Cell sorting and flow cytometry after vital staining with BioTracker ATP-red 1

Human breast cancer cell lines were first grown either as a 2D monolayer or as 3D-mammospheres. Then, they were stained with 10 µM of BioTracker ATP-Red 1 for at least 30 min. The cells were washed twice with Dulbecco’s phosphate-buffered saline (DPBS; Gibco, Life Technologies), collected and dissociated into a single-cell suspension, using a 25-g needle syringe and a 40 µm cell strainer (Fisher Scientific, Inc.), prior to analysis or sorting by flow-cytometry with the SONY SH800 Cell Sorter [19]. Briefly, ATP-high and ATP-low subpopulations of cells were isolated after vital staining with the probe ATP-Red 1. The ATP-high and ATP-low cell subpopulations were selected by gating, within the ATP-Red 1 signal (Excitation: 510 nm; Emission: 570 nm). Only cells with the least (bottom 5 or 10%) or the most (top 5 or 10%) signal were collected. Also, a population between the bottom and the top populations was sorted (bulk 5%). The cells outside the gates were discarded during sorting, due to the gate settings. However, such settings are required, to ensure high purity during sorting. Data were analyzed with FlowJo 10.1 software.