Pretreatment with GSK484 markedly diminished NET formation ex vivo and in vivo. (A) Immunofluorescence of H3Cit (red), MPO (green), and DAPI (blue) in primary neutrophils isolated from C57 BL/6J mice with ex vivo stimulation by LPS (200 μg mL-1), GSK484 (10 μmol/L), and PBS to detect the formation of NETs. Scale bar: 5 μm. (B) The quantification of cells that have undergone NET formation. (C) Representative images of immunofluorescence of H3Cit (red), MPO (green), and DAPI (blue) in liver sections from 10-week-old male C57 BL/6J mice with pretreatment of GSK484 (20 mg kg-1) and D-GalN/LPS–induced ALF according to the procedures as indicated in Figure 4. Scale bar: 5 μm. (D) The formation of NETs in panel C was quantified as the percentage of the H3Cit-positive area per field. ∗∗P < .01.