X mercaptoethanol

Production of MLL-AF9 transduced human CD34+ cells has been described.⁷⁸ Human CD34+ cells were isolated from human cord blood (New York Blood Center) using the EasySep™ Human Cord Blood CD34 Positive Selection Kit II (Stemcell Technologies). For pre-enrichment, Lymphoprep (Stemcell Technologies) and SepMate™ columns (Stemcell Technologies) were used. Viral supernatants were generated by co-transfection of HEK293-T cells with retroviral (pMSCV-MLL-AF9-IRES-GFP)⁷⁸ expression vector with packaging and envelope vectors (Human Retro: pUMVC and VSV-G) and X-tremeGene transfection reagent (Millipore). The viral supernatant was filtered through 0.45μm and was concentrated using Amicon Ultra centrifugal filters (Millipore). Human CD34+ cells were plated on retronectin-coated plates (Takara) and were spin infected at a 1:1 dilution of virus:media at 800g at 37°C for 1.5h. The cells were dissociated from the plates using enzyme-free dissociation buffer (Gibco) and were plated in fresh media. Cells were maintained in IMDM with 20% FBS, 1x penicillin/streptomycin (Gibco), 1xß-mercaptoethanol (Gibco), 6 μg/mL hIL3, 10 μg/mL hIL6, 10 μg/mL hSCF, 10 μg/mL TPO, and 10 μg/mL FLT3 (Stemcell Technologies) at 37°C and 5% CO2. Two days after transduction, GFP positive cells were sorted using FACSAria cell sorter (BD Bioscience).