Generation of CD18-mCFP KI mice. (A) Gene targeting procedure for CD18-mCFP KI mice. The mCFP sequence was knocked into the C terminus of the mouse integrin CD18 subunit. (B and C) Southern blot (B) and PCR (C) analyses were performed using genomic DNA isolated from the tails of WT, CD18-mCFP heterozygous (+/−), and homozygous (+/+) mice. (D) Western blot analyses for both CFP and CD18 were performed using spleen lysates from WT, heterozygous, and homozygous mice. For the Western blot analysis of CD18 integrin, WT mice and two homozygous mice with different ages (4 and 14 wk old) were used. A representative image is shown from experiments repeated three times. (E) The cell surface expression of CD18 was measured on neutrophils isolated from WT, heterozygous, and homozygous mice (blue; red, isotype control) by flow cytometry. A representative dataset is shown from experiments repeated three times. (F) The CD18-mCFP expression in splenocytes isolated from homozygous mice was visualized. Bars, 20 µm. (G) Granulocytes (Gr1⁺), macrophages (F4/80), monocytes (CD115), and T cells (CD3) were isolated from WT and CD18-mCFP mice, labeled with anti-CD18 antibody, and analyzed by flow cytometry to compare the cell surface expression of CD18. MFI, mean fluorescence intensity. (H) In vitro migration of naive CD4 T cells isolated from CD18-mCFP and WT mice was performed on ICAM-1– and CCL21-coated surfaces at 37°C. The cell migration velocity (Vel; µm/min), displacement rate (Ds; µm/min), and meandering index (Mi; µm/µm) shown in the figure were not significantly different. A dataset represents data from experiments repeated three times. (I) The local bacterial burden of WT and CD18-mCFP mice was evaluated at 6 and 24 h in the peritoneal lavage after sepsis challenge by cecal ligation and puncture. (G and I) Representative datasets are shown from experiments repeated three times. The results are expressed as the mean ± SEM.