Characterization of Murine Leukemic Lineages

Analyses of leukemic lineages and apoptosis were performed as described earlier [6 (link)]. Briefly, for analysis of lineages and LICs, bone marrow cells were stained with anti-mouse Mac-1-PE, anti-mouse Gr-1-APC, anti-mouse CD3e-PE, anti-mouse B220-PE, and anti-mouse c-Kit-APC monoclonal antibodies (eBioscience, USA). For detection of CD274 expression in Mac-1⁺/c-Kit⁺ LICs of murine AML model, anti-mouse CD274-biotin and streptavidin-PE (secondary antibody) were used (eBioscience, USA). Cell cycle status was determined in purified Mac-1⁺/c-Kit⁺ LICs with Pyronin Y and Hoeschst 33342 staining (Sigma, USA) as previously described [25 (link)]. Apoptosis analysis was conducted in purified Mac-1⁺/c-Kit⁺ LICs with anti-Annexin V-PE and 7-AAD staining (BD Pharmingen, USA) according to the manufacturer’s instructions.