To dissociate cells from spheroids, we recommend incubating the spheroids with a dissociation reagent, such as Accutase, 5 mM EDTA, 1X Trypsin/EDTA, or 1X, 5X, or 10X TrypLE. The incubation time required to dissociate cells will depend on the cell type, spheroid size, and length of culture time. Once the spheroids have started to dissociate, pipette to mix a few times, which will generate a single cell suspension.