In order to evaluate the mitochondrial activity, leukemic cells were treated with ibrutinib, nutlin-3 or their combination as described above, and then labeled with MitoTracker® Red CMXRos (Molecular Probes, Inc., Eugene, OR, USA) that stains the mitochondria of live cells depending on the mitochondrial membrane potential. Briefly, 24 h post treatment, cells were incubated for 30 min at 37 °C with 100 nM MitoTracker® Red CMXRos, washed with PBS (Gibco) and analyzed by flow cytometry (FACSCalibur, BD Biosciences, San Josè, CA, USA). Data were analyzed with the FlowJo software 9.9.6 (Tree Star).

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