Intravital Microscopy of Leukocyte Dynamics

Intravital microscopy was performed as previously described (Phillipson et al., 2009 (link)). Exposed cremasteric muscle was superfused with 1 µM fMLP and visualized with an intravital microscope (Axiolskip; ZEISS) connected to a video camera (5100 HS; Panasonic) using 25× (0.35 N, Fluotar; Leitz) and 40× (0.80 NA, Achroplan; ZEISS) objective lenses. The same five sections of single unbranched cremasteric venules (20–40 µm in diameter) were observed for a given experiment. Adhesion, crawling, and emigration were determined during video playback. Rolling flux and rolling velocity were determined concurrently with intravital analysis of cell adhesion, crawling, and emigration. Rolling leukocytes were defined as those cells moving at a velocity less than that of erythrocytes within a chosen vessel.

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Fine N., Dimitriou I.D., Rullo J., Sandí M.J., Petri B., Haitsma J., Ibrahim H., La Rose J., Glogauer M., Kubes P., Cybulsky M, & Rottapel R. (2016). GEF-H1 is necessary for neutrophil shear stress–induced migration during inflammation. The Journal of Cell Biology, 215(1), 107-119.

Publication 2016

Achroplan

Cell adhesion Cells Cremasteric Erythrocytes Intravital microscope Lenses Leukocytes Venules Vessel

Corresponding Organization : University Health Network

Other organizations : University of Calgary, Amsterdam UMC Location VUmc

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Variable analysis

independent variables

Superfusion of exposed cremasteric muscle with 1 µM fMLP

dependent variables

Adhesion
Crawling
Emigration
Rolling flux
Rolling velocity

control variables

Cremasteric muscle
Cremasteric venules (20–40 µm in diameter)
Intravital microscope (Axiolskip; ZEISS) connected to a video camera (5100 HS; Panasonic)
Objective lenses (25× (0.35 N, Fluotar; Leitz) and 40× (0.80 NA, Achroplan; ZEISS))

controls

The same five sections of single unbranched cremasteric venules were observed for a given experiment.

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