Cell Migration Assay with Microchemotaxis

Cell migration was assessed in a 48-well microchemotaxis chamber (Neuroprobe, Gaithersburg, MD, USA) as described previously [19 (link)]. The chamber consisted of acrylic top and bottom plates, each containing 48 matched wells. Twenty-six microliters of FBS-free medium containing tested substance (10 μg/ml) were filled in wells of the bottom plate. Wells filled with medium containing 0.0001 % of acetic acid served as control. Subsequently, the bottom plate was covered with a polycarbonate filter with 8-μm pore size (Neuroprobe, Gaithersburg, MD, USA) and the top plate was applied so that each well corresponded to that of the bottom plate. 1 × 10⁴ cells resuspended in 50 μl FBS-free medium were added to each well of the top plate and the whole chamber was incubated at 37 °C in humidified air with 5 % CO₂ for 8 h. After incubation, cells on the upper surface of the filter were removed over the wiper blade and the filters were then fixed with methanol and stained using Hemacolor staining kit (Merck, Darmstadt, Germany). The cells migrated across the filter were counted under a light microscope at high-power magnification (×100) to measure transmigration in each well. Four fields were counted in each well and the total number was calculated. Four wells were used for each group; experiments were repeated in triplicate.