The femora and tibiae were harvested from the mice immediately after their sacrifice with CO2. Bone marrow (BM) cells were flushed from bones into Iscove’s modified Dulbecco’s medium (IMDM; Invitrogen) containing 10% FCS, using a 21-guage needle and syringe. Low-density BM mononuclear cells (LDBMMNCs) were separated by Ficoll Hypaque density gradient (Sigma-Aldrich, St. Louis, MO) and washed with IMDM medium.
For flow analysis and cell sorting, BM cells from mice of the indicated genotype were stained with the following antibodies (all from BioLegend, San Diego, CA): Ter119 (#79748), CD45R/B220 (#79752), CD3e (#79751), Gr1 (#79750), CD11b (#79749), Ly-6A/E (Sca1, #108114), CD117 (c-Kit, 105826). Flow cytometric analyses were done using BD LSRII flow cytometer (BD Bioscience). For cell sorting, lineage negative cells were enriched using lineage depletion reagents (StemCell Technologies) according to the manufacturer’s instruction. The Linnegative and LSK populations were acquired by using the FACSAria II sorter (BD Biosciences).
For flow analysis and cell sorting, BM cells from mice of the indicated genotype were stained with the following antibodies (all from BioLegend, San Diego, CA): Ter119 (#79748), CD45R/B220 (#79752), CD3e (#79751), Gr1 (#79750), CD11b (#79749), Ly-6A/E (Sca1, #108114), CD117 (c-Kit, 105826). Flow cytometric analyses were done using BD LSRII flow cytometer (BD Bioscience). For cell sorting, lineage negative cells were enriched using lineage depletion reagents (StemCell Technologies) according to the manufacturer’s instruction. The Linnegative and LSK populations were acquired by using the FACSAria II sorter (BD Biosciences).