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. 2014 Jun 2;211(6):1123–1136. doi: 10.1084/jem.20132112

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© 2014 Mizuno et al.

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Figure 3. — Activation of PKA during the neutrophil recruitment cascade. (A) In vivo imaging of the lamina propria of the intestinal mucosa of PKAchu mice. A representative FRET/CFP ratio image from Video 3 and a scheme are shown. Cr, crypt; Ve, venule. Gamma, 1.14. The image is representative of a mouse in five independent experiments. (B) In vivo imaging of the lamina propria of the intestinal mucosa of a C57BL/6 mouse that was transplanted with bone marrow of PKAchu mice 7 wk before the experiment. Representative FRET/CFP ratio images and CFP/Qtracker 655 images from Video 4 and schemes during neutrophil extravasation are shown. Tracking of a neutrophil is shown by arrowheads. The same cells were also marked by yellow lines. Gamma, 1.44. The image is representative of a mouse in three independent experiments. Bars: (A) 30 µm; (B) 10 µm. (C) The PKA activity change was monitored in three representative neutrophils from three independently analyzed mice. The time of transition from the adhesion to crawling steps was set as zero. The rolling, adhesion, crawling, and transmigration steps are indicated by different colors. (D) PKA activity of neutrophils in the four steps of extravasation is plotted. 30 neutrophils in each step were randomly chosen in the CFP images and examined for their PKA activity in the corresponding FRET/CFP ratio image. To accumulate the incidence, three independent experiments were performed. Dots and bars indicate the PKA activity in each neutrophil and the mean values, respectively. ***, P < 0.001 (Student’s t test). (E) 90 neutrophils migrating in the interstitial tissue were randomly chosen in the CFP images and examined for their PKA activity and migration velocity during 5 min of time-lapse imaging. Results obtained from three mice were combined. The red line is an approximate curve showing the inverse correlation between PKA activity and migration velocity.