Uncoated transwell migration chambers

Cell invasion assays were performed using uncoated transwell migration chambers (BD Bioscience, San Jose, CA, USA) in 24-well cell culture plates. Cells (5 × 10⁴/well) were loaded into the invasion chamber in serum-free RPMI. The lower chamber contained RPMI supplemented with 10% FBS as a chemoattractant. The plates were incubated at 37 °C for 24 h, followed by staining with hematoxylin–eosin and mounting. Cell invasion was imaged using an upright microscope (×200). The wound-healing assay was performed using l-Dish 35-mm-high culture inserts (Ibidi, Martinsried, Germany) according to the manufacturer’s protocols. In brief, on the day before experimentation, the cells were seeded into each well of the culture inserts and incubated at 37 °C in a humidified atmosphere with 5% CO₂. After cell attachment, the culture inserts were gently removed using tweezers, and the cells were incubated in macrophage CM) for more than 12 h.

Cell migration and invasion assays were carried out using uncoated trans-well migration chambers (BD Bioscience, San Jose, CA, USA) in 24-well cell culture plates. Cells (5×10⁴ /well) were loaded into the migration and invasion chambers in serum-free RPMI media. The lower chambers contained RPMI supplemented with 10% FBS as a chemoattractant. Plates were incubated for 24 or 48 hours. Cells were then stained with calcein (2 μM, BD Biosciences, San Jose, CA, USA) or hematoxylin-eosin and mounted. Cell migration and invasion was quantified by fluorescence measurements with a VICTOR2 Multilabel Counter (Perkin Elmer, Boston, MA, USA) equipped with a 485/520 nm filter set.