Fms related tyrosine kinase 3 ligand

Primary AML and healthy donor samples were cultured in StemSpan medium (StemCell Technologies, Inc.), supplemented with 100 ng/mL stem cell factor, 100 ng/mL Fms-related tyrosine kinase 3 ligand, and 20 ng/mL thrombopoietin (all sourced from R&D systems). Serum-free conditions were used during drug treatment or cAMP assays. Unless stated otherwise, a concentration of 10 μM for compounds, and 1:100 dilution was used for DRD1-Ab, and the data were compared to 0.1% DMSO or IgG control for in vitro treatment assays. Shandon Kwik-Diff stain (#9990700; Thermo Fisher) was used to visualize cultured cells by light microscopy.

Mononuclear cells from primary human BM samples were isolated via density-gradient centrifugation using the Biocoll Separation Solution (Biochrom AG, Berlin, Germany) following the manufacturer’s instructions. CD34⁺ cells were purified via positive selection using the CD34⁺ MicroBeads kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and purity was confirmed to be at least 95%. BM mononuclear cells (BMMNC) were cultured at a density of 5 × 10⁵ cells/ml in serum-free media consisting of Iscove’s Modified Dulbecco’s Medium (IMDM) with L-alanyl-L-glutamine (IMDM GlutaMAX) with 20% BIT 9500 serum substitute (1% (w/v) bovine serum albumin, 10 μg/ml insulin, 200 μg/ml iron-saturated transferrin; StemCell Technologies, Vancouver, BC, Canada) and enriched with recombinant human stem cell factor (100 ng/ml), FMS-related tyrosine kinase-3 ligand (100 ng/ml), thrombopoetin (10 ng/ml), interleukin-6 (5 ng/ml), interleukin-3 (10 ng/ml; all from R&D Systems, Minneapolis, MN, USA), β-mercaptoethanol (10 μM; Gibco, Carlsbad, CA, USA) and low- density lipoproteins (4 μg/ml; Sigma-Aldrich, St Louis, MO, USA).