Macsquant vyb

Cells were preincubated with Fluo-4 AM (Molecular Probes) for 30 min at 37 °C. Then, cells were washed and resuspended in HBSS for flow cytometry analysis. 10,000 events per sample were collected in a MACSQuant-VYB and data analysis from flow cytometry was performed using FlowJo software.

Cells were seeded at 8 × 10⁴ cells per well in 24-well plates. The next day, cells were treated with indicated concentrations of ZZW-115 alone or in the presence of 1 μM Fer-1 for 72 h. After that, cells were incubated with 5 μM CellROX Green Reagent (C10444, Thermo, USA) or 10 μM MitoSOX Red (M36008, Thermo, USA) at 37 °C for 30 m in the dark. Then, the unincorporated dye was removed by washings with prewarmed PBS. Samples were then harvested by accutase, centrifuged at 1500 rpm for 5 m and the pellets were resuspended in 200 µL prewarmed HBSS (Gibco, Life Technologies) for flow cytometry. 10,000 events per sample were collected in a MACSQuant-VYB, and data were analyzed with FlowJo software.