Murine Leukemia Induction Protocol

BM cells were collected from tibia and femur from 8-week-old wild-type C57BL/6 J mice at 6 days after 5-fluorouracil (5-FU) treatment and resuspended in cold 1 × PBS buffer supplemented 2% FBS. BM cells were subjected to isolation of lineage-negative cells (Lin⁻) by harvesting the non-adherent fraction by selection with specific microbeads (CD11b⁻, Gr-1⁻, Ter119⁻, CD3⁻, B220⁻; Stemcell Technologies) following the manufacturer’s instructions. BM Lin⁻ cells were cultured in StemSpan SFEM (Stemcell Technologies) supplemented with murine SCF (10 ng/ml), TPO (50 ng/ml), and FLT3-L (50 ng/ml) overnight. The Lin⁻ cells were transduced with MSCV-GFP-IRES-MLL-AF9 (Addgene, Watertown, MA, USA) through two rounds of “spinoculation” as described previously [26 (link), 27 (link)]. After transduction, Lin⁻ cells were intravenously injected into lethally irradiated C57BL/6 J mice (Shanghai Model Organisms Center, Shanghai). The mice were humanely sacrificed, and green fluorescent protein⁺ (GFP⁺) cells were sorted by flow cytometry when they developed full-onset leukemia.