Quantifying Cell Death in 3D Cultures

Propidium iodide (10 µg/mL) was added to the 3D cell cultures previously incubated with vesicles or controls for 24 h, and incubated for 20 min at 37  °C, under a 5% CO₂ humidified atmosphere [34 (link)]. As dead cells are permeable to PI, an increase in PI fluorescence is observed in dead cells. Image acquisition was performed on a Zeiss Axio Observer microscope (White Plains, NY, USA) with a ×20 objective using ZEN software. The average PI fluorescence intensity of the spheroid area was measured with ZEN software. Phase contrast was used to define the spheroid area. A minimum of four spheroids were used per condition in each assay and three independent assays were performed.