ConA–mediated NET formation in vivo

Male mice (8 to 10 weeks old) were administered ConA at a dose of 15 mg/kg in sterile PBS by tail vein injection. After 12 hours, the mice were anesthetized with isoflurane, bled by cardiac puncture, and euthanized by rapid cervical dislocation. The blood was collected in tubes containing 50 μl of 5 mM EDTA. To obtain plasma, the blood was centrifuged at 3000g for 5 min at 4°C. RBCs were lysed with hypotonic ACK lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, and 100 nM Na₂EDTA). The remaining leukocytes were lysed in 1 ml of TRIzol for gene expression analysis.