Transwell with 3.0 m pore polycarbonate membrane insert

Neutrophils were obtained from peripheral blood from anonymous healthy donors over a Polymorphprep (Progen) gradient according to manufacturer procedures. Neutrophil purity was assessed by flow cytometry (>95% CD15+ cells) (CD15 Monoclonal Antibody, APC, Invitrogen Cat. No. 17-0158-41). Neutrophil migration assays were performed following the protocol previously described [57] (link) with some modifications. Briefly, 2 × 10⁵ neutrophils were added on top of the Transwell inserts (6.5 mm Transwell^® with 3.0 µm Pore Polycarbonate Membrane Insert, Corning 3415) in 200 μL of complete EGM-2MV medium (Lonza, CC-3202). The lower compartment was then filled with 600 μL of conditioned medium from untreated or CL264-treated M-MØ, RPMI-10% FBS as negative control, or 50 ng/mL recombinant human IL-8 (Immunotools) in RPMI-10% FBS as positive control. Migration was allowed for 1 h and migrated neutrophils were quantified by Flow Cytometry (CytoFLEX-S, Beckman-Coulter).

A 24 mm Transwell^® with 3.0 µm Pore Polycarbonate Membrane Insert (Corning Inc.) was used to assess the invasive capacity of SW982 cells, as previously described (24 (link)). In brief, 25 µl Matrigel (diluted with 3X serum-free medium; Sigma-Aldrich; Merck KGaA) was added to the upper chamber of the Transwell plate (Corning, Inc.) for 30 min at 37°C. A total of 0.5×10⁶ cells/ml were added into the Transwell upper chamber in serum-free DMEM, and media containing 20% FBS (Gibco; Thermo Fisher Scientific, Inc.) was added into the lower chamber. The plate was incubated at 37°C for 24 h. Subsequently, the Transwell insert was removed and washed twice with PBS, and 100% methanol was used for fixation for 30 min at room temperature, followed by drying. The membrane was stained with crystal violet for 20 min at room temperature, and the relative migration was determined by measuring the absorbance at 595 nm.