Astrios high speed cell sorter

For single cell cultures, single murine CD34^- CD48^- CD150⁺ Lineage^- SCA1⁺ KIT⁺ (LSK) cells (CD34^- SLAM cells) were sorted directly into a 96-well round-bottomed plate on an Astrios high speed cell sorter (Beckman Coulter). Single cells were deposited with an efficiency of 90%. These CD34^- SLAM cells comprise both CD34^- and CD34^low populations, as these have been reported to contain similar frequencies of long-term repopulating cells.^{12 (link)} These plates were preloaded with 100 mL of filtered (0.20 mm, Sartorius) CM supplemented with murine stem cell factor (SCF: 100 ng/mL) and murine interleukin-11 (IL-11; 20 ng/mL) (two growth factors; 2GF), both from R&D Systems (BioTechne). Additional cultures were further supplemented with Col1 (300 μg/mL; BioVendor) and human NGF (250 ng/mL; R&D Systems- BioTechne) (four growth factors; 4GF). Every 24 h, the number of cells per well was enumerated using a light microscope. After 5 days, cells that had divided at least once were harvested, pooled, stained with antibodies, and analyzed by flow cytometry. In some experiments, colony-forming ability of pools from divided cells was assessed using standard assays (M3434; Stemcell Technologies).