to terminate digestion and cells were triturated to form a single cell suspension. Cells were then washed twice with FACS buffer and resuspended at a concentration of 1×107 cells/mL. DNAse I was added to a final concentration of 100 units/mL and the suspension was incubated at room temperature for 10 minutes. Cells were stained immediately. 1×107 cells/mL were stained for 20 minutes at 4°C with the respective single conjugated antibodies or all four conjugated antibodies with dilutions consisting of 1:12 CD184, 1:20 CD271, 1:12 CD24, and 1:12 CD15. Cells were washed once in FACS buffer and resuspended in 500 µL for single color staining and 1–2 mL for four color staining. Cells were maintained at 4°C until ready for sorting using a 4-color sorting protocol on a FACS Aria cell sorter to obtain CD184+, CD271−, CD15+, CD24+ cells (BD Biosciences). A gating protocol was established in which CD184+ CD271− cells were separated into CD24+ and CD15+ containing populations. Cells were sorted directly into Neurobasal media at room temperature using a 100 µm nozzle, 20 psi sheath pressure, 2,000 events/sec; taking care not to subject the cells to excessive shear forces that would reduce viability. Cells were plated with ROCK Inhibitor
