LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) distinctly regulate neutrophil extravasation through hotspots I and II.

Animals

C57BL/6 and LysM-GFP 24 were purchased from Jackson Laboratory and maintained in a specific pathogen-free environment at the University of Rochester’s Animal Facility. CD11b-mYFP knock-in mice were generated at the Gene Targeting and Transgenic Core Facility at the University of Rochester using gene targeting techniques similar to those previously used in our laboratory to generate the CD18-mCFP and CD11a-mYFP knock-in mice and the K562 Mac-1 FRET cell line 9 , 21 , 25 . All mice were backcrossed to C57BL/6 for at least six generations.

LFA-1 FRET

(CD11a-mYFP/CD18-mCFP) mice were subsequently generated by mating the CD11a-mYFP and CD18-mYFP knock-in mice.

Mac-1 FRET

(CD11b-mYFP/CD18-mCFP) mice were generated by mating the CD11b-mYFP and CD18-mCFP knock-in mice. Two-photon intravital microscopy of blood vessels in the mouse cremaster muscle To visualize neutrophil motility during extravasation, two-photon intravital microscopy was performed using an Olympus FV1000-AOM multi-photon system equipped with a ×25 NA 1.05 water immersion objective. For two-photon excitation, a Spectra-Physics MaiTai HP Ti:Sa Deep See laser system was tuned to 900 nm for green fluorescent protein (GFP) and Texas Red and 840 nm for CFP, YFP, and Texas Red. The images were acquired at a resolution of 256 × 256 pixels with a pixel dwell time of 2 μs using step sizes of 1 μm to a depth of 25–30 μm every 30 s. To image the cremaster muscle, the mice were initially anesthetized via intraperitoneal injection of pentobarbital sodium (a dose of 65 mg/kg), and the hair on the skin of the imaging area was removed. To image the cremaster blood vessels, the right cremaster muscle was exteriorized and covered with pre-warmed physiological solution at 36 °C. The solution contained the following (in mM): NaCl, 131.9; KCl, 4.7; CaCl 2 , 2.0; MgSO 4 , 1.2; and NaHCO 3 , 18 (pH 7.4). The solution was equilibrated with gas containing 0% O 2 , 5% CO 2 , and 95% N 2 , to maintain the tissue PO 2