Immature cells were enriched from BM using lineage microbeads and the autoMACS Pro Separator and stained with antibodies against lineage markers; anti-Flt3, anti–c-KIT, anti–Sca-1, anti-CD150, and anti-CD48 (Thermo Fisher Scientific); and DAPI. Samples were sorted using a FACSAria II. Cells were cultured in U-bottom, 96-well plates (100 cells/well) in StemSpan SFEM II medium (StemCell Technologies) with penicillin/streptomycin (Invitrogen, Thermo Fisher Scientific), antibiotic/antimycotic (HyClone), and erythropoietin, thrombopoietin (TPO), IL-3, IL-6, FLT3, GMSCF (all 10 ng/mL; PeproTech), and SCF at 37°C and 5% CO2. Cell populations were enumerated by adding Countbright beads (Thermo Fisher Scientific); Lin– (anti-ter119, anti-IgG, anti-CD3e, anti-Nk1.1, anti-CD4, anti-CD19, anti–Gr-1, anti-B220, anti-CD8a, and anti-CD8b); anti-CD11b, anti–Sca-1, anti-CD117, anti-Flt3, anti-CD150, anti-CD48, and DAPI or Fixable Dye eFluor 450 to wells and analyzed on an LSR Fortessa.
Shah M., Kumar H., Qiu S., Li H., Harris M., He J., Abraham A., Crossman D.K., Paterson A., Welner R.S, & Bhatia R. (2023). Low c-Kit expression identifies primitive, therapy-resistant CML stem cells. JCI Insight, 8(1), e157421.
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