For the CFU assays, 8000 cord blood CD34+ (CB-CD34+) cells were incubated with compound for 72 h at 37 °C/5% CO2 in QBSF-60 (MSKCC Media Facility), 1 mM monothioglycerol, 2 mM glutamine, 20 ng/mL c-kit ligand, thrombopoietin and Flt3 ligand. After the incubation period, the compounds were washed out and the colony-forming assays were performed in triplicate in a 35 mm plate (1000 cells per well) using 1.2% methylcellulose (Dow Chemical), 30% FCS, 1 mM monothioglycerol (Sigma), 2 mM glutamine, 0.5 mM hemin (Sigma), 20 ng/mL interleukin-3 (Peprotech), granulocyte colony-stimulating factor (Amgen), c-kit ligand and 6 U/mL erythropoietin (Ortho Biotech). Samples were incubated at 37 °C/5% CO2. Colonies were scored 14 days after plating.
CAFC assays were performed by plating 2000 CB-CD34+ 72 h preincubated cells onto MS-5 monolayers in T12.5 tissue-culture flasks (Becton Dickinson) in duplicate. Weekly half of the medium and cells were removed and replaced with fresh medium. A cobblestone was defined as an instance of at least eight tightly packed phase-dark cells beneath the MS-5 stromal monolayer [17 (link)].
CAFC assays were performed by plating 2000 CB-CD34+ 72 h preincubated cells onto MS-5 monolayers in T12.5 tissue-culture flasks (Becton Dickinson) in duplicate. Weekly half of the medium and cells were removed and replaced with fresh medium. A cobblestone was defined as an instance of at least eight tightly packed phase-dark cells beneath the MS-5 stromal monolayer [17 (link)].