RvD4 decreases neutrophil and macrophage recruitment in the thrombus and inhibits NETosis. (A, top) Flow cytometry strategy gated on CD45+ leukocytes isolated from mouse thrombi (8 days after IVC stenosis). Monocytes (CD45+Ly6C+F4/80−), macrophages (CD45+Ly6C+F4/80+), and neutrophils (CD45+Ly6GhighLy6C−F4/80−) were identified in the thrombus (8 days after IVC stenosis). (Bottom) Diff-Quik staining of neutrophils, macrophages, and monocytes from mouse thrombus. Scale bar, 20 μm, ×40. (B, top) Percentage of neutrophils, macrophages, and monocytes obtained by flow cytometry in the thrombus 8 days after IVC stenosis of mice treated with vehicle or RvD4. (Bottom) Total neutrophil, macrophage, and monocyte counts in the thrombus. (C) Cell viability in the thrombus 8 days after IVC stenosis was measured using propidium iodide (PI) and annexin V staining. (D) Percentage of live cells (Annexin V−PI−), cells in early apoptosis (Annexin V+PI−), and cells in late apoptosis (Annexin V+PI+) in mouse thrombus (8 days after IVC stenosis) after treatment with vehicle or RvD4. (E-F) Neutrophils were isolated from peripheral blood of vehicle-treated or RvD4-treated mice 24 hours after treatment and kept unstimulated, or were stimulated with ionomycin for 4 hours. (E) Representative fluorescence microscopy images of ionomycin-treated neutrophils illustrating H4Cit+ neutrophils releasing NETs (white arrowheads). H4Cit, green; DNA, blue. (F) Quantification of the percentage of NET-releasing cells. n = 6. Scale bar, 20 µm. *P < .05; **P < .01; ***P < .001 with 2-tailed unpaired t test for comparison of vehicle to RvD4.