Orange cmra

B16F10 murine melanoma cells (a gift from John L. Francis, Center for Thrombosis Research, Florida Hospital, Orlando, Florida, USA; ref. 75 (link)) were cultured in RPMI 1640 medium (Sigma-Aldrich), 4T1/4T1-GFP murine breast cancer cells, MC-38-GFP murine colorectal cancer cells, and MDA-MB-231-CFP human breast cancer cells (ATCC) were cultured in DMEM (Sigma-Aldrich) in a 5% CO₂ humidified atmosphere at 37°C. Media were supplemented with 10% heat-inactivated FBS (Gibco), 2 mM l-glutamine, 25 mM HEPES, 50 U/ml penicillin, and 5 μg/ml streptomycin (Thermo Fisher Scientific), with addition of 0.4 mg/ml G418 or 5 μg/ml puromycin for 4T1-GFP and MC-38-GFP cells, respectively. Primary LMVECs were cultured in 2% gelatin–coated flasks (Sigma-Aldrich) in enriched DMEM (76 (link)). Cells were passaged using Versene (B16F10) (Thermo Fisher Scientific) or 0.05% trypsin-EDTA solution (all other cell lines) (Sigma-Aldrich). LMVECs were used within 10 and tumor cells within 20 passages and routinely tested for mycoplasma contamination (MycoAlert Mycoplasma Detection Kit, Lonza Group Ltd.). Exponentially growing B16F10 cells (50%–60% confluence) were stained with 12.5 μM solution of CellTracker Blue CMAC, Orange CMRA, or Green CMFDA dye (Thermo Fisher Scientific), following the manufacturer’s instructions.

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).

GECs,
GFs, PDLSCs, and BMSCs were seeded in 6-well plates at a density of
2 × 10⁵ cells/well, separately. Once the cells reached
80–90% confluency, they were prestained for 30 min with cell
tracker dye Violet BMQC, Green CMFDA, Orange CMRA, and Deep Red (Thermo
Fisher Scientific), as per the manufacturer’s instructions.
The cells were washed with PBS, collected by trypsinization, counted,
and diluted to a density of 2 × 10⁵ cells/mL. The
same quantity of each cell type was evenly mixed, and 200 μL
of the mixed cells was seeded onto the NFG-MS or E7-NFG-MS in 24-well
plates. After cultivation for 3 h, the nonadhered cells were removed
using cell strainers, and the adhered cells were collected by trypsinization,
fixed with 4% PFA, and resuspended in PBS for quantitative analysis
of the proportion of each type of cells by a flow cytometry assay.
Three samples were performed at each time point, and the experiment
was repeated three times.