Polycarbonate filter with 8 μm pores

Cell migration assays were performed as previously described using a 48‐well microchemotaxis Boyden chamber (Neuro Probe, Gaithersburg, MD, USA) and polycarbonate filter with 8‐μm pores (Neuro Probe).30 The lower side of the filter was precoated with 10 μg/mL collagen type I‐C (Nitta Gelatin, Osaka, Japan). After incubation for 3 hours, the number of cells that had migrated to the lower side was counted in 3 independent fields. These experiments were repeated a minimum of 4 times.

Cell migration was assessed using a 48-microchemotaxis chamber (Neuro Probe, Maryland, USA) and a polycarbonate filter with 8-μm pores (Neuro Probe). The lower side of the filter was precoated with 10 μg/mL collagen type I-C (Nitta Gelatin, Osaka, Japan). A medium containing 10% FBS was placed in the bottom chamber. Non-irradiated and irradiated cells were trypsinized, washed twice with serum-free medium and suspended in serum-free medium supplemented with 0.1% bovine serum albumin (BSA; FUJIFILM Wako Pure Chemical Corporation). Fifty microliters of cell suspension containing 1.1 × 10⁵ MDA-MB-231 cells were added to the upper wells, and incubated for 3 hours. The cells were fixed with 4% paraformaldehyde and stained with hematoxylin. The number of cells that migrated to the lower side through the pores was counted at ×40 magnification in three independent fields. At least three experiments were conducted.