Basement membrane matrix

Polycarbonate membrane cell culture inserts (CLS3422, Corning) were applied with Basement Membrane Matrix (Trevigen). After the matrix condensed at 37°C in cell incubator, each 24-well insert was seeded with 1×10⁵ of C4-2 cells in RPMI-1640 without FBS. RPMI-1640 with FBS was added to each well containing the insert. Ebastine or vehicle (ethanol or DMSO) was added to keep the same concentration inside and outside of the inserts. Each insert was fixed with methanol, and cells that permeated through the membrane were stained with 0.5% crystal violet. Images were captured, and the cell count was calculated using ImageJ (1.8.0).

Immunodeficient nude mice (Jackson Laboratory), 6–8-week-old, were injected with 10⁶ human breast cancer cells MDA-MB-231 (ATCC) suspended in 100 μL of 7 mg/mL basement membrane matrix (Trevigen) in the lower right abdominal mammary fat pad to generate tumors. In some instances, to trace the release and uptake of MDSC-derived EVs, the breast cancer cells were fluorescently pre-labeled with a green MemGlow dye (Cytoskeleton).