Single-cell classification in co-cultures

The same method described in the previous paragraph was used for the case of co-cultures. Specifically, we co-cultured MDA-MB-231 with MCF-7 and MDA-MB-231 with MCF-10A. MDA-MB-231 was grown separately in their native media and labeled with Vybrant™ DiD cell-labeling solution (emission wavelength: 665 nm) (Invitrogen™, USA) before co-culture. This allowed us to distinguish each cell type in the co-culture via fluorescence imaging. Therefore, colored brightfield images and fluorescence images were acquired. Both sets of images were then segmented and a dataset was produced for each co-culture, containing the 16 colorimetric features together with their type, which was extracted from the fluorescence image.