Figure 1.

Experimental staining scheme for specific early NETosis detection by imaging flow cytometry. Briefly, purified neutrophils were allowed to rest in the incubator for 30 min (30') prior to the treatment step. NETs agonists were added at the concentrations specified in Table 1 for various times between 2 min (2') and 60 min (60'). Stimulation was stopped by adding paraformaldehyde (PFA) for 30 min (30') at room temperature. Staining steps are underlined in the gray boxes and staining times for each step are in red. Total protocol time from neutrophil isolation to stained samples ready for imaging flow cytometer acquisition is 3 days. (ON, over night; RT, room temperature).