Boyden migration chamber

NB cells (1×10⁵ in 1 ml DMEM with 10% FBS) were added to the top of each Boyden migration chamber (8 μm, 12-well plate format; BD Biosciences, Palo Alto, CA). After 24 h cells were transfected with 100 nM FZD2 siRNA or scrambled siRNA using Lipofectamine and Plus reagent. After 24 h cells were incubated with 100 ng/ml recombinant Wnt3a or Wnt5a protein in DMEM with 1% FBS. After 48 h, medium was removed and membranes were washed twice with phosphate buffered saline (PBS). Cells from the upper side of the membrane were removed with cotton swabs. The membranes were excised using a scalpel, inverted and transferred to a PBS filled tissue culture well. Membranes were then fixed in methanol for 10 min at −20°C. After washing in PBS, membranes were stained with 1 μg/ml 4′-6-Diamidino-2-phenylindole (DAPI) in PBS for 10 min at room temperature and washed again in PBS. Membranes were then embedded in Cityfluor (Cityfluor, Leicester, UK) on glass slides. Representative sectors of migrated cancer cells were counted under a fluorescence microscope. Each experiment was performed in triplicate.

MCF7, SK-BR-3 and MDA-MB-231 breast cancer cell lines were starved over night in serum-free DMEM medium. Breast cancer cells (1×10⁵) in DMEM with 2% FBS were added to the top of each Boyden migration chamber (8 μm, 12-well plate format; BD Biosciences, Palo Alto, CA) with or without CSF-1R-blocking antibody (10μg/ml) followed by treatment with 200 ng/ml recombinant IL-34 or CSF-1 protein (R&D Systems, McKinley Place, MN, USA). After 48 h, medium was removed and membranes were processed as described [59 (link)].