Hg csf

To test myeloid potential, single cells were cultured in Terasaki plates in 20 l of Iscove's Modified Dulbecco's Medium (IMDM) with l-glutamine (Gibco), 20% HyClone Defined Fetal Bovine Serum (SH30070.03, GE Healthcare), penicillin-streptomycin (Invitrogen), and 0.1 M -mercaptoethanol (Sigma-Aldrich), supplemented with hSCF (20 ng/ml), hFlt3L (20 ng/ml), hIl-3 (20 ng/ml), hIl-5 (50 ng/ml), hIl-6 (20 ng/ml), hGM-CSF (50 ng/ml), and hG-CSF (20 ng/ml). For bulk cultures, 50 to 150 cells were cultured in 400 l of the same culture medium in 48-well plates. For combined erythroid, Downloaded from https://www.science.org on September 09, 2024 megakaryocyte, and myeloid readout, single cells were cultured in round-bottom 96-well plates in 50 l of StemSpan (STEMCELL Technologies) with hSCF (20 ng/ml), hFlt3L (20 ng/ml), hIl-3 (20 ng/ml), hIl-5 (50 ng/ml), hIl-6 (20 ng/ml), hGM-CSF (50 ng/ml), hG-CSF (20 ng/ml), hLDL (40 g/ml; Sigma-Aldrich), erythropoietin (0.5 U/ml), and thrombopoietin (100 ng/ml). Cells were cultured at 37°C, 5% CO 2 . Cytokines, suppliers, and concentrations used are found in table S6. Cytospins were prepared with a Shandon Cytospin at 1000 rpm with low acceleration, followed by May-Grünwald-Giemsa stain (VWR).