Peripheral blood was collected by retro-orbital bleeding, and bone marrow cells were isolated from the femurs and tibias of leukemic mice. Flow cytometry and cell cycle analyses were performed as we described previously [47 (link)]. Briefly, leukemia cells were stained with anti-mouse Mac-1-APC, anti-mouse Gr-1-PE, anti-mouse CD3-APC, anti-mouse B220-PE or anti-mouse c-Kit-PE monoclonal antibodies (eBioscience). The cell cycle stages were evaluated with either Ki-67/7-AAD staining (BD Pharmingen) or a 5-bromo-2′-deoxyuridine (BrdU) incorporation assay. For the analysis of apoptosis, leukemia cells were stained with PE-conjugated anti-Annexin V and 7-AAD (BD Pharmingen) according to the manufacturer's protocol. For the measurement of ROS, the cells were incubated with 1 μM 5-(and-6)-carboxy-2′,7′-dichlorofluorescein diacetate (carboxy-DCFDA, Invitrogen) for 30 minutes at 37°C, followed by flow cytometric analysis. For the examination of the BrdU incorporation assay, leukemic mice were subjected to three intraperitoneal injections of BrdU (Sigma; 3 mg/24 hours) in PBS. The BM cells were fixed, permeabilized and denatured, followed by antibody staining with anti–BrdU-APC according to the manufacturer's instructions (BD Pharmingen).
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