24 well invasion chamber

Cells (1.5 × 10⁵/well) were seeded into six-well plates in 2 ml EMEM+/+ and incubated for 24 h. The cells were detached with Accutase (Sigma-Aldrich) and washed in PBS−/−, starved for 4 h, and treated with vehicle or 5 μg/ml MPO. A 24-well invasion chamber (#354480 Corning) was used. The chamber was activated by the addition of warm (37 °C) EMEM−/− to the interior of the inserts (0.5 ml) and bottom of the wells (0.5 ml) for 2 h. Upon activation, the medium was removed, and 750 μl EMEM +/+ was added to the bottom well. The treated cell suspension was added to the insert and incubated for 24 h at 37 °C, and 5% CO₂. Afterwards, non-invaded cells were removed from the upper part of the membrane by wiping with cotton swab over the membrane of the chamber. The invaded cells were fixed and stained with Diff-Quick solution. A scalpel was used to remove the membrane from the insert and placed on a microscopy slide. Five photos of each membrane were captured at 100 × magnification using Olympus B×53 with an Olympus UC90 camera. CellSense Standard software was used. ImageJ software (R Foundation) cell counter function was used to count the invaded cells.

Cell migration was determined according to a previously reported method with modifications^{37 (link)} with a 24-well invasion chamber containing an upper and lower chamber with an 8 μm pore in between (Corning Life Science, Acton, MA). Briefly, cells were harvested and resuspended in DMEM containing 1% (v/v) fetal bovine serum. Subsequently, 100 μl of cell suspension (1 × 10⁵ cells in total) was added into the upper chamber while the lower chamber was filled with 600 μl of DMEM containing 10% (v/v) fetal bovine serum without or with 20 μM ATO. After 24 hours of incubation, adherent cells in the lower chamber were fixed with methanol and stained with 10% (w/v) Giemsa. The pictures of five randomly chosen areas were captured with a microscope at 100× magnification for quantification analysis.