Flt 3 ligand

PDXs were cultured in QBSF 60 medium (Quality Biological, USA) supplemented with Flt-3 ligand (20 ng/mL, ProSpec, USA), penicillin (100 U/mL), streptomycin (100 μg/mL) and L-glutamine (2 mM) (QBSF 60/F). Cisplatin and dexamethasone sensitivities were assessed by measuring mitochondrial activity via the Alamar Blue assay or cell viability by 7-AAD exclusion using flow cytometry as previously described [10 (link), 12 (link), 40 (link), 41 (link)]. Cell viability was expressed as a percentage of vehicle treated controls.

EL08.1D2 cells were cultured as previously described [28] (link). HSC were plated over 2000 irradiated EL08.1D2 cells in a medium consisting of 2∶1 (vol:vol) mix of Dulbecco’s Modification of Eagle’s Medium (DMEM) with 4.5 g/L glucose, L-glutamine 1640, and sodium pyruvate/Ham’s F12 Medium (all from Lonza, Verviers, Belgium) supplemented with 50 µM beta mercaptoethanol (β-ME), 50 µM ethanolamine, 20 mg/L ascorbic acid, 50 µg/L sodium selenite, 1% penicillin/streptomycin and 20% heat-inactivated human AB serum (all from Sigma, Poole, UK). Different interleukins such as 10 ng/ml IL-15, 5 ng/ml IL-3 (only for the first week; R&D System, Abingdon, UK), 20 ng/ml IL-7, 20 ng/ml c-kit ligand stem cell factor (SCF) and 10 ng/ml Flt3 ligand were added up to 21 days of culture and only 10 ng/ml IL-15 were added from day 21 to day 35 (all from Prospec, Israel). Cultures underwent weekly hemi-depletion. Lymphoid progenitors were characterized as CD45+CD7+ whereas myeloid progenitors as CD45+CD33+.