Inflammatory, agar-elicited neutrophils spontaneously die of NETosis ex vivo. (A) PMN cell death was evaluated by annexin V/7-AAD staining of CD11b⁺Gr-1⁺ cells. Early apoptotic cells (annexin V⁺/7-AAD⁻), apoptotic cells (annexin V⁺/7-AAD⁺), necrotic cells (annexin V⁻/7-AAD⁺), and live cells (annexin V⁻/7-AAD⁻) were identified. Representative forward scatter (FSC) and side scatter (SSC) plots are shown. (B) Activation of NADPH oxidase in PMNs from agar and in PMA-treated naive PMNs seeded onto poly-D-lysine–coated glasses. Activation of NADPH oxidase was qualitatively evaluated using the nitroblue tetrazolium (NBT) reduction test. Contrast-phase pictures obtained under a light microscope show that both agar-PMNs and naive PMNs treated with PMA produce ROS (blue precipitate). (C) Representative IF analysis shows a more efficient NETosis in agar-collected than PMA-treated spleen-derived PMNs (original magnification ×20). SYTOX green DNA dye was added to PMN after their fixation with 4% PFA. (D) Quantification of NET formation in agar-collected and PMA-treated neutrophils. Nuclear area of agar-PMN and PMA-treated neutrophils is plotted against the percentage of SYTOX-positive cells corresponding to a given nuclear area. PMNs undergoing NETosis have a broad range of nuclear areas, whereas apoptotic neutrophils show smaller nuclear areas, less distributed and composed within a narrow peak (top panel). In line, agar-PMNs showed higher mean nuclear area compared with PMA-treated spleen PMN (bottom panel). In this experiment, the SYTOX green DNA dye was added to PMNs after PFA fixation 18 hours later. Experiments comparing agar-PMNs with PMA-treated PMNs were repeated 6 times with PMNs from at least 3 donor mice each. Number of nuclei analyzed/experiment = 200.