Anti citrullinated h3

Circulating neutrophils were isolated from blood harvested by cardiac puncture from mice using anti–Ly-6G MicroBeads (Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. Bone marrow (BM)–derived leukocytes were harvested from the femurs and tibias of LDLR^−/− mice by flushing with 1.5 mmol/L ethylenediaminetetra-acetic acid in Hank’s Balanced Salt Solution. After centrifugation, 1 · 10⁶ cells were seeded on a poly-l-lysine–coated 22-mm coverslip (32 (link)). These cells were incubated in Roswell Park Memorial Institute Media 1640 with 20% each of murine plasma from wt mice fed NC or HFD, LDLR^−/− mice fed NC or HFD for 28 days or recombinant mouse CXCL1 (BioLegend, Inc., San Diego, California) for 2 h. Cells were fixed by 4% paraformaldehyde in phosphate-buffered saline (PBS[–]) and stained by anti–citrullinated H3 (Abcam, Cambridge, United Kingdom), PE-antimouse Ly-6G (BioLegend, Inc.) and 4′,6-diamidino-2-phenylindole, followed by observation with a fluorescence microscope. The obtained images were analyzed with Meta Morph. The ratio of citrullinated histone H3–positive, Ly-6G–positive neutrophils in total Ly-6G–positive neutrophils was calculated.