Ab137444

Cocultures were completed and macrophages were fixed as described above. Coverslips were washed once with PBS prior to staining with SYTOX Green (final concentration, 10 μM) (ThermoFisher) for double-stranded DNA (dsDNA), and Hoechst 33342 (final concentration, 5 μM ) (ThermoFisher) for condensed chromatin (nuclei). Additional staining for histones and MMPs was accomplished by blocking cells in 1% bovine serum albumin in PBS for 30 min at 37°C followed by a 1-h incubation at 37°C with antibodies for histone H3 (ab5103; Abcam, Cambridge, MA), neutrophil elastase (ab68672; Abcam), myeloperoxidase (ab9535; Abcam), matrix metalloproteinase 1 (MMP-1) (ab551168; Abcam), MMP-7 (ab5706; Abcam), MMP-8 (ab81286; Abcam), MMP-9 (ab38898; Abcam), or MMP-12 (ab137444; Abcam). Cells were then washed three times with 1% BSA in PBS, followed by a 30-min incubation with an Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (ThermoFisher) and two additional washes with 1% BSA in PBS prior to mounting coverslips onto glass microscope slides with Aqua Poly/Mount (Polysciences Inc., Warrington, PA). Macrophages were visualized with a Zeiss LSM 710 META inverted laser scanning confocal microscope, and extracellular traps were identified by dsDNA staining that extended into the extracellular environment.