Transwell precoated matrigel membrane filter inserts

Temsirolimus (#ab141999, Abcam) and RapaLink‐1 (#A8764, APExBIO) were used as mTOR inhibitors. Cell proliferation was assessed using XTT assays (Roche Applied Science). Cell migration was evaluated with in vitro wound healing. Cell invasion was examined with modified Boyden chambers consisting of Transwell precoated Matrigel membrane filter inserts with 8‐μm pores in 24‐well tissue culture plates (BD Biosciences). The experimental procedures were conducted as previously described.²³, ²⁴

A cellular migration assay was performed using modified Boyden Chambers consisting of Transwell pre-coated Matrigel membrane filter inserts with 8 µm pores in 24-well tissue culture plates (BD Biosciences). Briefly, PSCs were seeded at 5×10⁴ cells/well into the plates. Media supplemented with 10% FCS in the lower chamber served as the chemoattractant. The following day, pancreatic cancer cells were seeded at 2×10⁵ cells/insert into the culture inserts (8 µm pores) and placed on the 24-well plate containing human PSCs or SB525334. After a 24-h incubation at 37°C, the stained cells were counted by inverted microscopy. Briefly, cells were removed from the upper surface of the membranes with a cotton swab, and cells that migrated to the lower surface were stained with 0.2% (w/v) crystal violet in 2% ethanol for 15 min at room temperature and were washed with water. Dried membranes were cut out and mounted on glass slides in immersion oil. At least 10 random high-power fields from each of triplicate membranes were counted for each experimental condition at ×200 magnification. Each experiment was performed in triplicate.

Cell proliferation, migration and invasion assays were carried out as previously described.10, 11, 12Cell proliferation was determined by using an XTT assay (Roche Applied Sciences, Tokyo, Japan) performed according to the manufacturer's instructions. Cell migration activity was evaluated by wound healing assay. Cells were split into six‐well dishes, and the cell monolayer was scraped using a P‐20 micropipette tip. The initial gap length (0 h) and the residual gap length (24 h) after wounding were calculated from photomicrographs. A cell invasion assay was carried out using modified Boyden chambers consisting of Transwell‐pre‐coated Matrigel membrane filter inserts with 8‐mm pores in 24‐well tissue culture plates (BD Biosciences, Bedford, MA, USA). MEM containing 10% FBS in the lower chamber served as the chemoattractant. All experiments were performed in triplicate.