4.7. Under-Agarose Assay

Neutrophil chemotaxis was evaluated by using the under agarose assay essentially as described elsewhere [41]. Briefly, Petri dishes (35 × 10 mm) were coated with 10% FBS in PBS for 30 min at room temperature and washed twice with PBS. Then, the plates were filled with 3 mL of a 0.45% agarose dissolved in 50% HEPES-buffered complete HBSS and 50% supplemented with 20% FBS. The agarose was then allowed to solidify and two wells 3.5 mm diameter and 2.2 mm apart were cut into each gel. The gels were then equilibrated at 37 °C for 1 h and 10 µL of chemokine (1 pmol of fMLP or 10 pmol of IL-8) were loaded in the outer well, whereas 10 µL of 1 × 10⁷ cells/mL (1 × 10⁵ cells) not treated or pretreated for 15 min with 5 mg/mL of HES dissolved in complete HBSS were loaded in the inner well. After 1.5 h of incubation at 37 °C, images of migrating neutrophils were acquired every 20 s for 20 min, with a Canon EOS 700D digital reflex attached to an inverted microscope (Olympus IMT-2) equipped with a thermostatic chamber. The tracks of the migrating cells were then acquired by using ImageJ with multitracker plugin and the coordinates of the cells were analyzed by using a free software (Ibidi Chemotaxis and Migration Tool), in order to obtain quantitative parameters of migration such as FMI (forward migration index, parallel to the gradient and calculated as the ratio between the x coordinate of the cell’s end point of migration and the total distance accumulated), directionality (representing a measurement of the directness of cell trajectories, the ratio of the Euclidian distance and the total accumulated distance of a cell) and velocity (displacement/time).