Activation of ERK in neutrophils during adhesion to endothelial cells of the inflamed small intestine. (A) In vivo imaging of the lamina propria of the intestinal mucosa from an Eisuke mouse, which was subjected to LPS and fMLP 2 h before imaging. The representative FRET/CFP ratio image is shown in intensity-modulated display mode with 32-intensity in 8-ratio and a CFP image in grayscale from Video 1, with a schematic view of this region. Cr, crypt; Ve, venule. Gamma, 1.7. The image is representative of a mouse in five independent experiments. (B) ERK activities in three representative neutrophils from three independent experiments are plotted against time. The transition point from the adhesion to crawling steps was set as time 0. The rolling, adhesion, crawling, and transmigration steps are indicated by different colors. (C) ERK activities from three representative neutrophils were recorded with a shorter interval than in B, at one image every 1.5 s, to discriminate arrest and spreading phases of the adhesion step. Neutrophils were defined as arrest when they stopped rolling for >30 s. The start of spreading was defined when neutrophils changed from a round to an amoeboid shape. (D) Schematic of the four steps of extravasation. Arrowheads indicate the same neutrophil at different time points. (E) Time-lapse FRET/CFP and CFP images of neutrophil extravasation. The boxed region in A was magnified and shown in a time series. The arrowheads indicate the same neutrophil traced in the video. Bars: (A) 100 µm; (E) 10 µm. (F) ERK activity of neutrophils during neutrophil recruitment to the inflamed tissue. 30 neutrophils in each step were randomly selected in the CFP images and examined for their ERK activity in the corresponding FRET/CFP ratio image. Results obtained from three mice are combined. Black dots and red bars indicate the ERK activity in each neutrophil and the mean values, respectively. Difference from the rolling cells was evaluated by the Student’s t test: **, P < 0.01; ***, P < 0.001.