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.