Anti mouse cd117 microbeads

Bone marrow cells were harvested by crushing two tibias, two femurs, two pelvises, and one spine from each mouse. Bone marrow cells were enriched for immature cells using anti–mouse CD117 MicroBeads and an autoMACS machine (both Miltenyi Biotec) per manufacturer’s instructions. c-Kit–enriched populations were stained with antibodies against lineage markers (B220, CD3, Gr-1, Mac-1, and Ter119), c-Kit, Sca-1, CD150, CD16/32, and CD34 (eBioscience) as previously described (McGowan et al., 2011 (link)). Stained samples were either analyzed or sorted using a FACSAria II (BD). For all experiments in which c-Kit^hi or c-Kit^lo HSCs were purified, they were double-sorted to ensure >95% purity. To calculate the frequency of hematopoietic precursors, two femurs and two tibias were flushed into 1x PBS containing 2.5% fetal calf serum (Hyclone). Bone marrow aspirations were performed on mice under isoflurane anesthesia per IACUC-approved protocol. Aspirates were treated with ACK lysis buffer and stained in PBS/2.5% fetal calf serum with antibodies against lineage markers, c-Kit, Sca-1, CD150, CD16/32, Flk2, CD34, CD48, and CD41 for analysis on hematopoietic stem and progenitor cells. For myeloid progenitors, bone marrow cells were stained with antibodies against lineage markers, c-Kit, Sca-1, CD150, CD16/32, CD41, CD105, and CD71, as described by Pronk et al. (2007) (link).