Cytokine Assay and Ex vivo Transplantation of KMT2A-MLLT3 Leukemic Cells

For the cytokine assay, established mouse KMT2A-MLLT3 + Empty-GFP leukemic cells were washed once in cold PBS (GE Healthcare Life Sciences), resuspended in C10 medium supplemented with 25 ng/ml mSCF (Peprotech) and plated in 100 µl at a density of 50,000 cells per well in a 96-well U-bottom plate and 5 µl recombinant human MIF (Peprotech) was added at a final concentration of 100 or 500 ng/ml, and PBS (GE Healthcare Life Sciences) was used as control. Cells were counted on day 6 after seeding using CountBright beads (Life Technologies, Eugene, OR, USA) on a FACS Fortessa (BD). Data analysis was performed using the FlowJo software (FlowJo, LLC).
For the ex vivo transplantation assay, serially propagated dsRed⁺KMT2A-MLLT3 leukemia cells^{44 (link)} were used^{69 (link)}. Briefly, 5000 freshly isolated CD117⁺ quaternary transplant leukemia cells were treated ex vivo with or without 500 ng/ml MIF (Peprotech) in StemSpan™ SFEM (Stemcell Technologies) for 3 days before transplantation into sublethally irradiated (600 cGy) recipients. Blood sampling was performed 3 weeks post transplantation and at disease manifestation, BM cells from femurs and tibias were collected, and the percentage of dsRed⁺ leukemia cells were determined using flow cytometry.