AR-targeted disruption and analysis of peripheral neutrophils in ARKO mice. (A) Schematic representation of murine AR protein domains and corresponding exons in AR mRNA. The AR mRNA is composed of seven exons; exon 2 is removed in ARKO mice. DBD, DNA-binding domain. LBD, ligand-binding domain. (B) The purity of isolated bone marrow neutrophils from WT and ARKO mice is >90%. (C) Using the primers 5′-AATGGGACCTTGGATGGAGAAC-3′ and 5′-TCCCTGCTTCATAACATTTCCG-3′, an AR transcript of 305 bp will be obtained from WT AR, but only 153 bp when floxed exon 2 is deleted. Full-length AR mRNA is not expressed in neutrophils isolated from bone marrow of AR-deficient mice. (D) Intracellular AR protein is not detected in neutrophils isolated from bone marrow of AR-deficient mice. Nonspecific IgG was used as primary antibody for the control. (E) FACS analysis of peripheral blood from 8-wk-old WT and ARKO mice. FSC and SSC of peripheral leukocytes show significant decreases of high-SSC granulocytic population in ARKO mice (n = 4) compared with WT littermates (n = 4). (F) FACS analyses with neutrophil-specific antibodies (clone 7/4) and anti-CD11b antibodies. Neutrophils are drastically reduced in ARKO mice (n = 4) compared with WT littermates (n = 4). (G) Representative photomicrographs of Wright-Giemsa–stained cells on cytospins from blood after erythrocyte lysis. Neutrophils (arrow heads) are rarely found on leukocyte cytospins from ARKO mice compared with WT controls. Bar, 10 µm. (H) Manual differential counting of neutrophils was performed on blood smears from 9-wk-old sex-matched (WT), ARKO (KO), and Tfm male mice. Results are representative of four separate experiments (n = 4 per group). Data represent the mean ± SD. **, P < 0.01 compared with WT mice. ***, P < 0.001 compared with WT mice.