Mil 6

At day 2 post-infection, Sca1⁺/lin^- cells were plated at 5 x 10³ cells/mL in methyl-cellulose supplemented with mIL-3 (20 ng/mL), mIL-6 (20 ng/mL) and mSCF (100 ng/mL)(Stem-Cell Technologies, Vancouver, Canada). On day 10 after plating, the number of colony forming units (CFUs) was determined. After washing out from the methyl-cellulose, the cells were stained with specific antibodies for the detection of surface marker expression by FACS. 5 x 10³ cells/ml were replated in methyl-cellulose, thus permitting determination of the serial replating potential.

32Dcl3 (here after named as 32D) and BA/F3 cells (ACC-411, ACC-300, DSMZ, 2018–01) were cultured as described previously [25 (link), 26 (link)]. TF-1 cells (ACC-334, DSMZ, 2018–01) were cultured using RPMI 1640/10%FCS/GM-CSF (5 ng/ml). All cell lines were routinely tested for mycoplasma using PCR. Authentication of cell lines was performed using qRT-PCR for murine or human housekeeping gene as well as cell surface expression of characteristic receptor expression pattern (CD34, CD11b, Gr-1) using FACS analysis. Primary murine cells were cultured in serum-free BIT9500 cell culture medium (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with mIL-3 (10 ng/ml), mIL-6 (5 ng/ml) and mSCF (50 ng/ml). All cytokines were purchased form ImmunoTools, (Friesoythe, Germany). Further, lineage negative transgenic SCLtTA/Bcr-Abl BM cells were retrovirally infected using MSCV-ER-Hoxb8-Neo plasmid as described previously [27 (link)]. ER-HoxB8 derived immortalized progenitor cells were cultured in IMDM containing 10% FBS, 5% SCF-supernatant and 1% Pen-Strep and selected with G418 (1 mg/ml) for 1 week. FACS analysis for Gr-1, CD11b, B220, CD3 and Ter119 (BioLegend, Fell, Germany) were performed demonstrated the absence of mature cell surface markers.