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Cells were incubated in Hank's buffer with 2 M MitoSox-Red for 30 min at 37 C in a 5% CO 2 atmosphere, washed with PBS and the fluorescence assessed by flow cytometry or fluorescence microscopy

After 10 days, the number of wells containing spheres was determined for each condition and the software ELDA (Extreme limiting dilution analysis) was used to calculate the long-term cancer stem cell initiating frequency and significance [4]

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To know it is working, doctors applied different doses and measured levels in patients

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