Macsquantify software version 2

To test cell viability, cells were plated at a density of 5 × 10⁴ viable cells/ml and treated. Upon incubation, cells were labelled with propidium iodide (PI) solution (2 µg/ml) (Millipore Sigma, Darmstadt, Germany) and the count of viable cells (PI negative) was performed using the flow cytometer MACSQuant Analyzer (Miltenyi Biotec).
For cell cycle analyses, cells were fixed in 70% ethanol and stained with 2 µg/ml PI. Cell cycle distribution was measured by flow cytometry (MACSQuant Analyzer).
For assessment of apoptosis cells were stained with Annexin V and PI (Annexin V-FITC Kit, Miltenyi Biotec) following the manufacturer’s instructions and analysed by flow cytometry (MACSQuant Analyzer). Mitochondrial membrane depolarisation was assayed using the fluorescent probe tetramethylrhodamine ethyl ester-TMRE (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions and measured by flow cytometry (MACSQuant Analyzer).
All data were analysed using the MACSQuantify software version 2.6 (Miltenyi Biotec).

The percentages of cells at each phase of the cell cycle were determined by staining with propidium iodide (PI) according to the method of Lee et al. [18 (link)]. Briefly, trypsinized cells were centrifuged at 500 × g at 4°C for 7 min and then fixed with 70% ethanol overnight at -20°C. After washing with 1× phosphate-buffered saline (PBS), the cells were incubated with the Muse cell cycle reagent (Merck Millipore). Data from 10,000 cells were analyzed using the MACSQuant analyzer and MACSQuantify software version 2.5 (Miltenyi Biotec GmbH).