Spheroid-infiltrating cells flow cytometric staining
For flow cytometry analyses, 8 wells/condition were seeded. OUT and IN compartments were isolated by first pooling the 8 cocultures wells in eppendorf tubes. Spheroids were gently resuspended and left to sediment to the bottom of the tubes. Supernatant cell suspension constituted the non-infiltrating immune cells (=OUT). These steps were repeated twice with PBS in order to separate the spheroids from the non-infiltrating immune cells. Spheroids were then trypsinized to obtain a single cell suspension (=IN) further analyzed by flow cytometry.