Actin Dynamics during NET Formation

NETs were induced in peripheral blood neutrophils by PMA with or without anti-MYL6 antibody as above. Before and 30 min, 1 h, and 3 h after incubation, the samples were fixed with 4% paraformaldehyde for 15 min and permeated with 0.5% Triton X-100 for 5 min at room temperature (RT). Thereafter, the samples were reacted with 1:100 dilution of anti-β-actin monoclonal antibody (mAb; mAbcam 8226, mouse IgG1; Abcam) or equivalent concentrations of isotype control mouse IgG1 (Abcam) at 4°C overnight. After rinsing with PBS, the samples were reacted with 4 μg/mL Alexa Fluor 488-conjugated goat anti-mouse IgG1 antibody (Abcam) and 100 nM Acti-stain 555 phalloidin (Cytoskeleton, Denver, CO, USA) for 1 h at RT in the dark. In cells, actin exists in two different forms: nonpolymerized granular form (G-actin) and polymerized filamentous form (F-actin). Anti-β-actin antibody recognizes G-actin [18 (link)], whereas phalloidin binds specifically to F-actin. The samples were finally mounted with the mounting solution containing DAPI. This preliminary assay revealed that G-actin polymerization into F-actin occurred in early during NET formation (0–30 min after PMA stimulation), followed by F-actin degradation (Fig. S1). These findings were consistent with previous reports of Stojkov et al. [15 (link)] and Metzler et al. [16 (link)].