Boyden chamber transwell assay

MDA-MB-231-GFP cells were plated on fibronectin-coated 2D films or 3D scaffolds, placed in a live cell chamber (LiveCell™, Okolab, Ambridge, PA, USA) at 5% CO₂ and 37 °C and monitored by light microscopy (Olympus CKX41, Center Valley, PA, USA). Images of the same field were taken every hour for 48 h, and ImageJ software (NIH, Bethesda, MD, USA) [63 (link)] was used to analyze the photo series to track single cell (n = 3) movement. Chemotactic migration was quantified using a 24-well plate Boyden chamber transwell assay (8 μm pore size; Corning Costar, Tewksbury, MA, USA). 3D scaffolds were seeded with 4.0 × 10⁴ tumor cells, serum-starved for 4 h, and mounted into the upper chamber of the transwell. Medium in the lower chamber was supplemented with 10% (positive control) or 0% (negative control) FBS. After 30 h, cells present on the upper surface of the filter were removed using a sterile cotton swab, and cells that migrated through the chamber onto the lower surface were fixed and stained with Crystal Violet Dye (Sigma, St. Louis, MO, USA). The number of migrating cells was counted (n = 5 fields) and compared to the negative control. Experiments were performed in triplicate wells and repeated at least three times.

The in vitro migration assay was conducted using a Boyden chamber transwell assay (8 µm pore size; Corning Costar, cat. #3422) with a polycarbonate membrane. Cells were starved in serum-free media overnight, then introduced into the upper chamber (5×10⁴ cell/ml) for 6 or 24 hours. For the 24-h assay, mitomycin-C was added to block cell proliferation. The lower chamber was filled with growth media supplemented with 10% FBS. After incubation the cells were fixed and stained by Diff-Quik (Siemens). Migrated cells in three random fields were counted; all conditions were performed in triplicate.

Chemotactic migration was quantified by using a Boyden chamber transwell assay (8 μm pore size; uncoated filters; Corning, New York, NY, USA). Either control or Pirfenidone 1.5 mg/ml RPMI-1640 supplemented media was provided in the lower chamber and tumor cells in serum free RPMI-1640 were introduced to the upper chamber (5 × 10⁴). The cells were allowed to migrate for 16 h and then fixed with methanol and washed in PBS. Cells on the apical side of membranes were removed with cotton swaps. Remaining cells on the membrane were stained with crystal violet and imaged using a digital slide scanner (Nanozoomer 2.0 HT, Hamamatsu, Japan). ImageJ software (NIH, MD, USA) was used for image analyses. At least three independent experiments were performed with 4 technical replicates. Migration was plotted as percentage of Pirfenidone treated cells compared to PBS control.