Lsrfortessa software

The sorted LPCs (1 × 10⁵/well) were cultured in StemSpan (Stem cell Technologies, Vancouver, BC, Canada) supplemented with four growth factors (20 ng/mL recombinant human (rh) interleukin-3 (rhIL-3), 20 ng/mL rhIL-7, 20 ng/mL rh Flt3-ligand (rhFlt3-L), and 50 ng/mL rh stem cell factor (rhSCF)) (PeproTech, Locky Hill, NJ, USA). After 48 h of suspension culture with the vehicle (DMSO, Sigma), imatinib (5 μM, Novartis, Basel, Switzerland) [24 (link), 33 (link)], nilotinib (5 μM, Novartis) [33 (link)], and/or ruxolitinib (300 nM, Novartis) [34 (link), 35 (link)] treatments, an apoptosis assay was performed on the LPCs using the Annexin-V and 7-amino-actinomycin D (7-AAD) Apoptosis Detection Kit (Becton–Dickinson) as described in our previous reports [36 (link), 37 (link)]. The late apoptotic cells (Annexin-V⁺/7-AAD⁺) were analyzed using the BD LSRFortessa software (Becton–Dickinson). Aliquots of isotype-identical antibodies served as negative controls.

Bone marrow mononuclear cells were isolated by density centrifugation using lymphocyte separation medium (GE Healthcare, Milwaukee, WI, USA). Cell cycle analyses were performed by incubating with 10 μg/ml Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C for 45 min, and 1.0 μg/ml Pyronin Y (Sigma, St. Louis, MO, USA) at 37°C for an additional 15 min. Cells were stained with mouse anti-human CD34-PerCP-Cy5.5 and CD38-APC-conjugated monoclonal antibodies (Becton Dickinson) at room temperature for 15 min.
To detect apoptosis, cells were incubated with CD34-PE, CD38-APC and CD45-V500 and then incubated for 15 min with Annexin-V-FITC and 7-amino-actinomycin D (7-AAD) apoptosis detection kit (Becton Dickinson) according to the manufacturer's instruction. Multi-parameter flow cytometric analyses were done using a BD LSRFortessa (Becton Dickinson). Aliquots of unstained samples were used as negative controls. Data were analyzed using BD LSRFortessa software (Becton Dickinson).

The frozen BMMNCs of de novo Ph⁺ALL patients (N = 6) were thawed and stained with mouse anti-human CD58-FITC (Beckman-Coulter, Brea, CA, USA) and CD34-PE, CD19-APC-Cy7, CD45-PerCP, CD38-APC monoclonal antibodies (MoAbs, Becton–Dickinson, San Jose, CA, USA). The LPCs (CD34⁺CD38⁻CD58⁻) and other cell fractions (including CD34⁺CD38⁻CD58⁺, CD34⁺CD38⁺CD58⁻ and CD34⁺CD38⁺CD58⁺) in the viable BMMNCs were defined and sorted using a FACS Aria II (Becton–Dickinson) as previously reported¹² (Fig. 1). The purity of each fraction was >97%. Fluorescence-minus-one controls were used to identify positive events for CD34, CD38 and CD58. The data were analyzed using BD LSRFortessa software (Becton–Dickinson).Fig. 1

Representative flow cytometric analysis of a Ph⁺ALL patient sample sorted according to the distribution of CD34, CD38 and CD58 expression. In the viable bone marrow mononuclear cells (BMMNCs) of a de novo Ph⁺ALL patient, the LPCs (CD34⁺CD38⁻CD58⁻) and other cells (CD34⁺CD38⁻CD58⁺, CD34⁺CD38⁺CD58⁻ and CD34⁺CD38⁺CD58⁺) fractions were sorted simultaneously

ROS staining was done using an ROS staining kit (Byotimes, Shanghai, China) according to the manufacturer's protocol. Cells were incubated with 10μM 2′,7′-dichlorofluorescence diacetate (DCFH-DA, Byotimes) and mouse anti-human CD45-V500, CD34-PerCP-Cy5.5, CD38-APC-conjugated monoclonal antibodies (Becton Dickinson) at 37°C for 15 min. After crossing the cell membrane, DCFH-DA undergoes de-acetylation by intracellular esterase producing green fluorescent when oxidized by ROS. Mean fluorescence intensity of intracellular ROS was analyzed as intracellular ROS levels using BD LSRFortessa software (Becton Dickinson).