnetic particles. Unwanted labeled cells were then separated by using an EasySep™ magnet. Thus the desired Lin⁻ cells were obtained by negative selection. These cells were then enriched for cKit⁺ cells by using positive immunomagnetic selection kit (StemCell Technologies). LK cells were plated in RPMI1640 (GE Healthcare) in U-bottom, 96-well plate at a low density of 2 × 10⁴ cells/150 μL per well, until they are used for proliferation or migration assay in less than 24 hours following isolation. Representative flow cytometric dot plots evaluating the purity of LK cells enriched by this method is shown in Fig. 9.