Bio coat cell migration chamber

The modified Boyden chamber migration and wound healing assays were performed as described previously [44 (link), 45 (link)]. In brief, the modified Boyden chamber migration assay was performed for 12 h in 24-well Bio-Coat cell migration chambers (BD Biosciences, Franklin Lakes, NJ, USA). The lower surface of the membrane was coated with 30 μg/mL fibronectin for haptotactic migration. LM8 cells were applied to the upper chamber at a rate of 1 × 10⁴ cells with eribulin treatment for 12 h. Non-migratory cells were removed from the upper surface with cotton swabs. Migrated cells were fixed in 70% v/v methanol, stained with crystal violet, and counted. For the wound healing assay, confluent LM8 cells were scratched and throughly washed with phosphate buffered saline (PBS) to remove detached cells and debris. They were then treated with eribulin for 12 h. Images of wounds were measured with Fiji/ImageJ. Cell directionality was evaluated by fixing the cells during wound healing with 100% ice-cold methanol for 10 min at room temperature then blocking them for 30 min with PBS containing 1% BSA and 0.1% Tween 20. MTOCs were stained with anti-pericentrin antibody (1:1,000) at 4°C overnight. The cells were then incubated with anti-Rabbit IgG (Alexa Fluor^® 555) and Hoechst for 30 min at room temperature. More than 100 cells from three separate experiments were analyzed.

For migration assay, cells were maintained in serum-free RPMI-1640 medium for 18 h, harvested and resuspended in the same medium, and seeded into Bio-Coat cell migration chambers with 8 μm membrane pore sizes (BD Biosciences) at 1 × 10⁵ cells in 250 μl per chamber. The chambers were then inserted into the wells of a 24-well plate containing 750 μl of RPMI-1640 medium with 20% FBS alone or 20% FBS with 40 ng/ml human hepatocytes growth factor (HGF) (ImmunoTools, Friesoythe, Germany) and incubated at 37 °C with 5% CO₂ for 48 h. After this period, the cells remaining on the upper surface of the membrane were removed with cotton swab and the cells adhered to the lower surface were fixed with 70% ethanol for 15 min, stained with Giemsa (Sigma) for further 15 min, then washed twice with water. After drying, the membrane was removed with surgical blade and mounted on glass slides. Pictures were taken with Olympus BX50 microscope from five random microscopic fields at 200x magnification, and cells counted with image analysis software (ImageJ, NIH). For invasion assay, the same procedure was followed except for the coating of the migration chambers with 50 μl (0.3 mg/ml) of Matrigel matrix (BD Biosciences) before of the cells seeding.

Cells were cultured until they reached subconfluence, the monolayer was scratched with a 200 μL pipette tip. Cells were washed with PBS twice and cultured in FBS-free medium for additional 48 h. Cell migration into the wound area was monitored and analyzed using Leica LAS EZ software. Cell invasion experiments were performed using Bio-Coat cell migration chambers (BD Biosciences) with 8-μm-diameter. Cells resuspended in FBS-free medium were added to the Matrigel-coated upper chamber, and 20% FBS medium was placed in the lower chamber. After 48 h the invading cells on the upper surface were wiped with a cotton-tipped applicator, the invading cells on the under surface were fixed with 5% glutaraldehyde fixative and stained with Giemsa.