Direct co-culture of fibroblasts and BT20 cells

The CAFs, NFs, and INFs were directly co-cultured with BT20 cells as described previously [21]. CAFs, NFs, INFs or BT20 cells were incubated with serum-free DMEM/F12 containing 5 μM CellTracker Green CMFDA (5-chloromethylfluorescein diacetate; Invitrogen, Burlington, Ontario, Canada) for 45 minutes at 37°C. The solution was replaced with fresh, prewarmed medium for an additional 2 h. The cells were washed twice with PBS and then unstained fibroblasts or BT20 cells were seeded onto plates containing CMFDA-stained fibroblasts or BT20 cells, respectively. Finally, the co-cultures were incubated with medium (DMEM/F12, 1% FBS and 100 IU/mL penicillin with 100 μg/mL streptomycin) for 1 week. Using confocal microscopy, CMFDA-stained cells were easily distinguished from unstained cells.