Diff quick stain set

Invasion assays were performed using BD Biocoat Matrigel Invasion Chambers with 8-μm pore filters (BD Biosciences). The bottom wells of the companion plate were filled with 20% serum-containing medium to act as a chemoattractant for the cells. Cells were seeded at a density of 2.5 x 10⁴ per insert in serum-free medium. Invasion assays were incubated for approximately 16 hours at 37°C, 5% CO₂, stained with a Diff-Quick stain set (Fisher Scientific) and imaged with an Olympus BX53 light microscope (Olympus America). Invasive cells were counted in five representative high-power fields and all experiments were performed three times.
For invasion experiments involving cells treated with inhibitors, cells were pretreated with the respective dose of inhibitor two hours prior to being harvested for the assay. Inhibitors and vehicle controls were also added to the serum-free medium cell suspension prior to addition into the invasion chambers.

Cell invasion and migration assays were performed as described previously [38 (link)]. Briefly, cells, after treatment were seeded into trans‐well, inserts either uncoated (for migration assay) or coated (for invasion assay) with growth factor‐reduced matrigel for 24 h. After incubation, cells were stained with a Diff‐Quick stain set (Fisher Scientific, Pittsburg, PA, USA), and photographed under a fluorescent microscope.

Cell invasion and migration assay were performed using 24-well Transwell Permeable Supports with 8-lM pores (Corning, Lowell, MA). Briefly, after treatment of cells with NS398, Thiostrepton or a combination of the two inhibitors for 48 h, cells were harvested, counted again and 1.25 x 105 cells were suspended in serum-free medium and seeded into Transwell inserts either uncoated (for migration assay) or coated (for invasion assay) with growth factor-reduced Matrigel (BD Biosciences, Bedford, MA). Bottom wells were filled with complete media for 24 h. After incubation of 24 h, filters containing the cells were stained with Diff-Quick stain set (Fisher Scientific, Pittsburg, PA), photographed under a fluorescent microscope and manual cell counts were obtained [41 (link)].