Intravital Imaging of Neutrophil Dynamics

Lyz2-GFP⁺ mice (males, 10 weeks old) were anesthetized by ketamine/xylene injection and placed on the imaging stage at 37°C. Mice were injected in the ear pinna with 10 ng of IL-1β (R&D Systems #401ML-005) with or without the CD31^agonist (50 µM). Mice were transferred immediately to the microscope stage and imaged for 15–20 min for each time point at an X-Y pixel resolution of 512 × 512. Multiphoton imaging was performed with a Zeiss LSM7 MP system equipped with both a ×10/0.3 NA air and ×20/1.0 NA water-immersion objective lens (Zeiss) and a tunable titanium/sapphire solid-state two-photon excitation source (Chameleon Ultra II; Coherent Laser Group; Stephen et al., 2017 (link)). Videos were processed offline with the ImageJ software for the generation of time-superposition images and the GFP⁺ morphological parameters; the TrackMate plugin was implemented to calculate the speed of detaching neutrophils by applying a region of interest out of the vessel wall. TrackMate was also used to calculate the migratory path and the velocity of interstitial GFP^high neutrophils. Data are from two independent experiments in which one mouse was used per condition.