CXCR4 activation by CXCL12 attenuated the cell death of mouse neutrophils isolated from the injured lungs. Neutrophils were isolated from the mouse lungs at 24 h after LPS instillation. The isolated neutrophils were cultured in either media alone (RPMI 1640 supplemented with 10% FCS), media containing CXCL12, media containing CXCL12 and a specific CXCR4 antagonist or media with a specific CXCR4 antagonist alone for 24 h at 37 °C. Subsequently, cell death was assessed by trypan blue staining (a). Staining with Annexin V and 7-AAD was also performed to detect early apoptotic (Annexin V+7-AAD−; b, c) cells and late apoptotic/necrotic (Annexin V+7-AAD+; b, d) cells. Representative dot plots are shown. A total of six mice were used in each group. The values represent mean±s.e.m. *P<0.01 versus before culture group and †P<0.01 versus media only group using ANOVA with Scheffé's post hoc test. 7-AAD, 7-aminoactinomycin D; FCS, fetal calf serum.