Anti cd117 c kit microbeads

For competitive engraftment assays, the total BM cells were isolated from femurs and tibiae of WT and Camkk2 null (CD45.2 background). c-Kit-positive cells were enriched using anti-CD117/c-Kit microbeads (Miltenyi Biotec, Auburn, CA, USA). The KSL CD34^- stem cells (CD45.2) were then sorted and 1000 cells were injected with 5x10⁵ competitor whole BM (CD45.1) into lethally irradiated (split dose totaling 10Gy) recipient B6-CD45.1 (B6.SJL-Ptprca Pepcb/BoyJ) mice via the retro-orbital sinus. In the same experiments, KSL CD34^- cells were sorted from BM of Camkk2 null or control mice treated with 200cGy 90 days before transplant. For lineage analysis, peripheral blood cells were collected and prepared as described.^{51 (link)} In some experiments, transplanted mice were monitored for up to 4 months, and then mice received 450cGy TBI. Mice were finally monitored for chimerism.

Using anti-CD117/cKit microbeads (Miltenyi Biotec), BM cells from WT, HSC-DTR, Vav-DTR, and chimeric mice were enriched for c-Kit positive cells and sorted via flow cytometry into 5 mM EDTA in 1X PBS with 2% serum and then spun down. 100 HSCs, 200 MPPs, and 500 MyPros were plated in triplicates in IMDM media supplemented with 20% FBS, TPO (50 ng/mL), SCF (50 ng/mL), IL-6 (20 ng/mL), IL-3 (10 ng), IL-11 (20 ng/mL), 1X Primocin, and 1X Non-Essential Amino Acids. Live and nucleated cells were harvested and analyzed by flow cytometry after 7 days for Figs. 1e,f, 6a and Supplementary Fig. 3a. HSCs, MPPs, and MyPros were analyzed after 3 days in culture for Fig. 4e and Supplementary Fig. 2e,f.