Actin Polymerization Assay

Isolated PMNs (500.000 cells per measurement) were stimulated with fMLP (100 ng/ml) or left untreated for 5, 15, 30, 60, 120 and 180 sec. For every time point, regardless of stimulation, reaction was stopped by adding a Fixation Buffer (BioLegend) for 20 min at room temperature. Thereafter, cells were permeabilized and blocked to avoid non-specific binding. Following 20 min incubation, cells were intracellularly stained with FITC-conjugated Phalloidin (Abcam, 1:1000). As negative control, PMNs were left either unstimulated or unstained. Following 30 min incubation in the dark at 4°C, actin polymerization was determined by the level of Phalloidin expression, as measured by flow cytometry.