Blue cmac

CellTracker™ dyes were purchased from Thermofisher (Green CMFDA, C7025; Red CMTPX, C34552; Blue CMAC, C2110). Cells were seeded 48 h before staining into new flasks. The CellTracker™ dyes were diluted in serum-free medium to final concentrations (Table S3) and applied directly to the cells. Images were analyzed using ImageJ and Imaris version 9.6 software (Bitplane, Zurich, Switzerland).
Spheroids have been stained with ethidium homodimer (Thermofisher, Invitrogen, E1169) and calcein (Thermofisher, Invitrogen, C1430) through direct transfer from the culture plate into wells containing the staining solution at the concentration of 10 μM (ethidium homodimer) and 4 µM (calcein). Spheroids were left for ≥30 min in the staining solution till further use.

3D GBM spheroids were prepared as previously described. Brightfield microscopy images were taken using an EVOS M5000 microscope (Thermofisher Scientific). For confocal microscopy, astrocytes, HMC3 and U87MG were fluorescently labeled using CellTracker® Green CMFDA, Orange CMRA and Blue CMAC (Thermofisher Scientific), respectively, before seeding. After 3 or 5 days of culture, the 3D GBM spheroids were washed with Dulbeccos’s Phosphate Buffered Saline (DPBS, Thermofisher Scientific), fixed with 4% formaldehyde (Sigma-Aldrich) and again washed twice with DPBS. 3D GBM spheroids were kept in DPBS and imaged using a Nikon A1 Confocal Laser Microscopy System (Nikon Instruments, Tokyo, Japan).

Prior to cell harvesting, CRC cells, fibroblasts and endothelial cells were washed with PBS and incubated in serum free medium for 20 minutes supplemented with 40 µM blue CMAC, 5 µM Green CMFDA or 10 µM Red CMTPX (C2110, C7025 and C34552, Life Technologies), respectively. Afterwards, 3D co-cultures were established and fluorescent imaging was performed.