Anti cd34 conjugated magnetic beads

CD34+ cells were stained as described above and sorted by using a BDAria II and/or MoFlow FACS sorter (BD Biosystems). Alternatively, CD34+ cells were enriched using anti-CD34 conjugated magnetic beads (Miltenyi biotec) according to the manufacturer's instructions with slight modifications. Briefly, up to 10⁹ cells were incubated with constant mixing at 4° C with 100 μl of the corresponding magnetic beads in the presence of 100 μl of Fc-blocking solution in a total volume of 500 μl FACS blocking buffer. After 1 hour, cells were sorted by two consecutive rounds of column separation in order to increase purity by applying MACS separation magnets. Shortly, cells were passed through the first MS separation column allowing binding of labeled cells. Non-labeled cells were washed thoroughly with 3 ml FACS blocking buffer prior to elution of the labeled fraction. Eluted labeled cells were then subjected to a second purification step as described above.