Hsc007 methylcellulose

FACS-purified cells were sorted into FBS, spun down at 300 g, and then plated into either methylcellulose, BFU-E, or MegaCult media as per the manufacturer’s protocol. For methylcellulose colony assays, FACS-purified c-kit⁺ (5,000 per well) bone marrow cells were plated in HSC007 methylcellulose (R&D Systems). For BFU-E assays, FACS-purified MEP (3,500 per well) or c-kit⁺ cells treated with vehicle or Heparinase I, II, III (3,500 per well) were sorted and plated in HSC006 methylcellulose (R&D Systems) supplemented with 50 ng/ml SCF, 20 ng/ml IL-3, 20 ng/ml IL-6, and 10 U rhEPO. Colonies were then counted 7 d later. For MegaCult assays, FACS-purified c-kit⁺ (3,500 per well) or MEP (3,500) mouse bone marrow cells were sorted and plated in MegaCult media as per manufacturer protocol (STEMCELL Technologies). Human CD34⁺ cells from mobilized peripheral blood were allowed to recover in StemSpan II media with cytokines (SCF, IL-3, Flt,IL-6, and TPO all at 50 μg/ml) for 12 h before sorting, and human MEPs (3,000–4,500 per condition) were sorted and plated in MegaCult. For mouse samples, colonies with three or more megakaryocytes were scored 8 d later based on acetylcholinesterase positivity following manufacturer’s protocol. For human samples, slides were stained with the MegaCult Human Staining Kit as per manufacturer protocol.